67 results on '"Hsou-min"'
Search Results
2. Chloroplast import motor subunits FtsHi1 and FtsHi2 are located on opposite sides of the inner envelope membrane.
- Author
-
Chia-Yun, Lih-Jen, and Hsou-min
- Subjects
MOLECULAR motor proteins ,MALATE dehydrogenase ,MEMBRANE proteins ,ALKALINE protease ,ADENOSINE triphosphatase ,NEUROREHABILITATION - Abstract
Protein import into chloroplasts is powered by ATP hydrolysis in the stroma. Establishing the identity and functional mechanism of the stromal ATPase motor that drives import is critical for understanding chloroplast biogenesis. Recently, a complex consisting of Ycf2, FtsHi1, FtsHi2, FtsHi4, FtsHi5, FtsH12, and malate dehydrogenase was shown to be important for chloroplast protein import, and it has been proposed to act as the motor driving protein translocation across the chloroplast envelope into the stroma. To gain further mechanistic understanding of how the motor functions, we performed membrane association and topology analyses on two of its subunits, FtsHi1 and FtsHi2. We isolated cDNA clones encoding FtsHi1 and FtsHi2 preproteins to perform in vitro import experiments in order to determine the exact size of each mature protein. We also generated antibodies against the C-termini of the proteins, i.e., where their ATPase domains reside. Protease treatments and alkaline and high-salt extractions of chloroplasts with imported and endogenous proteins revealed that FtsHi1 is an integral membrane protein with its C-terminal portion located in the intermembrane space of the envelope, not the stroma, whereas FtsHi2 is a soluble protein in the stroma. We further complemented an FtsHi1-knockout mutant with a C-terminally tagged FtsHi1 and obtained identical results for topological analyses. Our data indicate that the model of a single membrane-anchored pulling motor at the stromal side of the inner membrane needs to be revised and suggest that the Ycf2-FtsHi complex may have additional functions. [ABSTRACT FROM AUTHOR]
- Published
- 2023
- Full Text
- View/download PDF
3. Increased ratio of galactolipid MGDG : DGDG induces jasmonic acid overproduction and changes chloroplast shape
- Author
-
Chun-Wei Yu, Hsou-min Li, and Yang-Tsung Lin
- Subjects
0106 biological sciences ,0301 basic medicine ,Chloroplasts ,Galactolipid ,Photosystem II ,Physiology ,Cyclopentanes ,macromolecular substances ,Plant Science ,Photosynthesis ,01 natural sciences ,Chloroplast membrane ,03 medical and health sciences ,chemistry.chemical_compound ,chloroplast ,digalactosyl diacylglycerol (DGDG) ,dgd1 ,Oxylipins ,Overproduction ,Diacylglycerol kinase ,Full Paper ,Galactolipids ,Research ,Jasmonic acid ,food and beverages ,Full Papers ,Chloroplast ,jasmonic acid (JA) ,monogalactosyl diacylglycerol (MGDG) ,030104 developmental biology ,Biochemistry ,chemistry ,membranes ,galactolipid ,010606 plant biology & botany - Abstract
Summary Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG‐synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown.We introduced an inducible microRNA (ami‐MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA‐biosynthesis and JA‐responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated.Expression of JA‐biosynthesis and JA‐responsive genes were reduced in amiR‐MGD1‐transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued.Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG.
- Published
- 2020
- Full Text
- View/download PDF
4. Tissue-Specific Regulation of Plastid Protein Import via Transit-Peptide Motifs
- Author
-
Chiung-Chih Chu, Krishna B S Swamy, and Hsou-min Li
- Subjects
0106 biological sciences ,0301 basic medicine ,Nuclear gene ,Amino Acid Motifs ,Arabidopsis ,Plant Science ,Protein Sorting Signals ,Plant Roots ,01 natural sciences ,In Brief ,Chloroplast Proteins ,03 medical and health sciences ,Gene Expression Regulation, Plant ,Transit Peptide ,Organelle ,Chromoplast ,Protein Isoforms ,Amino Acid Sequence ,Plastids ,Plastid ,biology ,fungi ,Peas ,food and beverages ,Leucoplast ,Cell Biology ,biology.organism_classification ,Cell biology ,Chloroplast ,Protein Transport ,030104 developmental biology ,Organ Specificity ,Mutation ,010606 plant biology & botany - Abstract
Plastids differentiate into various functional types (chloroplasts, leucoplasts, chromoplasts, etc.) that have distinct proteomes depending on the specific tissue. Most plastid proteins are encoded by the nuclear genome, synthesized as higher molecular mass preproteins with an N-terminal transit peptide, and then posttranslationally imported from the cytosol. Evidence for tissue-specific regulation of import into plastids, and subsequent modulation of plastid proteomes, has been lacking. We quantified protein import into isolated pea (Pisum sativum) leaf chloroplasts and root leucoplasts and identified two transit-peptide motifs that specifically enhance preprotein import into root leucoplasts. Using a plastid preprotein expressed in both leaves and roots of stable transgenic plants, we showed that losing one of the leucoplast motifs interfered with its function in root leucoplasts but had no effect on its function in leaf chloroplasts. We assembled a list of all Arabidopsis (Arabidopsis thaliana) plastid preproteins encoded by recently duplicated genes and show that, within a duplicated preprotein pair, the isoform bearing the leucoplast motif usually has greater root protein abundance. Our findings represent a clear demonstration of tissue-specific regulation of organelle protein import and suggest that it operates by selective evolutionary retention of transit-peptide motifs, which enhances import into specific plastid types.
- Published
- 2020
- Full Text
- View/download PDF
5. Protein Import Motors in Chloroplasts: On the Role of Chaperones
- Author
-
Steven M. Theg, Hsou-min Li, and Danny J. Schnell
- Subjects
0106 biological sciences ,0301 basic medicine ,food and beverages ,Cell Biology ,Plant Science ,Biology ,Plant cell ,01 natural sciences ,Malate dehydrogenase ,Transport protein ,Chloroplast ,03 medical and health sciences ,030104 developmental biology ,Biochemistry ,Temperature sensitive ,Protein translocation ,010606 plant biology & botany - Abstract
A recent article in The Plant Cell reported the identification of a Ycf2 (hypothetical chloroplast open reading frame2)-FtsHi (filamentous temperature sensitive inactive)-NAD+ malate dehydrogenase (MDH) complex and proposed it to be the motor for protein translocation into the chloroplast ([Kikuchi
- Published
- 2020
- Full Text
- View/download PDF
6. Protein Targeting to the Chloroplast Outer Membrane
- Author
-
Tu, Shuh-Long, Li, Hsou-min, and Garab, G., editor
- Published
- 1998
- Full Text
- View/download PDF
7. Chloroplast import of an intermembrane space protein is facilitated by translocon components Toc75 and Tic236
- Author
-
Hsou-min Li, Meng-Rong Chuang, and Lih-Jen Chen
- Subjects
Ecology ,Chemistry ,Botany ,Plant Science ,Translocon ,Biochemistry, Genetics and Molecular Biology (miscellaneous) ,Chloroplast membrane ,Transport protein ,Cell biology ,Chloroplast ,Tic236 ,intermembrane space ,Thylakoid ,QK1-989 ,Inner membrane ,protein transport ,mar1 ,Tic22 ,Bacterial outer membrane ,Intermembrane space ,Toc75 ,Ecology, Evolution, Behavior and Systematics ,chloroplast envelope ,Original Research - Abstract
Chloroplasts are divided into six subcompartments: the outer membrane, intermembrane space, and inner membrane of the envelope, the stroma, the thylakoid membrane, and the thylakoid lumen. Compared with our knowledge of protein import into other subcompartments, extremely little is known about how proteins are imported into the intermembrane space of the envelope. Tic22 was one of the first proteins identified as localizing to the intermembrane space and the only one for which import has been analyzed in some detail. However, conflicting results have been obtained concerning whether the general translocon is used to import Tic22 into the intermembrane space. Taking advantage of available translocon component mutants, we reanalyzed import of Tic22. We reveal reduced in vitro import of Tic22 preprotein (prTic22) into chloroplasts isolated from the Arabidopsis mar1 and tic236 mutants, which are functional knockdown mutants of the outer‐membrane channel Toc75 and the intermembrane space linker Tic236, respectively. Import competition experiments also showed that prTic22 import was reduced by excess amounts of a stroma‐targeted preprotein. Our results indicate that prTic22 uses at least part of the general translocon for import into the intermembrane space.
- Published
- 2021
8. TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import
- Author
-
Sihui Luo, Peilin Wang, Chanhong Kim, Hsou-min Li, Dogra, Chen L, Jingyun Fang, Bufan Li, and Ildoo Hwang
- Subjects
fungi ,Mutant ,food and beverages ,Biology ,Chloroplast outer membrane ,Translocon ,biology.organism_classification ,Cell biology ,Ubiquitin ligase ,Chloroplast ,biology.protein ,Arabidopsis thaliana ,Plastid ,Genetic screen - Abstract
SummaryThe chloroplast translocons TOC75 and TIC236 are homologs of the bacterial translocation and assembly module (Tam) A and TamB involved in protein export. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen aimed at finding crl suppressors revealed multiple TIC236 gain-of-function mutations (TIC236GFs). Despite the low sequence identity between TIC236 and bacterial TamB, each mutated TIC236GF residue is conserved in TamB. Consistently, a tic236- knockdown mutant exhibited multiple lesion phenotypes similar to crl, indicating a shared functionality of CRL and TIC236. Ensuing reverse genetic analyses revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Consistent with impaired plastid division exhibited by both crl and tic236-knockdown mutants, CRL interacts with the transit peptides of proteins essential in plastid division, and TIC236GF mutant proteins reinforce their import via increased TIC236 stability. Overall, our data shed new light on the links between plastid division, plant stress response and plastid protein import. We have also isolated and characterized the first GF mutants exhibiting increased protein import efficiency, which may inspire chloroplast engineering for agricultural advancement.
- Published
- 2021
- Full Text
- View/download PDF
9. Toc GTPases
- Author
-
Li, Hsou-min, Kesavulu, Muppuru M., Su, Pai-Hsiang, Yeh, Yi-Hung, and Hsiao, Chwan-Deng
- Published
- 2007
- Full Text
- View/download PDF
10. Developmental regulation of protein import into plastids
- Author
-
Chiung-Chih Chu and Hsou-min Li
- Subjects
0301 basic medicine ,fungi ,Plant Development ,food and beverages ,Leucoplast ,Cell Biology ,Plant Science ,General Medicine ,Protein Sorting Signals ,Biology ,Translocon ,Biochemistry ,Cell biology ,Chloroplast ,Chloroplast Proteins ,Protein Transport ,03 medical and health sciences ,030104 developmental biology ,Organ Specificity ,Transit Peptide ,Proteome ,Plastids ,Plastid - Abstract
The plastid proteome changes according to developmental stages. Accruing evidence shows that, in addition to transcriptional and translational controls, preprotein import into plastids is also part of the process regulating plastid proteomes. Different preproteins have distinct preferences for plastids of different tissues. Preproteins are also divided into at least three age-selective groups based on their import preference for chloroplasts of different ages. Both tissue and age selectivity are determined by the transit peptide of each preprotein, and a transit-peptide motif for older-chloroplast preference has been identified. Future challenges lie in identifying other motifs for tissue and age selectivity, as well as in identifying the receptor components that decipher these motifs. Developmental regulation also suggests that caution should be exercised when comparing protein import data generated with plastids isolated from different tissues or with chloroplasts isolated from plants of different ages.
- Published
- 2018
- Full Text
- View/download PDF
11. Chloroplast Galactolipids: The Link Between Photosynthesis, Chloroplast Shape, Jasmonates, Phosphate Starvation and Freezing Tolerance
- Author
-
Chun-Wei Yu and Hsou-min Li
- Subjects
0106 biological sciences ,0301 basic medicine ,Physiology ,Chemistry ,Jasmonic acid ,food and beverages ,Galactolipids ,Cell Biology ,Plant Science ,General Medicine ,Photosynthesis ,01 natural sciences ,Chloroplast membrane ,Cell biology ,Chloroplast ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Biosynthesis ,Thylakoid ,Jasmonate ,010606 plant biology & botany - Abstract
Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) together constitute approximately 80% of chloroplast lipids. Apart from facilitating the photosynthesis light reaction in the thylakoid membrane, these two lipids are important for maintaining chloroplast morphology and for plant survival under abiotic stresses such as phosphate starvation and freezing. Recently it was shown that severe growth retardation phenotypes of the DGDG-deficient mutant dgd1 were due to jasmonate overproduction, linking MGDG and DGDG homeostasis with phytohormone production and suggesting MGDG as a major substrate for jasmonate biosynthesis. Induction of jasmonate synthesis and jasmonic acid (JA) signaling was also observed under conditions of phosphate starvation. We hypothesize that when DGDG is recruited to substitute for phospholipids in extraplastidic membranes during phosphate deficiency, the altered MGDG to DGDG ratio in the chloroplast envelope triggers the conversion of galactolipids into jasmonates. The conversion may contribute to rebalancing the MGDG to DGDG ratio rapidly to maintain chloroplast shape, and jasmonate production can reduce the growth rate and enhance predator deterrence. We also hypothesize that other conditions, such as suppression of dgd1 phenotypes by trigalactosyldiacylglycerol (tgd) mutations, may all be linked to altered jasmonate production, indicating that caution should be exercised when interpreting phenotypes caused by conditions that may alter the MGDG to DGDG ratio at the chloroplast envelope.
- Published
- 2018
- Full Text
- View/download PDF
12. Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts
- Author
-
Hsou-min Li and Lih-Jen Chen
- Subjects
0106 biological sciences ,0301 basic medicine ,Toc complex ,Chloroplasts ,Immunoblotting ,Arabidopsis ,Plant Science ,Mitochondrion ,01 natural sciences ,03 medical and health sciences ,Genetics ,Arabidopsis thaliana ,Protein Precursors ,Plant Proteins ,biology ,Peas ,Native Polyacrylamide Gel Electrophoresis ,Cell Biology ,biology.organism_classification ,Translocon ,Chloroplast ,Protein Transport ,030104 developmental biology ,Membrane ,Biochemistry ,Protein Translocation Systems ,Biophysics ,Electrophoresis, Polyacrylamide Gel ,010606 plant biology & botany - Abstract
Summary Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC–TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC–TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC–TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures.
- Published
- 2017
- Full Text
- View/download PDF
13. Chloroplast Preproteins Bind to the Dimer Interface of the Toc159 Receptor during Import
- Author
-
Yi-Hung Yeh, Chwan-Deng Hsiao, Jun-Shian Chang, Hsou-min Li, and Lih-Jen Chen
- Subjects
0301 basic medicine ,Physiology ,Dimer ,food and beverages ,Plant Science ,Plasma protein binding ,GTPase ,Biology ,Chloroplast ,03 medical and health sciences ,chemistry.chemical_compound ,030104 developmental biology ,Protein structure ,Biochemistry ,chemistry ,Transit Peptide ,Genetics ,Biophysics ,Chloroplast Proteins ,Peptide sequence - Abstract
Most chloroplast proteins are synthesized in the cytosol as higher molecular weight preproteins and imported via the translocons in the outer (TOC) and inner (TIC) envelope membranes of chloroplasts. Toc159 functions as a primary receptor and directly binds preproteins through its dimeric GTPase domain. As a first step toward a molecular understanding of how Toc159 mediates preprotein import, we mapped the preprotein-binding regions on the Toc159 GTPase domain (Toc159G) of pea (Pisum sativum) using cleavage by bound preproteins conjugated with the artificial protease FeBABE and cysteine-cysteine cross-linking. Our results show that residues at the dimer interface and the switch II region of Toc159G are in close proximity to preproteins. The mature portion of preproteins was observed preferentially at the dimer interface, whereas the transit peptide was found at both regions equally. Chloroplasts from transgenic plants expressing engineered Toc159 with a cysteine placed at the dimer interface showed increased cross-linking to bound preproteins. Our data suggest that, during preprotein import, the Toc159G dimer disengages and the dimer interface contacts translocating preproteins, which is consistent with a model in which conformational changes induced by dimer-monomer conversion in Toc159 play a direct role in facilitating preprotein import.
- Published
- 2017
- Full Text
- View/download PDF
14. TIC236 gain-of-function mutations unveil the link between plastid division and plastid protein import.
- Author
-
Jun Fang, Bingqi Li, Lih-Jen Chen, Dogra, Vivek, Shengji Luo, Wangpin Wu, Pengcheng Wang, Inhwan Hwang, Hsou-min Li, and Chanhong Kim
- Subjects
GAIN-of-function mutations ,UBIQUITIN ligases ,MEMBRANE proteins ,CHLOROPLAST membranes ,GENETIC testing ,COMMERCIAL products - Abstract
TIC236 is an essential component of the translocon for protein import into chloroplasts, as evidenced by the embryonic lethality of the knockout mutant. Here, we unveil a TIC236-allied component, the chloroplast outer membrane protein CRUMPLED LEAF (CRL), absence of which impairs plastid division and induces autoimmune responses in Arabidopsis thaliana. A forward genetic screen exploring CRL function found multiple dominant TIC236 gain-of-function (tic236-gf) mutations that abolished crl-induced phenotypes. Moreover, CRL associates with TIC236, and a tic236-knockdown mutant exhibited multiple lesions similar to the crl mutant, supporting their shared functionality. Consistent with the defective plastid division phenotype of crl, CRL interacts with the transit peptides of proteins essential in plastid division, with tic236-gf mutations reinforcing their import via increased TIC236 stability. Ensuing reverse genetic analyses further revealed genetic interaction between CRL and SP1, a RING-type ubiquitin E3 ligase, as well as with the plastid protease FTSH11, which function in TOC and TIC protein turnover, respectively. Loss of either SP1 or FTSH11 rescued crl mutant phenotypes to varying degrees due to increased translocon levels. Collectively, our data shed light on the links between plastid protein import, plastid division, and plant stress responses. [ABSTRACT FROM AUTHOR]
- Published
- 2022
- Full Text
- View/download PDF
15. TIC236 links the outer and inner membrane translocons of the chloroplast
- Author
-
Jie-Ru Wen, Lih-Jen Chen, Yih-Lin Chen, Po-Kai Huang, Hsou-min Li, and Chiung-Chih Chu
- Subjects
0106 biological sciences ,0301 basic medicine ,Nuclear gene ,Chloroplasts ,Arabidopsis ,01 natural sciences ,Evolution, Molecular ,03 medical and health sciences ,Chloroplast Proteins ,Transit Peptide ,Inner membrane ,Protein Precursors ,Multidisciplinary ,Chemistry ,Arabidopsis Proteins ,Escherichia coli Proteins ,Peas ,Membrane Proteins ,Membrane Transport Proteins ,Intracellular Membranes ,Cell biology ,Transport protein ,Chloroplast ,Cytosol ,Protein Transport ,030104 developmental biology ,Multiprotein Complexes ,Mutation ,Intermembrane space ,010606 plant biology & botany ,Bacterial Outer Membrane Proteins ,Protein Binding - Abstract
The two-membrane envelope is a defining feature of chloroplasts. Chloroplasts evolved from a Gram-negative cyanobacterial endosymbiont. During evolution, genes of the endosymbiont have been transferred to the host nuclear genome. Most chloroplast proteins are synthesized in the cytosol as higher-molecular-mass preproteins with an N-terminal transit peptide. Preproteins are transported into chloroplasts by the TOC and TIC (translocons at the outer- and inner-envelope membranes of chloroplasts, respectively) machineries1,2, but how TOC and TIC are assembled together is unknown. Here we report the identification of the TIC component TIC236; TIC236 is an integral inner-membrane protein that projects a 230-kDa domain into the intermembrane space, which binds directly to the outer-membrane channel TOC75. The knockout mutation of TIC236 is embryonically lethal. In TIC236-knockdown mutants, a smaller amount of the inner-membrane channel TIC20 was associated with TOC75; the amount of TOC-TIC supercomplexes was also reduced. This resulted in a reduced import rate into the stroma, though outer-membrane protein insertion was unaffected. The size and the essential nature of TIC236 indicate that-unlike in mitochondria, in which the outer- and inner-membrane translocons exist as separate complexes and a supercomplex is only transiently assembled during preprotein translocation3,4-a long and stable protein bridge in the intermembrane space is required for protein translocation into chloroplasts. Furthermore, TIC236 and TOC75 are homologues of bacterial inner-membrane TamB5 and outer-membrane BamA, respectively. Our evolutionary analyses show that, similar to TOC75, TIC236 is preserved only in plants and has co-evolved with TOC75 throughout the plant lineage. This suggests that the backbone of the chloroplast protein-import machinery evolved from the bacterial TamB-BamA protein-secretion system.
- Published
- 2018
16. Chloroplast Hsp93 Directly Binds to Transit Peptides at an Early Stage of the Preprotein Import Process
- Author
-
Lih-Jen Chen, Po-Kai Huang, Po-Ting Chan, Pai-Hsiang Su, and Hsou-min Li
- Subjects
0106 biological sciences ,0301 basic medicine ,Chloroplasts ,Physiology ,Immunoprecipitation ,Arabidopsis ,Plant Science ,Bioinformatics ,01 natural sciences ,Chloroplast Proteins ,03 medical and health sciences ,Transit Peptide ,Heat shock protein ,Genetics ,Heat-Shock Proteins ,biology ,Arabidopsis Proteins ,Chemistry ,digestive, oral, and skin physiology ,Peas ,Articles ,Translocon ,Transport protein ,Cell biology ,Chloroplast ,Protein Transport ,030104 developmental biology ,Chaperone (protein) ,biology.protein ,Peptides ,Molecular Chaperones ,010606 plant biology & botany - Abstract
Three stromal chaperone ATPases, cpHsc70, Hsp90C, and Hsp93, are present in the chloroplast translocon, but none has been shown to directly bind preproteins in vivo during import, so it remains unclear whether any function as a preprotein-translocating motor and whether they have different functions during the import process. Here, using protein crosslinking followed by ionic detergent solubilization, we show that Hsp93 directly binds to the transit peptides of various preproteins undergoing active import into chloroplasts. Hsp93 also binds to the mature region of a preprotein. A time course study of import, followed by coimmunoprecipitation experiments, confirmed that Hsp93 is present in the same complexes as preproteins at an early stage when preproteins are being processed to the mature size. In contrast, cpHsc70 is present in the same complexes as preproteins at both the early stage and a later stage after the transit peptide has been removed, suggesting that cpHsc70, but not Hsp93, is important in translocating processed mature proteins across the envelope.
- Published
- 2015
- Full Text
- View/download PDF
17. Increased ratio of galactolipid MGDG : DGDG induces jasmonic acid overproduction and changes chloroplast shape.
- Author
-
Yu, Chun‐Wei, Lin, Yang‐Tsung, and Li, Hsou‐min
- Subjects
CHLOROPLASTS ,JASMONIC acid ,OVERPRODUCTION ,STUNTED growth ,CHLOROPLAST membranes ,PHOTOSYSTEMS ,QUANTUM efficiency - Abstract
Summary: Galactolipids monogalactosyl diacylglycerol (MGDG) and digalactosyl diacylglycerol (DGDG) constitute c. 50% and c. 30% of chloroplast membrane lipids, respectively. They are important for photosynthesis and stress tolerance. Mutations in DGD1, the major DGDG‐synthesizing enzyme, severely reduce DGDG content and induce jasmonic acid (JA) overproduction, resulting in stunted growth. However, how DGDG reduction leads to JA overproduction is unknown.We introduced an inducible microRNA (ami‐MGD1) into an Arabidopsis dgd1 mutant to reduce MGDG synthesis, thereby further diminishing galactolipid content, but partially restoring the MGDG : DGDG ratio. Galactolipid and Chl contents, expression of JA‐biosynthesis and JA‐responsive genes, photosystem II (PSII) maximum quantum efficiency, and chloroplast shape were investigated.Expression of JA‐biosynthesis and JA‐responsive genes were reduced in amiR‐MGD1‐transformed dgd1 plants. Stunted growth caused by JA overproduction was also partially rescued, but Chl reduction and PSII impairment remained similar to the original dgd1 mutant. Altered chloroplast shape, which is another defect observed in dgd1 but is not caused by JA overproduction, was also partially rescued.Our results reveal that an increased MGDG : DGDG ratio is the primary cause of JA overproduction. The ratio is also important for determining chloroplast shapes, whereas reduced Chl and photosynthesis are most likely a direct consequence of insufficient DGDG. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
18. Structural characterizations of the chloroplast translocon protein Tic110
- Author
-
Lih-Jen Chen, Yi-Hung Yeh, Chwan-Deng Hsiao, Hsou-min Li, Chiung-Chih Chu, and Jia-Yin Tsai
- Subjects
Cyanidioschyzon merolae ,Tic110 ,Sequence alignment ,Plant Science ,Crystallography, X-Ray ,Chloroplast Proteins ,chloroplast ,Sequence Analysis, Protein ,Botany ,Genetics ,Amino Acid Sequence ,Peptide sequence ,translocon ,biology ,Algal Proteins ,Membrane Proteins ,Cell Biology ,Original Articles ,biology.organism_classification ,Translocon ,Transport protein ,Protein Structure, Tertiary ,Transmembrane domain ,Chloroplast DNA ,CMQ342C ,Rhodophyta ,Biophysics ,HEAT repeats ,Sequence Alignment - Abstract
Tic110 is a major component of the chloroplast protein import translocon. Two functions with mutually exclusive structures have been proposed for Tic110: a protein-conducting channel with six transmembrane domains and a scaffold with two N-terminal transmembrane domains followed by a large soluble domain for binding transit peptides and other stromal translocon components. To investigate the structure of Tic110, Tic110 from Cyanidioschyzon merolae (CmTic110) was characterized. We constructed three fragments, CmTic110A , CmTic110B and CmTic110C , with increasing N-terminal truncations, to perform small-angle X-ray scattering (SAXS) and X-ray crystallography analyses and Dali structural comparison. Here we report the molecular envelope of CmTic110B and CmTic110C determined by SAXS, and the crystal structure of CmTic110C at 4.2 Å. Our data indicate that the C-terminal half of CmTic110 possesses a rod-shaped helix-repeat structure that is too flattened and elongated to be a channel. The structure is most similar to the HEAT-repeat motif that functions as scaffolds for protein-protein interactions.
- Published
- 2013
19. The Amino-Terminal Domain of Chloroplast Hsp93 Is Important for Its Membrane Association and Functions in Vivo
- Author
-
Hsou-min Li and Chiung-Chih Chu
- Subjects
Vesicle-associated membrane protein 8 ,Chloroplasts ,Physiology ,Molecular Sequence Data ,Mutant ,Arabidopsis ,Plant Science ,Substrate Specificity ,Chloroplast Proteins ,Structure-Activity Relationship ,Heat shock protein ,Genetics ,Heat-Shock Proteins ,Sequence Deletion ,Adenosine Triphosphatases ,biology ,Arabidopsis Proteins ,Genetic Complementation Test ,Peas ,Membrane Proteins ,Endopeptidase Clp ,Intracellular Membranes ,Translocon ,biology.organism_classification ,Protein Structure, Tertiary ,Chloroplast ,Phenotype ,Membrane ,Biochemistry ,Cell Biology and Signal Transduction ,Chaperone (protein) ,Proteolysis ,biology.protein ,Protein Binding - Abstract
Chloroplast 93-kD heat shock protein (Hsp93/ClpC), an Hsp100 family member, is suggested to have various functions in chloroplasts, including serving as the regulatory chaperone for the ClpP protease in the stroma and acting as a motor component of the protein translocon at the envelope. Indeed, although Hsp93 is a soluble stromal protein, a portion of it is associated with the inner envelope membrane. The mechanism and functional significance of this Hsp93 membrane association have not been determined. Here, we mapped the region important for Hsp93 membrane association by creating various deletion constructs and found that only the construct with the amino-terminal domain deleted, Hsp93-ƊN, had reduced membrane association. When transformed into Arabidopsis (Arabidopsis thaliana), most atHsp93V-ƊN proteins did not associate with membranes and atHsp93V-ƊΝN failed to complement the pale-green and protein import-defective phenotypes of an hsp93V knockout mutant. The residual atHsp93V-ƊN at the membranes had further reduced association with the central protein translocon component Tic110. However, the degradation of chloroplast glutamine synthetase, a potential substrate for the ClpP protease, was not affected in the hsp93V mutant or in the atHSP93V-ƊN transgenic plants. Hsp93-ƊN also had the same ATPase activity as that of full-length Hsp93. These data suggest that the association of Hsp93 with the inner envelope membrane through its amino-terminal domain is important for the functions of Hsp93 in vivo.
- Published
- 2012
- Full Text
- View/download PDF
20. Pea Chloroplast DnaJ-J8 and Toc12 Are Encoded by the Same Gene and Localized in the Stroma
- Author
-
Lih Jen Chen, Hsou-min Li, and Chi Chou Chiu
- Subjects
endocrine system ,Chloroplasts ,Physiology ,Molecular Sequence Data ,Arabidopsis ,Receptors, Cell Surface ,Plant Science ,Genes, Plant ,Chloroplast membrane ,Stroma ,Genetics ,Arabidopsis thaliana ,Amino Acid Sequence ,Plant Proteins ,Sequence Homology, Amino Acid ,biology ,Arabidopsis Proteins ,Peas ,food and beverages ,biology.organism_classification ,Cell biology ,Chloroplast ,Mutagenesis, Insertional ,Protein Transport ,Chloroplast stroma ,Chloroplast DNA ,RNA, Plant ,Cell Biology and Signal Transduction ,Mutation ,Intermembrane space ,Sequence Alignment - Abstract
Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.
- Published
- 2010
- Full Text
- View/download PDF
21. Stromal Hsp70 Is Important for Protein Translocation into Pea and Arabidopsis Chloroplasts
- Author
-
Hsou-min Li and Pai-Hsiang Su
- Subjects
Chloroplasts ,Genotype ,Immunoprecipitation ,Molecular Sequence Data ,Arabidopsis ,Chromosomal translocation ,Plant Science ,Plasma protein binding ,Models, Biological ,Anthocyanins ,HSP70 Heat-Shock Proteins ,Photosynthesis ,Protein Precursors ,Research Articles ,Plant Proteins ,biology ,Arabidopsis Proteins ,Endoplasmic reticulum ,Peas ,food and beverages ,Membrane Proteins ,Cell Biology ,Translocon ,Cell biology ,Transport protein ,Chloroplast ,Protein Transport ,Cross-Linking Reagents ,Seedlings ,Chaperone (protein) ,Mutation ,biology.protein ,Molecular Chaperones ,Protein Binding - Abstract
Hsp70 family proteins function as motors driving protein translocation into mitochondria and the endoplasmic reticulum. Whether Hsp70 is involved in protein import into chloroplasts has not been resolved. We show here Arabidopsis thaliana knockout mutants of either of the two stromal cpHsc70s, cpHsc70-1 and cpHsc70-2, are defective in protein import into chloroplasts during early developmental stages. Protein import was found to be affected at the step of precursor translocation across the envelope membranes. From solubilized envelope membranes, stromal cpHsc70 was specifically coimmunoprecipitated with importing precursors and stoichiometric amounts of Tic110 and Hsp93. Moreover, in contrast with receptors at the outer envelope membrane, cpHsp70 is important for the import of both photosynthetic and nonphotosynthetic proteins. These data indicate that cpHsc70 is part of the chloroplast translocon for general import and is important for driving translocation into the stroma. We further analyzed the relationship of cpHsc70 with the other suggested motor system, Hsp93/Tic40. Chloroplasts from the cphsc70-1 hsp93-V double mutant had a more severe import defect than did the single mutants, suggesting that the two proteins function in parallel. The cphsc70-1 tic40 double knockout was lethal, further indicating that cpHsc70-1 and Tic40 have an overlapping essential function. In conclusion, our data indicate that chloroplasts have two chaperone systems facilitating protein translocation into the stroma: the cpHsc70 system and the Hsp93/Tic40 system.
- Published
- 2010
- Full Text
- View/download PDF
22. Tic40 is important for reinsertion of proteins from the chloroplast stroma into the inner membrane
- Author
-
Chi Chou Chiu and Hsou-min Li
- Subjects
Chloroplasts ,Molecular Sequence Data ,Mutant ,Arabidopsis ,Plant Science ,Biology ,membrane protein insertion ,Stroma ,Gene Expression Regulation, Plant ,Genetics ,Inner membrane ,Protein Precursors ,proline ,inner membrane ,Arabidopsis Proteins ,Membrane Proteins ,food and beverages ,Intracellular Membranes ,Original Articles ,Cell Biology ,Transport protein ,Cell biology ,Chloroplast ,Protein Transport ,Cytosol ,Chloroplast stroma ,Biochemistry ,Membrane protein ,RNA, Plant ,Protein Biosynthesis ,post-import ,Mutation ,Tic40 ,Molecular Chaperones - Abstract
Chloroplast inner-membrane proteins Tic40 and Tic110 are first imported from the cytosol into the chloroplast stroma, and subsequently reinserted from the stroma into the inner membrane. However, the mechanism of reinsertion remains unclear. Here we show that Tic40 itself is involved in this reinsertion process. When precursors of either Tic40 or a Tic110 C-terminal truncate, tpTic110-Tic110N, were imported into chloroplasts isolated from a tic40-null mutant, soluble Tic40 and Tic110N intermediates accumulated in the stroma of tic40-mutant chloroplasts, due to a slower rate of reinsertion. We further show that a larger quantity of soluble Tic21 intermediates also accumulated in the stroma of tic40-mutant chloroplasts. In contrast, inner-membrane insertion of the triose-phosphate/phosphate translocator was not affected by the tic40 mutation. Our data suggest that multiple pathways exist for the insertion of chloroplast inner-membrane proteins.
- Published
- 2008
- Full Text
- View/download PDF
23. Reduced Biosynthesis of Digalactosyldiacylglycerol, a Major Chloroplast Membrane Lipid, Leads to Oxylipin Overproduction and Phloem Cap Lignification in Arabidopsis[OPEN]
- Author
-
Yang-Tsung Lin, Ivo Feussner, Cornelia Herrfurth, Hsou-min Li, and Lih-Jen Chen
- Subjects
0106 biological sciences ,Chloroplasts ,Arabidopsis ,Plant Science ,Cyclopentanes ,Phloem ,Genes, Plant ,01 natural sciences ,Chloroplast membrane ,Lignin ,03 medical and health sciences ,chemistry.chemical_compound ,Membrane Lipids ,Gene Expression Regulation, Plant ,Arabidopsis thaliana ,Oxylipins ,Plastid ,Inflorescence ,Photosynthesis ,Research Articles ,Alleles ,030304 developmental biology ,0303 health sciences ,biology ,Indoleacetic Acids ,Jasmonic acid ,Galactolipids ,Allene-oxide cyclase ,Cell Biology ,Chloroplast outer membrane ,Ethylenes ,biology.organism_classification ,Biosynthetic Pathways ,Up-Regulation ,Chloroplast ,Phenotype ,Biochemistry ,chemistry ,Mutation ,lipids (amino acids, peptides, and proteins) ,010606 plant biology & botany ,Signal Transduction - Abstract
DIGALACTOSYLDIACYLGLYCEROL SYNTHASE1 (DGD1) is a chloroplast outer membrane protein responsible for the biosynthesis of the lipid digalactosyldiacylglycerol (DGDG) from monogalactosyldiacylglycerol (MGDG). The Arabidopsis thaliana dgd1 mutants have a greater than 90% reduction in DGDG content, reduced photosynthesis, and altered chloroplast morphology. However, the most pronounced visible phenotype is the extremely short inflorescence stem, but how deficient DGDG biosynthesis causes this phenotype is unclear. We found that, in dgd1 mutants, phloem cap cells were lignified and jasmonic acid (JA)-responsive genes were highly upregulated under normal growth conditions. The coronative insensitive1 dgd1 and allene oxide synthase dgd1 double mutants no longer exhibited the short inflorescence stem and lignification phenotypes but still had the same lipid profile and reduced photosynthesis as dgd1 single mutants. Hormone and lipidomics analyses showed higher levels of JA, JA-isoleucine, 12-oxo-phytodienoic acid, and arabidopsides in dgd1 mutants. Transcript and protein level analyses further suggest that JA biosynthesis in dgd1 is initially activated through the increased expression of genes encoding 13-lipoxygenases (LOXs) and phospholipase A-Iγ3 (At1g51440), a plastid lipase with a high substrate preference for MGDG, and is sustained by further increases in LOX and allene oxide cyclase mRNA and protein levels. Our results demonstrate a link between the biosynthesis of DGDG and JA.
- Published
- 2015
24. Protein import into isolated pea root leucoplasts
- Author
-
Hsou-min Li and Chiung-Chih Chu
- Subjects
Starch ,Transgene ,food and beverages ,Leucoplast ,Plant Science ,Biology ,lcsh:Plant culture ,Translocon ,root ,Chloroplast ,chemistry.chemical_compound ,Biochemistry ,chemistry ,leucoplasts ,Chromoplast ,Organelle ,Botany ,lcsh:SB1-1110 ,Plastid ,protein import ,plastid ,translocon ,Original Research - Abstract
Leucoplasts are important organelles for the synthesis and storage of starch, lipids and proteins. However, molecular mechanism of protein import into leucoplasts and how it differs from that of import into chloroplasts remain unknown. We used pea seedlings for both chloroplast and leucoplast isolations to compare within the same species. We further optimized the isolation and import conditions to improve import efficiency and to permit a quantitative comparison between the two plastid types. The authenticity of the import was verified using a mitochondrial precursor protein. Our results show that, when normalized to Toc75, most translocon proteins are less abundant in leucoplasts than in chloroplasts. A precursor shown to prefer the receptor Toc132 indeed had relatively more similar import efficiencies between chloroplasts and leucoplasts compared to precursors that preferred Toc159. Furthermore we found two precursors that exhibited very high import efficiency into leucoplasts. Their transit peptides may be candidates for delivering transgenic proteins into leucoplasts and for analyzing motifs important for leucoplast import.
- Published
- 2015
25. Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts
- Author
-
Hsou-min Li and Kuan-Yu Chen
- Subjects
Chloroplast ,Toc complex ,Membrane ,Biochemistry ,Density gradient ,Genetics ,Cell Biology ,Plant Science ,Plasma protein binding ,Biology ,Translocon ,Polyacrylamide gel electrophoresis ,Transport protein - Abstract
The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.
- Published
- 2006
- Full Text
- View/download PDF
26. Signal Peptide-Dependent Targeting of a Riceα-Amylase and Cargo Proteins to Plastids and Extracellular Compartments of Plant Cells
- Author
-
Yung-Reui Chen, Min-Huei Chen, Su-May Yu, Li-Fen Huang, and Hsou-min Li
- Subjects
Signal peptide ,biology ,Physiology ,Nicotiana tabacum ,fungi ,food and beverages ,Plant Science ,biology.organism_classification ,medicine.disease_cause ,Subcellular localization ,Cell wall ,Chloroplast ,Biochemistry ,Protein targeting ,Genetics ,medicine ,Amyloplast ,Plastid - Abstract
α-Amylases are important enzymes for starch degradation in plants. However, it has been a long-running debate as to whether α-amylases are localized in plastids where starch is stored. To study the subcellular localization of α-amylases in plant cells, a rice (Oryza sativa) α-amylase, αAmy3, with or without its own signal peptide (SP) was expressed in transgenic tobacco (Nicotiana tabacum) and analyzed. Loss-of-function analyses revealed that SP was required for targeting of αAmy3 to chloroplasts and/or amyloplasts and cell walls and/or extracellular compartments of leaves and suspension cells. SP was also required for in vitro transcribed and/or translated αAmy3 to be cotranslationally imported and processed in canine microsomes. αAmy3, present in chloroplasts of transgenic tobacco leaves, was processed to a product with M r similar to αAmy3 minus its SP. Amino acid sequence analysis revealed that the SP of chloroplast localized αAmy3 was cleaved at a site only one amino acid preceding the predicted cleavage site. Function of the αAmy3 SP was further studied by gain-of-function analyses. β-Glucuronidase (GUS) and green fluorescence protein fused with or without the αAmy3 SP was expressed in transgenic tobacco or rice. The αAmy3 SP directed translocation of GUS and green fluorescence protein to chloroplasts and/or amyloplasts and cell walls in tobacco leaves and rice suspension cells. The SP of another rice α-amylase, αAmy8, similarly directed the dual localizations of GUS in transgenic tobacco leaves. This study is the first evidence of SP-dependent dual translocations of proteins to plastids and extracellular compartments, which provides new insights into the role of SP in protein targeting and the pathways of SP-dependent protein translocation in plants.
- Published
- 2004
- Full Text
- View/download PDF
27. Chloroplast Protein Translocon Components atToc159 and atToc33 Are Not Essential for Chloroplast Biogenesis in Guard Cells and Root Cells
- Author
-
Tien-Shin Yu and Hsou-min Li
- Subjects
Physiology ,fungi ,Wild type ,food and beverages ,Plant Science ,Biology ,Translocon ,biology.organism_classification ,Chloroplast membrane ,Chloroplast ,Biochemistry ,Guard cell ,Thylakoid ,Genetics ,Arabidopsis thaliana ,Plastid - Abstract
Protein import into chloroplasts is mediated by a protein import apparatus located in the chloroplast envelope. Previous results indicate that there may be multiple import complexes in Arabidopsis. To gain further insight into the nature of this multiplicity, we analyzed the Arabidopsis ppi1 and ppi2 mutants, which are null mutants of the atToc33 and atToc159 translocon proteins, respectively. In the ppi2 mutant, in contrast to the extremely defective plastids in mesophyll cells, chloroplasts in guard cells still contained starch granules and thylakoid membranes. The morphology of root plastids in both mutants was similar to that in wild type. After prolonged light treatments, root plastids of both mutants and the wild type differentiated into chloroplasts. Enzymatic assays indicated that the activity of a plastid enzyme was reduced only in leaves but not in roots. These results indicated that both theppi1 and ppi2 mutants had functional root and guard cell plastids. Therefore, we propose that import complexes are cell type specific rather than substrate or plastid specific.
- Published
- 2001
- Full Text
- View/download PDF
28. Leaf-Specific Upregulation of Chloroplast Translocon Genes by a CCT Motif–Containing Protein, CIA 2
- Author
-
Lih-Jen Chen, Chih Wen Sun, Hsou-min Li, and Li-Chung Lin
- Subjects
Chloroplasts ,Amino Acid Motifs ,Molecular Sequence Data ,Nuclear Localization Signals ,Arabidopsis ,Plant Science ,Biology ,environment and public health ,Chloroplast membrane ,Chloroplast Proteins ,Gene Expression Regulation, Plant ,Genes, Reporter ,Amino Acid Sequence ,RNA, Messenger ,Transgenes ,Cloning, Molecular ,Protein Precursors ,Nuclear protein ,Plastid ,Gene ,Plant Proteins ,Genetics ,Arabidopsis Proteins ,Reverse Transcriptase Polymerase Chain Reaction ,DNA, Chloroplast ,Membrane Proteins ,Nuclear Proteins ,food and beverages ,Cell Biology ,Translocon ,Up-Regulation ,Transport protein ,Plant Leaves ,Chloroplast ,Protein Transport ,Organ Specificity ,RNA, Plant ,Mutation ,lipids (amino acids, peptides, and proteins) ,Transcription Factors ,Research Article - Abstract
Chloroplasts are a major destination of protein traffic within leaf cells. Protein import into chloroplasts is mediated by a set of translocon complexes at the chloroplast envelope. Current data indicate that the expression of translocon genes is regulated in a tissue-specific manner, possibly to accommodate the higher import demand of chloroplasts in leaves and the lower demand of plastids in other tissues. We have designed a transgene-based positive screen to isolate mutants disrupted in protein import into plastids. The first locus we isolated, CIA2, encodes a protein containing a motif conserved within the CCT family of transcription factors. Biochemical analysis indicates that CIA2 is responsible for specific upregulation of the translocon genes atToc33 and atToc75 in leaves. Identification of CIA2 provides new insights into the tissue-specific regulation of translocon gene expression.
- Published
- 2001
- Full Text
- View/download PDF
29. A Novel Chloroplastic Outer Membrane-targeting Signal That Functions at Both Termini of Passenger Polypeptides
- Author
-
Lih-Jen Chen and Hsou-min Li
- Subjects
C-terminus ,Translocase of the outer membrane ,food and beverages ,Cell Biology ,Biology ,Biochemistry ,Fusion protein ,Chloroplast ,Cytosol ,Biophysics ,Outer membrane efflux proteins ,Bacterial outer membrane ,Intermembrane space ,Molecular Biology - Abstract
Several components in the machinery mediating the import of nuclear-encoded chloroplastic precursor proteins have been identified. One of the components, OEP34, is an outer membrane protein and is synthesized at its mature size in the cytosol without a distinguishable chloroplast-targeting signal. To address the question of how components in the transport machinery are imported to chloroplasts themselves, we first identified the chloroplastic outer membrane-targeting signal of OEP34. Using an Arabidopsishomologue of the originally isolated pea OEP34, we show that the outer membrane-targeting signal of OEP34 is located within a 10-amino acid hydrophobic core of the C-terminal membrane anchor. Interestingly, this signal can target a passenger protein to the chloroplastic outer membrane no matter whether it is placed at the N or C terminus of a passenger protein. Proper insertion of fusion proteins into the outer membrane requires in addition the C-terminal hydrophilic region following the hydrophobic core. Furthermore, passenger proteins fused to the C terminus of the targeting/insertion signal were most likely imported into the intermembrane space of the envelope.
- Published
- 1997
- Full Text
- View/download PDF
30. Evolution of Chloroplast J Proteins
- Author
-
Lih Jen Chen, Hsou-min Li, Pai-Hsiang Su, and Chi Chou Chiu
- Subjects
Proteomics ,Chloroplasts ,Plant Evolution ,Arabidopsis Thaliana ,Plant Cell Biology ,Protein domain ,Molecular Sequence Data ,Arabidopsis ,lcsh:Medicine ,Sequence alignment ,Plant Science ,Biology ,Cyanobacteria ,Biochemistry ,Chloroplast ,Conserved sequence ,Evolution, Molecular ,Chloroplast Proteins ,Model Organisms ,Phylogenetics ,Plant and Algal Models ,Gene Expression Regulation, Plant ,Stress, Physiological ,Molecular Cell Biology ,HSP70 Heat-Shock Proteins ,lcsh:Science ,Conserved Sequence ,Phylogeny ,Genetics ,Multidisciplinary ,Arabidopsis Proteins ,lcsh:R ,food and beverages ,Proteins ,Plants ,biology.organism_classification ,Chaperone Proteins ,lcsh:Q ,Genome, Plant ,Research Article ,Molecular Chaperones - Abstract
Hsp70 chaperones are involved in multiple biological processes and are recruited to specific processes by designated J domain-containing cochaperones, or J proteins. To understand the evolution and functions of chloroplast Hsp70s and J proteins, we identified the Arabidopsis chloroplast J protein constituency using a combination of genomic and proteomic database searches and individual protein import assays. We show that Arabidopsis chloroplasts have at least 19 J proteins, the highest number of confirmed J proteins for any organelle. These 19 J proteins are classified into 11 clades, for which cyanobacteria and glaucophytes only have homologs for one clade, green algae have an additional three clades, and all the other 7 clades are specific to land plants. Each clade also possesses a clade-specific novel motif that is likely used to interact with different client proteins. Gene expression analyses indicate that most land plant-specific J proteins show highly variable expression in different tissues and are down regulated by low temperatures. These results show that duplication of chloroplast Hsp70 in land plants is accompanied by more than doubling of the number of its J protein cochaperones through adding new J proteins with novel motifs, not through duplications within existing families. These new J proteins likely recruit chloroplast Hsp70 to perform tissue specific functions related to biosynthesis rather than to stress resistance.
- Published
- 2013
31. Developmental regulation of protein import into plastids.
- Author
-
Chu, Chiung-Chih and Li, Hsou-min
- Abstract
The plastid proteome changes according to developmental stages. Accruing evidence shows that, in addition to transcriptional and translational controls, preprotein import into plastids is also part of the process regulating plastid proteomes. Different preproteins have distinct preferences for plastids of different tissues. Preproteins are also divided into at least three age-selective groups based on their import preference for chloroplasts of different ages. Both tissue and age selectivity are determined by the transit peptide of each preprotein, and a transit-peptide motif for older-chloroplast preference has been identified. Future challenges lie in identifying other motifs for tissue and age selectivity, as well as in identifying the receptor components that decipher these motifs. Developmental regulation also suggests that caution should be exercised when comparing protein import data generated with plastids isolated from different tissues or with chloroplasts isolated from plants of different ages. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
- View/download PDF
32. CUE1: A Mesophyll Cell-Specific Positive Regulator of Light-Controlled Gene Expression in Arabidopsis
- Author
-
Hsou-min Li, Richard A. Dixon, Joanne Chory, and Kevin M. Culligan
- Subjects
Chalcone synthase ,Genetics ,Phytochrome ,Epidermis (botany) ,fungi ,Mutant ,food and beverages ,Cell Biology ,Plant Science ,Biology ,biology.organism_classification ,Cell biology ,Chloroplast ,Arabidopsis ,Gene expression ,biology.protein ,Derepression ,Research Article - Abstract
Light plays a key role in the development and physiology of plants. One of the most profound effects of light on plant development is the derepression of expression of an array of light-responsive genes, including the genes encoding the chlorophyll a/b binding proteins (CAB) of photosystem II. To understand the mechanism by which light signals nuclear gene expression, we developed a genetic selection to identify mutants with reduced CAB transcription. Here, we describe a new Arabidopsis locus, CUE1 (for CAB underexpressed). Mutations at this locus result in defects in expression of several light-regulated genes, specifically in mesophyll but not in bundle-associated or epidermis cells. Reduced accumulation of CAB and other photosynthesis-related mRNAs in the mesophyll was correlated with defects in chloroplast development in these cells, resulting in a reticulate pattern with veins greener than the interveinal regions of leaves. Moreover, chalcone synthase mRNA, although known to be regulated by both phytochrome and a blue light receptor, accumulated normally in the leaf epidermis. Dark basal levels of CAB expression were unaffected in etiolated cue1 seedlings; however, induction of CAB transcription by pulses of red and blue light was reduced, suggesting that CUE1 acts downstream from both phytochrome and blue light photoreceptors. CUE1 appears to play a role in the primary derepression of mesophyll-specific gene expression in response to light, because cue1 mutants are severely deficient at establishing photoautotrophic growth. Based on this characterization, we propose that CUE1 is a cell-specific positive regulator linking light and intrinsic developmental programs in Arabidopsis leaf mesophyll cells.
- Published
- 1995
- Full Text
- View/download PDF
33. Targeting of proteins into chloroplasts
- Author
-
Kenneth Keegstra, Sharyn E. Perry, Barry D. Bruce, Melissa Hurley, and Hsou-min Li
- Subjects
Chloroplast ,Biochemistry ,Physiology ,Cytoplasm ,Protein targeting ,Genetics ,medicine ,Cell Biology ,Plant Science ,General Medicine ,Biology ,medicine.disease_cause ,Cell biology - Abstract
Most chloroplastic proteins are synthesized in the cytoplasm and are transported to their proper location as a posttranslational event. In the present paper we briefly review some aspects of this transport process. Because chloroplasts contain six different locations, one interesting aspect of protein targeting into chloroplasts that we consider is how precursor proteins are targeted to these various locations. One step shared by many proteins is transport across the envelope membranes. Although this process has been well studied, the components of the apparatus that mediate this transport step are mostly unidentified. Strategies to identify components of this transport apparatus are considered
- Published
- 1995
- Full Text
- View/download PDF
34. Differential age-dependent import regulation by signal peptides
- Author
-
Po-Ting Chan, Hsou-min Li, and Yi-Shan Teng
- Subjects
Signal peptide ,Chloroplasts ,QH301-705.5 ,Plant Cell Biology ,Arabidopsis ,Sequence alignment ,Protein Sorting Signals ,Biology ,Chloroplast ,General Biochemistry, Genetics and Molecular Biology ,Transit Peptide ,Molecular Cell Biology ,Amino Acid Sequence ,Protein Precursors ,Biology (General) ,Peptide sequence ,Gene ,Plant Proteins ,General Immunology and Microbiology ,General Neuroscience ,Peas ,Plants, Genetically Modified ,Cell biology ,Transport protein ,Protein Transport ,Biochemistry ,Membranes and Sorting ,Chloroplast Proteins ,General Agricultural and Biological Sciences ,Sequence Alignment ,Chlamydomonas reinhardtii ,Research Article - Abstract
The import of certain proteins into chloroplasts is dependent on the age of the organelle, with age-dependent import being controlled by a specific motif within the signal peptide., Gene-specific, age-dependent regulations are common at the transcriptional and translational levels, while protein transport into organelles is generally thought to be constitutive. Here we report a new level of differential age-dependent regulation and show that chloroplast proteins are divided into three age-selective groups: group I proteins have a higher import efficiency into younger chloroplasts, import of group II proteins is nearly independent of chloroplast age, and group III proteins are preferentially imported into older chloroplasts. The age-selective signal is located within the transit peptide of each protein. A group III protein with its transit peptide replaced by a group I transit peptide failed to complement its own mutation. Two consecutive positive charges define the necessary motif in group III signals for older chloroplast preference. We further show that different members of a gene family often belong to different age-selective groups because of sequence differences in their transit peptides. These results indicate that organelle-targeting signal peptides are part of cells' differential age-dependent regulation networks. The sequence diversity of some organelle-targeting peptides is not a result of the lack of selection pressure but has evolved to mediate regulation., Author Summary It is well known that some genes are preferentially transcribed in young tissues and others are specifically expressed in aging tissue, but protein import into organelles is generally thought to be constitutive and independent of age. In this study, we find that, contrary to expectation, in higher plants the import of proteins into chloroplasts is indeed dependent on the age of the organelle. We find that chloroplast precursor proteins can be divided into three age-selective groups, with each having a preference for chloroplasts of a different age. The age-selective signal is located within the signal peptide of each protein that controls organelle import, and we further identify a motif that is necessary to make a signal peptide target older chloroplasts preferentially. We show that different members of a gene family often belong to different age-selective groups, and that changing the sequence motifs within a protein's signal peptide can change its age selectivity. These results indicate that organelle-targeting signal peptides are one set of tools available to multicellular organisms for differential age-specific regulation.
- Published
- 2012
35. Determining the Location of an Arabidopsis Chloroplast Protein Using In Vitro Import Followed by Fractionation and Alkaline Extraction
- Author
-
Chiung-Chih Chu and Hsou-min Li
- Subjects
Chloroplast ,Membrane ,Biochemistry ,Membrane protein ,Thylakoid ,Organelle ,food and beverages ,Biology ,Intermembrane space ,Integral membrane protein ,Transport protein - Abstract
Chloroplasts have one of the most complicated structures among organelles. They have three membrane systems, the outer and inner envelope membranes and the thylakoid membrane, which enclose three aqueous spaces: the intermembrane space between the two envelope membranes, the stroma, and the thylakoid lumen. Each of the chloroplast's sub-organellar compartments houses a distinct set of proteins that perform distinct functions. Determining the sub-organellar location of a protein in the chloroplast is vital for understanding or verifying the function of the protein. Here, we present protocols for determining the sub-organellar location of a chloroplast protein. The protein of interest is synthesized and labeled with [(35)S]methionine by an in vitro translation system, and imported into isolated chloroplasts. The location of the protein is then identified by fractionation of the chloroplasts through differential and sucrose step-gradient centrifugations. The various sub-chloroplast fractions are analyzed by SDS-PAGE and autoradiography, so no specific antibody against the protein of interest is required. For membrane proteins, an alkaline extraction protocol is provided to further determine whether the protein is a peripheral or an integral membrane protein. The fractionation and extraction procedures presented can also be used in conjunction with immunoblotting, if an antibody against the protein of interest is available, enabling analyses of endogenous proteins.
- Published
- 2011
- Full Text
- View/download PDF
36. Protein transport into chloroplasts
- Author
-
Chi Chou Chiu and Hsou-min Li
- Subjects
Signal peptide ,Chloroplasts ,Physiology ,Receptors, Cytoplasmic and Nuclear ,Plant Science ,Biology ,Chloroplast membrane ,Cytosol ,Plant Cells ,Endomembrane system ,Plastid ,Photosynthesis ,Molecular Biology ,Plant Proteins ,Cell Nucleus ,Cell Biology ,Intracellular Membranes ,Plants ,Translocon ,Cell biology ,Transport protein ,Chloroplast ,Protein Transport ,Biochemistry ,Metabolic Networks and Pathways - Abstract
Most proteins in chloroplasts are encoded by the nuclear genome and synthesized as precursors with N-terminal targeting signals called transit peptides. Novel machinery has evolved to specifically import these proteins from the cytosol into chloroplasts. This machinery consists of more than a dozen components located in and around the chloroplast envelope, including a pair of GTPase receptors, a β-barrel-type channel across the outer membrane, and an AAA+-type motor in the stroma. How individual components assemble into functional subcomplexes and the sequential steps of the translocation process are being mapped out. An increasing number of noncanonical import pathways, including a pathway with initial transport through the endomembrane system, is being revealed. Multiple levels of control on protein transport into chloroplasts have evolved, including the development of two receptor subfamilies, one for photosynthetic proteins and one for housekeeping proteins. The functions or expression levels of some translocon components are further adjusted according to plastid type, developmental stage, and metabolic conditions.
- Published
- 2010
37. Targeting of proteins to the outer envelope membrane uses a different pathway than transport into chloroplasts
- Author
-
Kenneth Keegstra, Thomas Moore, and Hsou-min Li
- Subjects
Chloroplasts ,Molecular Sequence Data ,Tic complex ,DNA, Single-Stranded ,Plant Science ,Biology ,Cell Fractionation ,Protein Structure, Secondary ,Adenosine Triphosphate ,Transit Peptide ,Cloning, Molecular ,Plant Proteins ,Plants, Medicinal ,Valinomycin ,Base Sequence ,Membrane Proteins ,food and beverages ,Biological Transport ,Fabaceae ,Intracellular Membranes ,Cell Biology ,Chloroplast outer membrane ,Cell Compartmentation ,Chloroplast ,Cytosol ,Membrane ,Membrane protein ,Biochemistry ,Nigericin ,Bacterial outer membrane ,Research Article - Abstract
The chloroplastic envelope is composed of two membranes, inner and outer, each with a distinct set of polypeptides. Like proteins in other chloroplastic compartments, most envelope proteins are synthesized in the cytosol and post-translationally imported into chloroplasts. Considerable knowledge has been obtained concerning protein import proteins. We isolated a cDNA clone from pea that encodes a 14-kilodalton outer envelope membrane protein. The precursor form of this protein does not possess a cleavable transit peptide and its import into isolated chloroplasts does not require either ATP or a thermolysin-sensitive component on the chloroplastic surface. These findings, together with similar observations made with a spinach chloroplastic outer membrane protein, led us to propose that proteins destined for the outer membrane of the chloroplastic envelope follow an import pathway distinct from that followed by proteins destined for other chloroplastic compartments.
- Published
- 1991
- Full Text
- View/download PDF
38. Precursor binding to an 880-kDa Toc complex as an early step during active import of protein into chloroplasts
- Author
-
Kuan-Yu, Chen and Hsou-min, Li
- Subjects
Molecular Weight ,Protein Transport ,Sucrose ,Chloroplasts ,chloroplast ,translocon complex ,Multiprotein Complexes ,Peas ,blue-native polyacrylamide gel electrophoresis ,Original Articles ,protein import ,Plant Proteins ,Protein Binding - Abstract
The import of protein into chloroplasts is mediated by translocon components located in the chloroplast outer (the Toc proteins) and inner (the Tic proteins) envelope membranes. To identify intermediate steps during active import, we used sucrose density gradient centrifugation and blue-native polyacrylamide gel electrophoresis (BN-PAGE) to identify complexes of translocon components associated with precursor proteins under active import conditions instead of arrested binding conditions. Importing precursor proteins in solubilized chloroplast membranes formed a two-peak distribution in the sucrose density gradient. The heavier peak was in a similar position as the previously reported Tic/Toc supercomplex and was too large to be analyzed by BN-PAGE. The BN-PAGE analyses of the lighter peak revealed that precursors accumulated in at least two complexes. The first complex migrated at a position close to the ferritin dimer (approximately 880 kDa) and contained only the Toc components. Kinetic analyses suggested that this Toc complex represented an earlier step in the import process than the Tic/Toc supercomplex. The second complex in the lighter peak migrated at the position of the ferritin trimer (approximately 1320 kDa). It contained, in addition to the Toc components, Tic110, Hsp93, and an hsp70 homolog, but not Tic40. Two different precursor proteins were shown to associate with the same complexes. Processed mature proteins first appeared in the membranes at the same fractions as the Tic/Toc supercomplex, suggesting that processing of transit peptides occurs while precursors are still associated with the supercomplex.
- Published
- 2006
39. Stable megadalton TOC- TIC supercomplexes as major mediators of protein import into chloroplasts.
- Author
-
Chen, Lih‐Jen and Li, Hsou‐min
- Subjects
- *
CHLOROPLASTS , *POLYACRYLAMIDE gel electrophoresis , *ARABIDOPSIS thaliana , *MITOCHONDRIA , *IMMUNOPRECIPITATION - Abstract
Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC-TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC-TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC-TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
40. Import pathways of chloroplast interior proteins and the outer-membrane protein OEP14 converge at Toc75
- Author
-
Danny J. Schnell, Hsou-min Li, Lih Jen Chen, Shih-Long Tu, Yi Shin Su, and Matthew D. Smith
- Subjects
Signal peptide ,Toc complex ,Chloroplasts ,Arabidopsis ,Plant Science ,Biology ,Protein Sorting Signals ,Translocon complex ,Thermolysin ,Protein Precursors ,Research Articles ,Plant Proteins ,Liposome ,Arabidopsis Proteins ,Cell Membrane ,Peas ,Membrane Proteins ,Cell Biology ,Intracellular Membranes ,Chloroplast ,Protein Transport ,Membrane ,Biochemistry ,Liposomes ,Biophysics ,Bacterial outer membrane - Abstract
Most chloroplast outer-membrane proteins are synthesized at their mature size without cleavable targeting signals. Their insertion into the outer membrane is insensitive to thermolysin pretreatment of chloroplasts and does not require ATP. It has therefore been assumed that insertion of outer-membrane proteins proceeds through a different pathway from import into the interior of chloroplasts, which requires a thermolysin-sensitive translocon complex and ATP. Here, we show that a model outer-membrane protein, OEP14, competed with the import of a chloroplast interior protein, indicating that the two import pathways partially overlapped. Cross-linking studies showed that, during insertion, OEP14 was associated with Toc75, a thermolysin-resistant component of the outer-membrane protein–conducting channel that mediates the import of interior-targeted precursor proteins. Whereas almost no OEP14 inserted into protein-free liposomes, OEP14 inserted into proteoliposomes containing reconstituted Toc75 with a high efficiency. Taken together, our data indicate that Toc75 mediates OEP14 insertion, and therefore plays a dual role in the targeting of proteins to the outer envelope membrane and interior of chloroplasts.
- Published
- 2004
41. Insertion of atToc34 into the chloroplastic outer membrane is assisted by at least two proteinaceous components in the import system
- Author
-
Li-Yun Tsai, Shuh-Long Tu, and Hsou-min Li
- Subjects
Chloroplasts ,GTP' ,Arabidopsis Proteins ,Mutant ,Apyrase ,Arabidopsis ,Peas ,Thermolysin ,Membrane Proteins ,Cell Biology ,Biology ,Biochemistry ,Chloroplast ,Cytosol ,Adenosine Triphosphate ,Transit Peptide ,Biophysics ,Outer membrane efflux proteins ,Guanosine Triphosphate ,Bacterial outer membrane ,Molecular Biology ,Plant Proteins - Abstract
Toc34 is a member of the outer membrane translocon complex that mediates the initial stage of protein import into chloroplasts. Toc34, like most outer membrane proteins, is synthesized in the cytosol at its mature size without a cleavable transit peptide. The majority of outer membrane proteins do not require thermolysin-sensitive components on the chloroplastic surface or ATP for their insertion into the outer membrane. However, different results have been obtained concerning the factors required for Toc34 insertion into the outer membrane. Using an Arabidopsis homologue of pea Toc34, atToc34, we show that the insertion of atToc34 was greatly reduced by thermolysin pretreatment of chloroplasts as assayed either by protease digestion or by alkaline extraction. The insertion was also dependent on the presence of ATP or GTP. A mutant of atToc34 with the GTP-binding domain deleted still required ATP for optimal insertion, indicating that ATP was used by other protein components in the import system. The ATP-supported insertion was observed even in thermolysin-pretreated chloroplasts, suggesting that the protein component responsible for ATP-stimulated insertion is a different protein from the thermolysin-sensitive component that assists atToc34 insertion.
- Published
- 1999
42. A mutant deficient in the plastid lipid DGD is defective in protein import into chloroplasts
- Author
-
Lih-Jen Chen and Hsou-min Li
- Subjects
Signal peptide ,Chloroplasts ,Galactolipids ,Hydrolysis ,Arabidopsis ,food and beverages ,Biological Transport ,Cell Biology ,Plant Science ,Chloroplast outer membrane ,Membrane transport ,Biology ,Chloroplast ,Cytosol ,Adenosine Triphosphate ,Biochemistry ,ATP hydrolysis ,Genetics ,Plastids ,Plastid ,Glycolipids ,Diacylglycerol kinase ,Plant Proteins - Abstract
Summary Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol with N-terminal extensions called transit peptides. Transit peptides function as the import signal to chloroplasts. The import process requires several protein components in the envelope and stroma and also requires the hydrolysis of ATP. Lipids have been implicated in the import process based on theories or experiments within vitromodel systems. We show here that chloroplasts isolated from anArabidopsismutant deficient in the plastid lipid digalactosyl diacylglycerol (DGD) were normal in importing a chloroplast outer membrane protein, but were defective in importing precursor proteins targeted to the interior of chloroplasts. The impairment includes the binding, or docking, step of the import process that is supported by 100 μM ATP.
- Published
- 1998
43. An Arabidopsis mutant defective in the plastid general protein import apparatus
- Author
-
Paul Jarvis, Lih Jen Chen, Hsou-min Li, Charles A. Peto, Joanne Chory, and Christian Fankhauser
- Subjects
Toc complex ,Chlorophyll ,Chloroplasts ,Tic complex ,Molecular Sequence Data ,Arabidopsis ,Biology ,environment and public health ,Gene Expression Regulation, Plant ,Amino Acid Sequence ,Plastid ,Plastid envelope ,Plant Proteins ,Genetics ,Multidisciplinary ,Arabidopsis Proteins ,fungi ,food and beverages ,Membrane Proteins ,Biological Transport ,Intracellular Membranes ,Translocon ,biology.organism_classification ,Plants, Genetically Modified ,Cell biology ,Chloroplast ,Plant Leaves ,Phenotype ,Membrane protein ,Mutation ,lipids (amino acids, peptides, and proteins) ,Sequence Alignment - Abstract
Elaborate mechanisms have evolved for the translocation of nucleus-encoded proteins across the plastid envelope membrane. Although putative components of the import apparatus have been identified biochemically, their role in import remains to be proven in vivo. An Arabidopsis mutant lacking a new component of the import machinery [translocon at the outer envelope membrane of chloroplasts (Toc33), a 33-kilodalton protein] has been isolated. The functional similarity of Toc33 to another translocon component (Toc34) implies that multiple different translocon complexes are present in plastids. Processes that are mediated by Toc33 operate during the early stages of plastid and leaf development. The data demonstrate the in vivo role of a translocon component in plastid protein import.
- Published
- 1998
44. Structural characterizations of chloroplast translocon protein Tic110
- Author
-
Lih-Jen Chen, Yi-Hung Yeh, Chwan-Deng Hsiao, Jia-Yin Tsai, Hsou-min Li, and Chiung-Chih Chu
- Subjects
Chloroplast ,Structural Biology ,Chemistry ,Biophysics ,Translocon - Published
- 2013
- Full Text
- View/download PDF
45. Molecular cloning of a chloroplastic protein associated with both the envelope and thylakoid membranes
- Author
-
Hsou-min Li, Kenneth Keegstra, and Yasuko Kaneko
- Subjects
Chloroplasts ,Molecular Sequence Data ,Plant Science ,Molecular cloning ,Biology ,Genetics ,Amino Acid Sequence ,Cloning, Molecular ,Polyacrylamide gel electrophoresis ,Peptide sequence ,Plant Proteins ,Cloning ,Plants, Medicinal ,Base Sequence ,food and beverages ,Fabaceae ,General Medicine ,Compartment (chemistry) ,Intracellular Membranes ,Immunohistochemistry ,Chloroplast ,Molecular Weight ,Kinetics ,Membrane ,Biochemistry ,Thylakoid ,Electrophoresis, Polyacrylamide Gel ,Agronomy and Crop Science - Abstract
Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.
- Published
- 1994
46. Genetic Dissection of Signal Transduction Pathways that Regulate Cab Gene Expression
- Author
-
Ronald E. Susek, Hsou-min Li, Hector Cabrera, Lothar Altschmied, and Joanne Chory
- Subjects
Genetics ,Chloroplast ,Genetic dissection ,Greening ,Gene expression ,food and beverages ,Signal transduction ,Meristem ,Biology ,Gene - Abstract
In developing leaves, photosynthetically competent chloroplasts arise from small, undifferentiated proplastids that are present in meristematic cells. This process, called greening, involves the coordinate regulation of many nuclear and chloroplast genes (Mullet, 1988). The cues for the initiation of this developmental program are both extrinsic and intrinsic.
- Published
- 1993
- Full Text
- View/download PDF
47. Targeting of Proteins into and Across the Chloroplastic Envelope
- Author
-
Kenneth Keegstra, Jerry S. Marshall, Hsou-min Li, Sharyn E. Perry, and Jennifer Ostrom
- Subjects
Chloroplast ,Nuclear gene ,Cytoplasm ,Chemistry ,Thylakoid ,Organelle ,food and beverages ,Plastid ,Lipid bilayer ,Genome ,Cell biology - Abstract
Chloroplasts are functionally complex organelles that perform a diverse array of metabolic processes in addition to their well known role in photosynthesis. Consistent with their functional complexity, chloroplasts are structurally complex organelles, possessing three different lipid bilayer membranes enclosing three different aqueous compartments (Fig. 1). Because of the limited coding capacity of the plastid genome, most of the proteins in each compartment are encoded by nuclear genes and synthesized in the cytoplasm (Fig. 1). Understanding how these precursor proteins are targeted from the cytoplasm to their proper location within chloroplasts is an intriguing problem that has received considerable attention in recent years. In this chapter, we will briefly summarize relevant work from other laboratories as well as some recent results from our laboratory, all of which are aimed at understanding the import and proper localization of cytoplasmically synthesized chloroplastic proteins.
- Published
- 1992
- Full Text
- View/download PDF
48. Chapter 15 In Vitro Reconstitution of Protein Transport into Chloroplasts
- Author
-
Kenneth Keegstra, Hsou-min Li, and Sharyn E. Perry
- Subjects
Gel electrophoresis ,Chloroplast ,Electrophoresis ,Biochemistry ,food and beverages ,Compartment (chemistry) ,Cell fractionation ,Biology ,Membrane transport ,In vitro ,Transport protein - Abstract
Publisher Summary This chapter discusses various steps required for the in vitro reconstitution of protein transport into chloroplasts, the first being the production of precursor proteins. This is normally accomplished with an in vitro translation system, but other methods are also possible. The second step is isolation and purification of intact chloroplasts. Then, the precursor proteins and chloroplasts are incubated together under conditions that will allow transport to occur. The transport reaction is terminated by separating the chloroplasts, containing imported proteins, from the reaction mixture that contains residual precursors. It is possible to fractionate the recovered chloroplasts to determine which chloroplastic compartment contains the imported protein. Finally, the extent of transport is determined by measuring the amount of mature protein accumulated inside the chloroplasts. Transport has been reconstituted with many different precursors and with chloroplasts from several different species. The procedures that are used to study the transport of precursors into pea chloroplasts are discussed in the chapter. The products are then analyzed by electrophoretic separation of products and quantitative analysis of import products.
- Published
- 1991
- Full Text
- View/download PDF
49. Dimerization is important for the GTPase activity of chloroplast translocon components
- Author
-
Chwan-Deng Hsiao, Y.-H. Yeh, S.-S. Chern, Hsou-min Li, and Y.-J. Sun
- Subjects
Chloroplast ,Structural Biology ,Chemistry ,GTPase ,Translocon ,Cell biology - Published
- 2008
- Full Text
- View/download PDF
50. Metal-ion-center assembly of ferredoxin and plastocyanin in isolated chloroplasts
- Author
-
Steven M. Theg, Cynthia M. Bauerle, Hsou-min Li, and Kenneth Keegstra
- Subjects
Gel electrophoresis ,chemistry.chemical_classification ,Multidisciplinary ,biology ,Stereochemistry ,macromolecular substances ,environment and public health ,Cofactor ,Chloroplast ,Protein structure ,chemistry ,Membrane protein ,Biochemistry ,Metalloprotein ,biology.protein ,Plastocyanin ,Ferredoxin ,Research Article - Abstract
Most chloroplastic proteins are cytosolically synthesized and posttranslationally transported to their proper locations. Two examples of this group of proteins are ferredoxin and plastocyanin, both of which are metal-containing components of the photosynthetic electron-transport chain. The import process for these two proteins includes the insertion of the metal ions to produce the holo forms of the proteins. We show here that in vitro translated precursor proteins of ferredoxin and plastocyanin are synthesized as apo forms and are assembled into their respective holo forms after being imported into isolated chloroplasts. We also provide evidence that only mature-sized proteins are competent to be assembled into holo forms.
- Published
- 1990
Catalog
Discovery Service for Jio Institute Digital Library
For full access to our library's resources, please sign in.