90 results on '"Gietl C"'
Search Results
2. Ricinosomes: an organelle for developmentally regulated programmed cell death in senescing plant tissues
- Author
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Gietl, C. and Schmid, M.
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- 2001
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3. Independent spontaneous mitochondrial malate dehydrogenase null mutants in soybean are the result of deletions
- Author
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Imsande, J., Pittig, J., Palmer, R.G., Wimmer, C., and Gietl, C.
- Subjects
Genetics -- Case studies ,Heredity -- Research ,Soybean -- Genetic aspects ,Biological sciences - Abstract
Research describing the mitochondrial malate dehydrogenase-1 gene of soybean is presented. In particular spontaneous mutations to a null phenotype are investigated.
- Published
- 2001
4. Import of glyoxysomal malate dehydrogenase precursor into glyoxysomes: A heterologous in-vitro system
- Author
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Gietl, C. and Hock, B.
- Published
- 1986
5. Sequence homologies between glyoxysomal and mitochondrial malate dehydrogenase
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Gietl, C., Lottspeich, F., and Hock, B.
- Published
- 1986
6. Biogenesis of Plant Microbodies
- Author
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Hock, B., Gietl, C., Sautter, C., Fahimi, H. Dariush, editor, and Sies, Helmut, editor
- Published
- 1987
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7. AthPEX10, ariuclear gene essential for peroxisome and storage organelle formation during Arabidopsis embryogenesis
- Author
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Schumann, U., Wanner, G., Veenhuis, M., Schmid, M., Gietl, C., Schumann, U., Wanner, G., Veenhuis, M., Schmid, M., and Gietl, C.
- Abstract
Times Cited: 66
- Published
- 2003
8. AthPEX10, ariuclear gene essential for peroxisome and storage organelle formation during Arabidopsis embryogenesis
- Author
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Schumann, U, Wanner, G, Veenhuis, M, Schmid, M, Gietl, C, Schumann, U, Wanner, G, Veenhuis, M, Schmid, M, and Gietl, C
- Abstract
In yeasts and mammals, PEX10 encodes an integral membrane protein with a C3HC4 RING finger motif in its C-terminal domain and is required for peroxisome biogenesis and matrix protein import. in humans, its dysfunction in peroxisome biogenesis leads to severe Zellweger Syndrome and infantile Refsum disease. Here we show that dysfunction of a homologous gene in Arabidopsis leads to lethality at the heart stage of embryogenesis, impairing the biogenesis of peroxisomes, lipid bodies, and protein bodies. In a T-DNA insertion mutant disrupting the fourth exon of the Ath-PEX10 gene, ultrastructural analyses fail to detect peroxisomes characteristic for wild-type embryogenesis. Storage triacyl glycerides are not assembled into lipid bodies (oil bodies; oleosomes) surrounded by the phospholipid-protein monolayer membrane. Instead, the dysfunctional monolayer membranes, which derive from the bilayer membrane of the endoplasmic reticulum, accumulate in the cytosol. Concomitantly the transfer of the storage proteins from their site of synthesis at the endoplasmic reticulum to the vacuoles is disturbed. The mutant can be rescued by transformation with wild-type AthPEX10 cDNA. Transformants of wild-type Hansenula polymorpha cells with the AthPEX10 cDNA did produce the encoded protein without targeting it to peroxisomes. Additionally, the cDNA could not complement a Hansenula pex10 mutant unable to form peroxisomes. The ultrastructural knockout phenotype of AthPEX10p suggests that this protein in Arabidopsis is essential for peroxisome, oleosome, and protein transport vesicle formation.
- Published
- 2003
9. FUNCTIONAL-ANALYSIS OF THE N-TERMINAL PREPEPTIDES OF WATERMELON MITOCHONDRIAL AND GLYOXYSOMAL MALATE-DEHYDROGENASES
- Author
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LEHNERER, M, KEIZERGUNNIK, [No Value], VEENHUIS, M, GIETL, C, and Electron Microscopy
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HANSENULA POLYMORPHA ,PROTEINS ,CLEAVAGE ,IMPORT ,INTERMEDIATE PEPTIDASE ,ISOENZYMES ,ORGANELLES ,PRECURSOR CLEAVAGE ,THIOLASE ,MEMBRANES ,CITRULLUS VULGARIS ,PRECURSOR ,PEROXISOMES ,ENZYMES - Abstract
Mitochondrial and glyoxysomal malate dehydrogenase (mMDH; gMDH; L-malate : NAD(+) oxidoreductase; EC 1.1.1.37) of watermelon (Citrullus vulgaris) cotyledons are synthesized with N-terminal cleavable presequences which are shown to specify sorting of the two proteins. The two presequences differ in length (27 or 37 amino acids) and primary structure. Precursor proteins of the two isoenzymes with site-directed mutations in their presequences and hybrid precursor proteins with reciprocally exchanged presequences were analyzed for proper import using two approaches, namely in vitro using isolated watermelon organelles or in vivo after synthesis in the heterologous host, Hansenula polymorpha. The mitochondrial presequence is essential and sufficient to target the mature glyoxysomal isoenzyme into mitochondria (Gietl et al., 1994). As to the function of the mitochondrial presequence a substitution of (-3)R (considered important for one step precursor cleavage in yeast and mammals) with (-3)L permitted import into mitochondria but cleavage of the transit peptide and conversion into active mature enzyme was impeded. Substitution of (-13)R(-12)S (in a sequence reminiscent of the octapeptide motif serving as a substrate for the mammalian and yeast intermediate peptidase) into (-13)L(-12)F permitted mitochondrial import and processing like the wild type transit peptide. Purified rat mitochondrial processing protease, which can effect single step cleavage of mitochondrial protein precursors, cleaves in vitro translated watermelon mMDH precursor into its mature form. The glyoxysomal presequence is essential and sufficient to target the mature mitochondrial isoenzyme into peroxisomes of Hansenula polymorpha, but these peroxisomes lack a processing enzyme to cleave the presequence (Gietl et al., 1994). We here show that isolated watermelon organelles also import the hybrid proteins in vitro and process the glyoxysomal presequence. Site directed mutations within the conserved RI-X(5)-HL-motif impede efficiency of import and cleavage by watermelon organelles.
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- 1994
10. The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum
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Schmid, M., Simpson, D. J., Sarioglu, H., Lottspeich, F., Gietl, C., Schmid, M., Simpson, D. J., Sarioglu, H., Lottspeich, F., and Gietl, C.
- Abstract
Times Cited: 61
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- 2001
11. KDEL-tailed cysteine endopeptidases involved in programmed cell death, intercalation of new cells, and dismantling of extensin scaffolds
- Author
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Helm, M., primary, Schmid, M., additional, Hierl, G., additional, Terneus, K., additional, Tan, L., additional, Lottspeich, F., additional, Kieliszewski, M. J., additional, and Gietl, C., additional
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- 2008
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12. Vibration Response Imaging (VRI) bei Patienten mit Herzinsuffizienz und Lungenstauung
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Gmelin, C, primary, Gietl, C, additional, Barner, A, additional, Gat, M, additional, and Worth, H, additional
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- 2007
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13. Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes
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Schmid, M., Simpson, D., Gietl, C., Schmid, M., Simpson, D., and Gietl, C.
- Abstract
Times Cited: 89
- Published
- 1999
14. Normokapnie bei COPD – Akut oder chronisch?
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Gietl, C, primary, Bauer, JU, additional, and Laier-Groeneveld, G, additional
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- 2005
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15. Mature and translocatable forms of glyoxysomal malate dehydrogenase have different activities and stabilities but similar crystal structures
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Cox, B., primary, Chit, M.M., additional, Weaver, T., additional, Bailey, J., additional, Gietl, C., additional, Bell, E., additional, and Banaszak, L., additional
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- 2005
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16. Normokapnie bei COPD – Akut oder chronisch?
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Gietl, C, primary, Bauer, JU, additional, and Laier-Groeneveld, G, additional
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- 2004
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17. The plant PTS1 receptor : similarities and differences to its human and yeast counterparts
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Wimmer, C., Schmid, M., Veenhuis, M., Gietl, C., Wimmer, C., Schmid, M., Veenhuis, M., and Gietl, C.
- Abstract
Times Cited: 36
- Published
- 1998
18. A cysteine endopeptidase with a C-terminal KDEL motif isolated from castor bean endosperm is a marker enzyme for the ricinosome, a putative lytic compartment
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Schmid, M., Simpson, D., Kalousek, F., Gietl, C., Schmid, M., Simpson, D., Kalousek, F., and Gietl, C.
- Abstract
Times Cited: 58
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- 1998
19. A Cysteine Endopeptidase Isolated from Castor Bean Endosperm Microbodies Processes the Glyoxysomal Malate Dehydrogenase Precursor Protein
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Gietl, C., primary, Wimmer, B., additional, Adamec, J., additional, and Kalousek, F., additional
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- 1997
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20. The methylotrophic yeast Hansenula polymorpha contains an inducible import pathway for peroxisomal matrix proteins with an N-terminal targeting signal (PTS2 proteins).
- Author
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Faber, K N, primary, Haima, P, additional, Gietl, C, additional, Harder, W, additional, Ab, G, additional, and Veenhuis, M, additional
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- 1994
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21. Mutational analysis of the N-terminal topogenic signal of watermelon glyoxysomal malate dehydrogenase using the heterologous host Hansenula polymorpha.
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Gietl, C, primary, Faber, K N, additional, van der Klei, I J, additional, and Veenhuis, M, additional
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- 1994
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22. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast,Hansenula polymorpha
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van der Klei, I.J., primary, Faber, K.N., additional, Keizer-Gunnink, I., additional, Gietl, C., additional, Harder, W., additional, and Veenhuis, M., additional
- Published
- 1993
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23. Glyoxysomal malate dehydrogenase from watermelon is synthesized with an amino-terminal transit peptide.
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Gietl, C, primary
- Published
- 1990
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24. Untitled.
- Author
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Gietl, C. and Schmid, M.
- Abstract
This review describes aspects of programmed cell death (PCD). Present research maps the enzymes involved and explores the signal transduction pathways involved in their synthesis. A special organelle (the ricinosome) has been discovered in the senescing endosperm of germinating castor beans ( Ricinus communis) that develops at the beginning of PCD and delivers large amounts of a papain-type cysteine endopeptidase (CysEP) in the final stages of cellular disintegration. Castor beans store oil and proteins in a living endosperm surrounding the cotyledons. These stores are mobilized during germination and transferred into the cotyledons. PCD is initiated after this transfer is complete. The CysEP is synthesized in the lumen of the endoplasmic reticulum (ER) where it is retained by its C-terminal KDEL peptide as a rather inactive pro-enzyme. Large number of ricinosomes bud from the ER at the same time as the nuclear DNA is characteristically fragmented during PCD. The mitochondria, glyoxysomes and ribosomes are degraded in autophagic vacuoles, while the endopeptidase is activated by removal of the propeptide and the KDEL tail and enters the cytosol. The endosperm dries and detaches from the cotyledons. A homologous KDEL-tailed cysteine endopeptidase has been found in several senescing tissues; it has been localized in ricinosomes of withering day-lily petals and dying seed coats. Three genes for a KDEL-tailed cysteine endopeptidase have been identified in Arabidopsis. One is expressed in senescing ovules, the second in the vascular vessels and the third in maturing siliques. These genes open the way to exploring PCD in plants. [ABSTRACT FROM AUTHOR]
- Published
- 2001
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25. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenula polymorpha
- Author
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van der Klei, I.J., Faber, K.N., Keizer-Gunnink, I., Gietl, C., Harder, W., and Veenhuis, M.
- Abstract
We have studied the fate of the watermelon (Citrullus vulgarisSchrad.) glyoxysomal enzyme, malate dehydrogenase (gMDH), after synthesis in the methylotrophic yeast, Hansenula polymorpha. The gene encoding the precursor form of gMDH (pre‐gMDH) was cloned in an H. polymorphaexpression vector downstream of the inducible H. polymorphaalcohol oxidase promoter. During methylotrophic growth, pre‐gMDH was synthesized and imported into peroxisomes, where it was enzymatically active. The apparent molecular mass of the protein located in H. polymorphaperoxisomes was equal to that of pre‐gMDH (41 kDa), indicating that N‐terminal processing of the transit peptide had not occurred in the yeast.
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- 1993
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26. Mobilization of T-DNA from Agrobacterium to plant cells involves a protein that binds single-stranded DNA.
- Author
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Gietl, C, Koukolíková-Nicola, Z, and Hohn, B
- Abstract
Crude protein extracts of induced and uninduced octopine wild-type strain of Agrobacterium tumefaciens, as well as several mutants of the virulence loci virA, -B, -G, -C, -D, and -E, were probed with single- and double-stranded synthetic oligodeoxynucleotides of different sequence and length in an electrophoretic retardation assay. Four complexes involving sequence-nonspecific, single-stranded-DNA-binding proteins were recognized. One inducible complex is determined by the virE locus, two Ti-plasmid-dependent complexes are constitutively expressed, and a fourth one is controlled by chromosomal genes. The protein-DNA complexes were characterized by sucrose density gradient centrifugation and by determination of the length of single-stranded DNA required for their formation. It is hypothesized that the single-stranded-DNA-binding proteins are involved in the production of T-DNA intermediates or have a carrier or protective function during T-DNA transfer.
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- 1987
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27. Watermelon glyoxysomal malate dehydrogenase is sorted to peroxisomes of the methylotrophic yeast, Hansenuia polymorpha
- Author
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Klei, I. J. Van der, Faber, K. N., Keizer-Gunnink, I., and Gietl, C.
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- 1993
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28. Emergence and Epidemiology of Bovine Babesiosis Due to Babesia divergens on a Northern German Beef Production Farm.
- Author
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Springer A, Höltershinken M, Lienhart F, Ermel S, Rehage J, Hülskötter K, Lehmbecker A, Wohlsein P, Barutzki D, Gietl C, Baumgärtner W, Hoedemaker M, and Strube C
- Abstract
Babesia divergens , transmitted by the tick Ixodes ricinus , is the most common cause of bovine babesiosis in northern Europe and plays a role as a zoonotic pathogen. However, several studies have indicated a decline of B. divergens prevalence in Europe during the last decades. Here, we investigate the epidemiology of bovine babesiosis on a beef production farm in northern Germany, which had not been affected by babesiosis until an initial outbreak in 2018. In June 2018, 21 adult cattle died, showing classical symptoms of babesiosis. Babesia divergens merozoites were detected in blood smears of clinically affected animals and the species was confirmed by PCR and sequencing of a part of the 18S rRNA gene. In 2018, screening of the farm's entire stock by PCR revealed that Babesia -positive animals were present in only one of five herds grazing on different pastures. In the following year, further babesiosis cases occurred in multiple herds. In March 2020, 95 cattle were tested for anti- B. divergens antibodies and 36 of them (37.89%) had positive titres. To investigate the local Babesia prevalence in ticks, 1,430 questing I. ricinus ticks (555 larvae, 648 nymphs, 227 adults) were collected on the farm's pastures and subjected to PCR for Babesia detection. Babesia divergens DNA could not be detected, but Babesia microti showed an overall prevalence of 0.49% (7/1,430; 0.88% [2/227] of adult ticks, 0.77% [5/648] of nymphs, 0.00% [0/555] of larvae). Babesia venatorum was detected in 0.42% (6/1,430) of ticks (0.44% [1/227] of adult ticks, 0.77% [5/648] of nymphs, 0.00% [0/555] of larvae) and B. capreoli in 0.07% (1/1,430) of ticks (0.00% [0/227] of adult ticks, 0.15% [1/648] of nymphs, 0.00% [0/555] of larvae). Despite the fact that no B. divergens -positive ticks were found, the collected data suggest a geographical spread of the pathogen on the farm. Bovine babesiosis remains a disease of veterinary importance in Europe and may cause considerable economic losses when (re-)emerging in non-endemic areas, especially as awareness for the disease among veterinarians and farmers declines., (Copyright © 2020 Springer, Höltershinken, Lienhart, Ermel, Rehage, Hülskötter, Lehmbecker, Wohlsein, Barutzki, Gietl, Baumgärtner, Hoedemaker and Strube.)
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- 2020
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29. The role of KDEL-tailed cysteine endopeptidases of Arabidopsis (AtCEP2 and AtCEP1) in root development.
- Author
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Höwing T, Dann M, Müller B, Helm M, Scholz S, Schneitz K, Hammes UZ, and Gietl C
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- Apoptosis physiology, Arabidopsis Proteins genetics, Cysteine Endopeptidases genetics, Gene Expression Regulation, Developmental physiology, Gene Expression Regulation, Plant physiology, Gene Knockout Techniques, Plants, Genetically Modified, Seedlings growth & development, Arabidopsis growth & development, Arabidopsis Proteins metabolism, Cysteine Endopeptidases metabolism, Organogenesis, Plant physiology, Plant Roots growth & development
- Abstract
Plants encode a unique group of papain-type cysteine endopeptidases (CysEP) characterized by a C-terminal KDEL endoplasmic reticulum retention signal (KDEL-CysEP) and an unusually broad substrate specificity. The three Arabidopsis KDEL-CysEPs (AtCEP1, AtCEP2, and AtCEP3) are differentially expressed in vegetative and generative tissues undergoing programmed cell death (PCD). While KDEL-CysEPs have been shown to be implicated in the collapse of tissues during PCD, roles of these peptidases in processes other than PCD are unknown. Using mCherry-AtCEP2 and EGFP-AtCEP1 reporter proteins in wild type versus atcep2 or atcep1 mutant plants, we explored the participation of AtCEP in young root development. Loss of AtCEP2, but not AtCEP1 resulted in shorter primary roots due to a decrease in cell length in the lateral root (LR) cap, and impairs extension of primary root epidermis cells such as trichoblasts in the elongation zone. AtCEP2 was localized to root cap corpses adherent to epidermal cells in the rapid elongation zone. AtCEP1 and AtCEP2 are expressed in root epidermis cells that are separated for LR emergence. Loss of AtCEP1 or AtCEP2 caused delayed emergence of LR primordia. KDEL-CysEPs might be involved in developmental tissue remodeling by supporting cell wall elongation and cell separation., Competing Interests: The authors have declared that no competing interests exist.
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- 2018
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30. Involvement of Arabidopsis thaliana endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 (AtCEP1) in powdery mildew-induced and AtCPR5-controlled cell death.
- Author
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Höwing T, Dann M, Hoefle C, Hückelhoven R, and Gietl C
- Subjects
- Arabidopsis microbiology, Microscopy, Fluorescence, Arabidopsis enzymology, Cell Death, Cysteine Endopeptidases metabolism, Endoplasmic Reticulum enzymology, Mycoses enzymology, Plant Diseases microbiology
- Abstract
Programmed cell death (PCD) is a prerequisite for successful development and it limits the spread of biotrophic pathogens in a rapid hypersensitive response at the site of infection. KDEL-tailed cysteine endopeptidases (KDEL CysEP) are a subgroup of papain-type cysteine endopeptidases expressed in tissues undergoing PCD. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. We have previously shown that AtCEP1 is a factor of basal resistance to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum, and is expressed in spatiotemporal association with the late fungal development on Arabidopsis leaves. The endoplasmic reticulum-localized proenzyme of AtCEP1 was further visualized at the haustorial complex encased with callose. The AtCPR5 gene (CONSTITUTIVE EXPRESSION OF PR GENES 5) is a regulator of expression of pathogenesis related genes. Loss of AtCPR5 leads to spontaneous expression of chlorotic lesions which was associated with enhanced expression of AtCEP1. We used the atcpr5-2 mutant plants and the atcep1 atcpr5-2 double mutants harboring a non-functional reporter (PCEP1::pre-pro-3xHA-EGFP-KDEL) for visualization of AtCEP1 promoter activity. We found the specific up-regulation of AtCEP1 in direct neighborhood of spreading leaf lesions thus likely representing cells undergoing PCD. Furthermore, we found a strong resistance of atcpr5 mutant plants against infection with E. cruciferarum. Loss of AtCEP1 had no obvious influence on the strong resistance of atcpr5-2 mutant plants against infection with E. cruciferarum. However, the area of necrotic leaf lesions associated with E. cruciferarum colonies was significantly larger in atcpr5-2 as compared to atcep1 atcpr5-2 double mutant plants. The presence of AtCEP1 thus contributes to AtCPR5-controlled PCD at the sites of powdery mildew infection.
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- 2017
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31. Expression analysis of KDEL-CysEPs programmed cell death markers during reproduction in Arabidopsis.
- Author
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Zhou LZ, Höwing T, Müller B, Hammes UZ, Gietl C, and Dresselhaus T
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- Arabidopsis growth & development, Arabidopsis physiology, Arabidopsis Proteins metabolism, Biomarkers metabolism, Cysteine Endopeptidases metabolism, Fertilization, Flowers genetics, Flowers growth & development, Flowers physiology, Gene Expression Regulation, Plant, Reproduction, Seeds genetics, Seeds growth & development, Seeds physiology, Xylem genetics, Xylem growth & development, Xylem physiology, Apoptosis, Arabidopsis genetics, Arabidopsis Proteins genetics, Cysteine Endopeptidases genetics
- Abstract
Key Message: CEP cell death markers. Programmed cell death (PCD) is essential for proper plant growth and development. Plant-specific papain-type KDEL-tailed cysteine endopeptidases (KDEL-CysEPs or CEPs) have been shown to be involved in PCD during vegetative development as executors for the last step in the process. The Arabidopsis genome encodes three KDEL-CysEPs: AtCEP1, AtCEP2 and AtCEP3. With the help of fluorescent fusion reporter lines, we report here a detailed expression analysis of KDEL-CysEP (pro)proteins during reproductive processes, including flower organ and germline development, fertilization and seed development. AtCEP1 is highly expressed in different reproductive tissues including nucellus cells of mature ovule and the connecting edge of anther and filament. After fertilization, AtCEP1 marks integument cell layers of the seeds coat as well as suspensor and columella cells of the developing embryo. Promoter activity of AtCEP2 is detected in the style of immature and mature pistils, in other floral organs including anther, sepal and petal. AtCEP2 mainly localizes to parenchyma cells next to xylem vessels. Although there is no experimental evidence to demonstrate that KDEL-CysEPs are involved in PCD during fertilization, the expression pattern of AtCEPs, which were previously shown to represent cell death markers during vegetative development, opens up new avenues to investigate PCD in plant reproduction.
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- 2016
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32. Ex vivo processing for maturation of Arabidopsis KDEL-tailed cysteine endopeptidase 2 (AtCEP2) pro-enzyme and its storage in endoplasmic reticulum derived organelles.
- Author
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Hierl G, Höwing T, Isono E, Lottspeich F, and Gietl C
- Subjects
- Arabidopsis cytology, Arabidopsis genetics, Arabidopsis physiology, Arabidopsis Proteins genetics, Cell Wall metabolism, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases genetics, Enzyme Activation, Enzyme Precursors chemistry, Enzyme Precursors genetics, Hydrogen-Ion Concentration, Hypocotyl cytology, Hypocotyl enzymology, Hypocotyl genetics, Hypocotyl physiology, Oligopeptides genetics, Organ Specificity, Plant Epidermis cytology, Plant Epidermis enzymology, Plant Epidermis genetics, Plant Epidermis physiology, Plant Leaves cytology, Plant Leaves enzymology, Plant Leaves genetics, Plant Leaves physiology, Plant Roots cytology, Plant Roots enzymology, Plant Roots genetics, Plant Roots physiology, Plants, Genetically Modified, Protein Sorting Signals genetics, Protein Transport, Recombinant Fusion Proteins, Sequence Deletion, Substrate Specificity, Apoptosis physiology, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Cysteine Endopeptidases metabolism, Endoplasmic Reticulum enzymology, Enzyme Precursors metabolism, Oligopeptides metabolism
- Abstract
Ricinosomes are specialized ER-derived organelles that store the inactive pro-forms of KDEL-tailed cysteine endopeptidases (KDEL-CysEP) associated with programmed cell death (PCD). The Arabidopsis genome encodes three KDEL-CysEP (AtCEP1, AtCEP2, and AtCEP3) that are differentially expressed in vegetative and generative tissues undergoing PCD. These Arabidopsis proteases have not been characterized at a biochemical level, nor have they been localized intracellularly. In this study, we characterized AtCEP2. A 3xHA-mCherry-AtCEP2 gene fusion including pro-peptide and KDEL targeting sequences expressed under control of the endogenous promoter enabled us to isolate AtCEP2 "ex vivo". The purified protein was shown to be activated in a pH-dependent manner. After activation, however, protease activity was pH-independent. Analysis of substrate specificity showed that AtCEP2 accepts proline near the cleavage site, which is a rare feature specific for KDEL-CysEPs. mCherry-AtCEP2 was detected in the epidermal layers of leaves, hypocotyls and roots; in the root, it was predominantly found in the elongation zone and root cap. Co-localization with an ER membrane marker showed that mCherry-AtCEP2 was stored in two different types of ER-derived organelles: 10 μm long spindle shaped organelles as well as round vesicles with a diameter of approximately 1 μm. The long organelles appear to be ER bodies, which are found specifically in Brassicacae. The round vesicles strongly resemble the ricinosomes first described in castor bean. This study provides a first evidence for the existence of ricinosomes in Arabidopsis, and may open up new avenues of research in the field of PCD and developmental tissue remodeling.
- Published
- 2014
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33. Endoplasmic reticulum KDEL-tailed cysteine endopeptidase 1 of Arabidopsis (AtCEP1) is involved in pathogen defense.
- Author
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Höwing T, Huesmann C, Hoefle C, Nagel MK, Isono E, Hückelhoven R, and Gietl C
- Abstract
Programmed cell death (PCD) is a genetically determined process in all multicellular organisms. Plant PCD is effected by a unique group of papain-type cysteine endopeptidases (CysEP) with a C-terminal KDEL endoplasmic reticulum (ER) retention signal (KDEL CysEP). KDEL CysEPs can be stored as pro-enzymes in ER-derived endomembrane compartments and are released as mature CysEPs in the final stages of organelle disintegration. KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated hydroxyprolines of the extensins that form the basic scaffold of the cell wall. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2, and AtCEP3) are expressed. Cell- and tissue-specific activities of these three genes suggest that KDEL CysEPs participate in the abscission of flower organs and in the collapse of tissues in the final stage of PCD as well as in developmental tissue remodeling. We observed that AtCEP1 is expressed in response to biotic stress stimuli in the leaf. atcep1 knockout mutants showed enhanced susceptibility to powdery mildew caused by the biotrophic ascomycete Erysiphe cruciferarum. A translational fusion protein of AtCEP1 with a three-fold hemaglutinin-tag and the green fluorescent protein under control of the endogenous AtCEP1 promoter (PCEP1::pre-pro-3xHA-EGFP-AtCEP1-KDEL) rescued the pathogenesis phenotype demonstrating the function of AtCEP1 in restriction of powdery mildew. The spatiotemporal AtCEP1-reporter expression during fungal infection together with microscopic inspection of the interaction phenotype suggested a function of AtCEP1 in controlling late stages of compatible interaction including late epidermal cell death. Additionally, expression of stress response genes appeared to be deregulated in the interaction of atcep1 mutants and E. cruciferarum. Possible functions of AtCEP1 in restricting parasitic success of the obligate biotrophic powdery mildew fungus are discussed.
- Published
- 2014
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34. Regulatory focus in the life story: prevention and promotion as expressed in three layers of personality.
- Author
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Manczak EM, Zapata-Gietl C, and McAdams DP
- Subjects
- Female, Humans, Longitudinal Studies, Male, Memory, Episodic, Middle Aged, Models, Psychological, Personality Inventory, Quality of Life psychology, Goals, Personality physiology, Sense of Coherence physiology
- Abstract
Regulatory Focus Theory (Higgins, 1997) outlines the ways in which people attempt to achieve the presence of positive outcomes (promotion) or preserve the absence of negative outcomes (prevention), suggesting that individuals may differ in chronic orientations. The present work examines regulatory outlooks within a multilayered model of personality composed of dispositional traits, characteristic goals, and life stories (narrative identity). Foregrounding the concept of narrative identity, the current study investigates how narrative themes of prevention and promotion relate to regulatory focus as expressed in dispositional traits and characteristic goals and explores the relative contribution of each layer of personality to psychological well-being and physical health. The findings suggest that dispositional traits, personal goals, and life narratives cohere loosely around the central themes of prevention and promotion. Moreover, promotion focus across layers of personality was related to higher levels of self-reported quality of life, compared to prevention focus. Illustrating the incremental validity of different layers of personality, promotion focus in life stories independently predicted psychological health above and beyond promotion focus in dispositional traits.
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- 2014
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35. Calmodulin-like protein AtCML3 mediates dimerization of peroxisomal processing protease AtDEG15 and contributes to normal peroxisome metabolism.
- Author
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Dolze E, Chigri F, Höwing T, Hierl G, Isono E, Vothknecht UC, and Gietl C
- Subjects
- Arabidopsis genetics, Arabidopsis metabolism, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Calmodulin metabolism, Chromosomes, Artificial, Yeast genetics, Dimerization, Intracellular Calcium-Sensing Proteins genetics, Intracellular Calcium-Sensing Proteins metabolism, Intracellular Calcium-Sensing Proteins physiology, Peptide Hydrolases metabolism, Phylogeny, Recombinant Proteins, Sequence Alignment, Serine Endopeptidases genetics, Serine Endopeptidases metabolism, Serine Endopeptidases physiology, Arabidopsis Proteins metabolism, Peroxisomes metabolism
- Abstract
Matrix enzymes are imported into peroxisomes and glyoxysomes, a subclass of peroxisomes involved in lipid mobilization. Two peroxisomal targeting signals (PTS), the C-terminal PTS1 and the N-terminal PTS2, mediate the translocation of proteins into the organelle. PTS2 processing upon import is conserved in higher eukaryotes, and in watermelon the glyoxysomal processing protease (GPP) was shown to catalyse PTS2 processing. GPP and its ortholog, the peroxisomal DEG protease from Arabidopsis thaliana (AtDEG15), belong to the Deg/HtrA family of ATP-independent serine proteases with Escherichia coli DegP as their prototype. GPP existes in monomeric and dimeric forms. Their equilibrium is shifted towards the monomer upon Ca(2+)-removal and towards the dimer upon Ca(2+)-addition, which is accompanied by a change in substrate specificity from a general protease (monomer) to the specific cleavage of the PTS2 (dimer). We describe the Ca(2+)/calmodulin (CaM) mediated dimerization of AtDEG15. Dimerization is mediated by the CaM-like protein AtCML3 as shown by yeast two and three hybrid analyses. The binding of AtCML3 occurs within the first 25 N-terminal amino acids of AtDEG15, a domain containing a predicted CaM-binding motif. Biochemical analysis of AtDEG15 deletion constructs in planta support the requirement of the CaM-binding domain for PTS2 processing. Phylogenetic analyses indicate that the CaM-binding site is conserved in peroxisomal processing proteases of higher plants (dicots, monocots) but not present in orthologs of animals or cellular slime molds. Despite normal PTS2 processing activity, an atcml3 mutant exhibited reduced 2,4-DB sensitivity, a phenotype previously reported for the atdeg15 mutant, indicating similarly impaired peroxisome metabolism.
- Published
- 2013
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36. Programmed cell death in Ricinus and Arabidopsis: the function of KDEL cysteine peptidases in development.
- Author
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Hierl G, Vothknecht U, and Gietl C
- Subjects
- Acids metabolism, Arabidopsis growth & development, Cell Wall metabolism, Endoplasmic Reticulum enzymology, Endosperm growth & development, Endosperm metabolism, Glycoproteins metabolism, Plant Cells enzymology, Plant Proteins metabolism, Proteolysis, Ricinus growth & development, Seeds enzymology, Seeds growth & development, Substrate Specificity, Vacuoles metabolism, Arabidopsis cytology, Arabidopsis enzymology, Cell Death, Cysteine Endopeptidases metabolism, Ricinus cytology, Ricinus enzymology
- Abstract
Programmed cell death (PCD) in plants is a prerequisite for development as well as seed and fruit production. It also plays a significant role in pathogen defense. A unique group of papain-type cysteine endopeptidases, characterized by a C-terminal endoplasmic reticulum (ER) retention signal (KDEL CysEP), is involved in plant PCD. Genes for these endopeptidases have been sequenced and analyzed from 25 angiosperms and gymnosperms. They have no structural relationship to caspases involved in mammalian PCD and homologs to this group of plant cysteine endopeptidases have not been found in mammals or yeast. In castor beans (Ricinus communis), the CysEP is synthesized as pre-pro-enzyme. The pro-enzyme is transported to the cytosol of cells undergoing PCD in ER-derived vesicles called ricinosomes. These vesicles release the mature CysEP in the final stages of organelle disintegration triggered by acidification of the cytoplasm resulting from the disruption of the vacuole. Mature CysEP digests the hydroxyproline (Hyp)-rich proteins (extensins) that form the basic scaffold of the plant cell wall. The KDEL CysEPs accept a wide variety of amino acids at the active site, including the glycosylated Hyp residues of the extensins. In Arabidopsis, three KDEL CysEPs (AtCEP1, AtCEP2 and AtCEP3) are expressed in tissues undergoing PCD. In transgenic Arabidopsis plants expressing β-glucuronidase under the control of the promoters for these three genes, cell- and tissue-specific activities were mapped during seedling, flower and seed development. KDEL CysEPs participate in the collapse of tissues in the final stage of PCD and in tissue re-modeling such as lateral root formation., (Copyright © Physiologia Plantarum 2012.)
- Published
- 2012
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37. The Arabidopsis calmodulin-like proteins AtCML30 and AtCML3 are targeted to mitochondria and peroxisomes, respectively.
- Author
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Chigri F, Flosdorff S, Pilz S, Kölle E, Dolze E, Gietl C, and Vothknecht UC
- Subjects
- Amino Acid Sequence, Arabidopsis genetics, Arabidopsis ultrastructure, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Bacterial Proteins genetics, Bacterial Proteins metabolism, Biological Transport, Active, Calmodulin chemistry, Calmodulin genetics, Intracellular Calcium-Sensing Proteins chemistry, Intracellular Calcium-Sensing Proteins genetics, Luminescent Proteins genetics, Luminescent Proteins metabolism, Mitochondria metabolism, Molecular Sequence Data, Peroxisomes metabolism, Phylogeny, Plants, Genetically Modified, Protein Conformation, Recombinant Fusion Proteins genetics, Recombinant Fusion Proteins metabolism, Sequence Homology, Amino Acid, Signal Transduction, Nicotiana genetics, Nicotiana metabolism, Nicotiana ultrastructure, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Calmodulin metabolism, Intracellular Calcium-Sensing Proteins metabolism
- Abstract
Calmodulin (CaM) is a ubiquitous sensor/transducer of calcium signals in eukaryotic organisms. While CaM mediated calcium regulation of cytosolic processes is well established, there is growing evidence for the inclusion of organelles such as chloroplasts, mitochondria and peroxisomes into the calcium/calmodulin regulation network. A number of CaM-binding proteins have been identified in these organelles and processes such as protein import into chloroplasts and mitochondria have been shown to be governed by CaM regulation. What have been missing to date are the mediators of this regulation since no CaM or calmodulin-like protein (CML) has been identified in any of these organelles. Here we show that two Arabidopsis CMLs, AtCML3 and AtCML30, are localized in peroxisomes and mitochondria, respectively. AtCML3 is targeted via an unusual C-terminal PTS1-like tripeptide while AtCML30 utilizes an N-terminal, non-cleavable transit peptide. Both proteins possess the typical structure of CaMs, with two pairs of EF-hand motifs separated by a short linker domain. They furthermore display common characteristics, such as calcium-dependent alteration of gel mobility and calcium-dependent exposure of a hydrophobic surface. This indicates that they can function in a similar manner as canonical CaMs. The presence of close homologues to AtCML3 and AtCML30 in other plants further indicates that organellar targeting of these CMLs is not a specific feature of Arabidopsis. The identification of peroxisomal and mitochondrial CMLs is an important step in the understanding how these organelles are integrated into the cellular calcium/calmodulin signaling pathways.
- Published
- 2012
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38. Different functions of the C3HC4 zinc RING finger peroxins PEX10, PEX2, and PEX12 in peroxisome formation and matrix protein import.
- Author
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Prestele J, Hierl G, Scherling C, Hetkamp S, Schwechheimer C, Isono E, Weckwerth W, Wanner G, and Gietl C
- Subjects
- Amino Acid Motifs genetics, Amino Acid Sequence, Arabidopsis genetics, Arabidopsis Proteins genetics, Biological Transport, Carbon Dioxide metabolism, Extracellular Matrix Proteins metabolism, Gene Expression Regulation, Plant, Glyoxysomes metabolism, Glyoxysomes ultrastructure, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Membrane Proteins genetics, Membrane Transport Proteins genetics, Metabolomics methods, Microscopy, Confocal, Microscopy, Electron, Models, Biological, Molecular Sequence Data, Mutation, Peroxins, Peroxisome-Targeting Signal 1 Receptor, Peroxisomes ultrastructure, Photosynthesis, Plants, Genetically Modified, RING Finger Domains genetics, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear metabolism, Reverse Transcriptase Polymerase Chain Reaction, Sequence Homology, Amino Acid, Zinc Fingers genetics, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Membrane Proteins metabolism, Membrane Transport Proteins metabolism, Peroxisomes metabolism
- Abstract
The integral peroxisomal membrane proteins PEX10, PEX2, and PEX12 contain a zinc RING finger close to the C terminus. Loss of function of these peroxins causes embryo lethality at the heart stage in Arabidopsis. Preventing the coordination of Zn(2+) ions by amino acid substitutions in PEX10, PEX2, and PEX12 and overexpressing the resulting conditional sublethal mutations in WT uncovered additional functions of PEX10. Plants overexpressing DeltaZn-mutant PEX10 display deformed peroxisomal shapes causing diminished contact with chloroplasts and possibly with mitochondria. These changes correlated with impaired metabolite transfer and, at high CO(2), recoverable defective photorespiration plus dwarfish phenotype. The N-terminal PEX10 domain is critical for peroxisome biogenesis and plant development. A point mutation in the highly conserved TLGEEY motif results in vermiform peroxisome shape without impairing organelle contact. Addition of an N-terminal T7 tag to WT PEX0 resulted in partially recoverable reduced growth and defective inflorescences persisting under high CO(2). In contrast, plants overexpressing PEX2-DeltaZn-T7 grow like WT in normal atmosphere, contain normal-shaped peroxisomes, but display impaired peroxisomal matrix protein import. PEX12-DeltaZn-T7 mutants exhibit unimpaired import of matrix protein and normal-shaped peroxisomes when grown in normal atmosphere. During seed germination, glyoxysomes form a reticulum around the lipid bodies for mobilization of storage oil. The formation of this glyoxysomal reticulum seemed to be impaired in PEX10-DeltaZn but not in PEX2-DeltaZn-T7 or PEX12-DeltaZn-T7 plants. Both cytosolic PEX10 domains seem essential for peroxisome structure but differ in metabolic function, suggesting a role for this plant peroxin in addition to the import of matrix protein via ubiquitination of PEX5.
- Published
- 2010
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39. The DEG15 serine protease cleaves peroxisomal targeting signal 2-containing proteins in Arabidopsis.
- Author
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Schuhmann H, Huesgen PF, Gietl C, and Adamska I
- Subjects
- 2,4-Dichlorophenoxyacetic Acid analogs & derivatives, 2,4-Dichlorophenoxyacetic Acid pharmacology, Amino Acid Sequence, Arabidopsis drug effects, Arabidopsis metabolism, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Green Fluorescent Proteins analysis, Molecular Sequence Data, Mutagenesis, Site-Directed, Protein Structure, Tertiary, Recombinant Fusion Proteins analysis, Sequence Alignment, Serine Endopeptidases chemistry, Serine Endopeptidases genetics, Substrate Specificity, Arabidopsis enzymology, Arabidopsis Proteins metabolism, Peroxisomes metabolism, Protein Sorting Signals, Serine Endopeptidases physiology
- Abstract
Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. Recently, candidates for the responsible processing protease were identified from plants (DEG15) and mammals (TYSND1). We demonstrate that plants lacking DEG15 show an expressed phenotype potentially linked to reduced beta-oxidation, indicating the impact of protein processing on peroxisomal functions in higher eukaryotes. Mutational analysis of Arabidopsis (Arabidopsis thaliana) DEG15 revealed that conserved histidine, aspartic acid, and serine residues are essential for the proteolytic activity of this enzyme in vitro. This indicates that DEG15 and related enzymes are trypsin-like serine endopeptidases. Deletion of a plant-specific stretch present in the protease domain diminished, but did not abolish, the proteolytic activity of DEG15 against the PTS2-containing glyoxysomal malate dehydrogenase. Fluorescence microscopy showed that a DEG15-green fluorescent protein fusion construct is targeted to peroxisomes in planta. In vivo studies with isolated homozygous deg15 knockout mutants and complemented mutant lines suggest that this enzyme mediates general processing of PTS2-containing proteins.
- Published
- 2008
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40. Normocapnia following noninvasive ventilation in acute exacerbations and chronic state of obstructive pulmonary disease.
- Author
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Laier-Groeneveld G, Gietl C, and Bauer JU
- Subjects
- Bicarbonates blood, Carbon Dioxide metabolism, Female, Humans, Hydrogen-Ion Concentration, Hypercapnia etiology, Hypercapnia metabolism, Hypercapnia physiopathology, Inhalation, Male, Partial Pressure, Pulmonary Disease, Chronic Obstructive metabolism, Pulmonary Disease, Chronic Obstructive physiopathology, Pulmonary Disease, Chronic Obstructive therapy, Tidal Volume, Treatment Outcome, Hypercapnia therapy, Pulmonary Disease, Chronic Obstructive complications, Respiration, Artificial methods
- Abstract
We attempted to decrease PCO2 during noninvasive ventilation (NIV) and studied he effects of this therapy both in acute exacerbations of chronic obstructive pulmonary disease (COPD) and in its chronic state. Ninety six patients (63% male) with COPD and hypercapnia above 6.7 kPa were investigated. The mode and setting of the ventilator had to be chosen to achieve normocapnia. The subgroup of acute exacerbated COPD was separated by pH (<7.35=acute), by HCO3- (<26 mmol/l=acute), and by history (acute=history of recent deterioration). Ventilator settings were the following: tidal volume-972+/-137 ml and frequency-20+/-2.2 (volume preset). Inspiratory pressure was 33.6+/-14.2 mbar and frequency-19.7+/-5.1 (pressure preset). The preference of volume preset ventilators resulted from insufficient maximal pressures of the pressure preset devices. Eighty three percent of the patients became normocapnic while on NIV after 6.8+/-5.7 days. The mean PCO2 decreased from 64+/-13 mmHg to 41+/-6 mmHg (P<0.001). After 4 weeks, 72% of the patients were normocapnic while breathing spontaneously (P<0.001). The subgroups of acute exacerbation were the following: pH 28%, HCO3- 3.1%, and history 68%. All three indicators together were present in 2% of patients. Normocapnia under ventilation and during spontaneous breathing was independent from the subgroup. In conclusion, the study showed that normocapnia can be achieved in COPD under the ventilator and while breathing spontaneously in chronic and acute disease.
- Published
- 2007
41. Dual specificities of the glyoxysomal/peroxisomal processing protease Deg15 in higher plants.
- Author
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Helm M, Lück C, Prestele J, Hierl G, Huesgen PF, Fröhlich T, Arnold GJ, Adamska I, Görg A, Lottspeich F, and Gietl C
- Subjects
- Arabidopsis enzymology, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Enzyme Precursors chemistry, Enzyme Precursors genetics, Enzyme Precursors metabolism, Glyoxysomes genetics, Heat-Shock Proteins chemistry, Malate Dehydrogenase genetics, Mutation, Periplasmic Proteins chemistry, Peroxisomes genetics, Plant Proteins genetics, Plant Proteins metabolism, Plants, Genetically Modified, Serine Endopeptidases chemistry, Serine Endopeptidases genetics, Substrate Specificity genetics, Arabidopsis Proteins metabolism, Citrullus enzymology, Glyoxysomes enzymology, Heat-Shock Proteins metabolism, Periplasmic Proteins metabolism, Peroxisomes enzymology, Protein Processing, Post-Translational, Serine Endopeptidases metabolism, Serine Endopeptidases physiology
- Abstract
Glyoxysomes are a subclass of peroxisomes involved in lipid mobilization. Two distinct peroxisomal targeting signals (PTSs), the C-terminal PTS1 and the N-terminal PTS2, are defined. Processing of the PTS2 on protein import is conserved in higher eukaryotes. The cleavage site typically contains a Cys at P1 or P2. We purified the glyoxysomal processing protease (GPP) from the fat-storing cotyledons of watermelon (Citrullus vulgaris) by column chromatography, preparative native isoelectric focusing, and 2D PAGE. The GPP appears in two forms, a 72-kDa monomer and a 144-kDa dimer, which are in equilibrium with one another. The equilibrium is shifted on Ca(2+) removal toward the monomer and on Ca(2+) addition toward the dimer. The monomer is a general degrading protease and is activated by denatured proteins. The dimer constitutes the processing protease because the substrate specificity proven for the monomer (Phi-Arg/Lys downward arrow) is different from the processing substrate specificity (Cys-Xxx downward arrow/Xxx-Cys downward arrow) found with the mixture of monomer and dimer. The Arabidopsis genome analysis disclosed three proteases predicted to be in peroxisomes, a Deg-protease, a pitrilysin-like metallopeptidase, and a Lon-protease. Specific antibodies against the peroxisomal Deg-protease from Arabidopsis (Deg15) identify the watermelon GPP as a Deg15. A knockout mutation in the DEG15 gene of Arabidopsis (At1g28320) prevents processing of the glyoxysomal malate dehydrogenase precursor to the mature form. Thus, the GPP/Deg15 belongs to a group of trypsin-like serine proteases with Escherichia coli DegP as a prototype. Nevertheless, the GPP/Deg15 possesses specific characteristics and is therefore a new subgroup within the Deg proteases.
- Published
- 2007
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42. Requirement of the C3HC4 zinc RING finger of the Arabidopsis PEX10 for photorespiration and leaf peroxisome contact with chloroplasts.
- Author
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Schumann U, Prestele J, O'Geen H, Brueggeman R, Wanner G, and Gietl C
- Subjects
- Amino Acid Sequence, Arabidopsis chemistry, Arabidopsis genetics, Arabidopsis growth & development, Arabidopsis Proteins chemistry, Arabidopsis Proteins genetics, Cell Respiration, DNA, Plant genetics, Gene Expression Regulation, Plant, Genome, Plant genetics, Glyoxysomes metabolism, Membrane Transport Proteins chemistry, Membrane Transport Proteins genetics, Microscopy, Electron, Molecular Sequence Data, Mutation genetics, Oxidation-Reduction, Peroxins, Photochemistry, Plant Leaves chemistry, Plant Leaves genetics, Plant Leaves metabolism, Plant Leaves ultrastructure, Plants, Genetically Modified, Seedlings genetics, Seedlings metabolism, Transcription, Genetic genetics, Transgenes genetics, Zinc Fingers, Arabidopsis metabolism, Arabidopsis Proteins metabolism, Chloroplasts metabolism, Membrane Transport Proteins metabolism, Peroxisomes metabolism
- Abstract
Plant peroxisomes perform multiple vital metabolic processes including lipid mobilization in oil-storing seeds, photorespiration, and hormone biosynthesis. Peroxisome biogenesis requires the function of peroxin (PEX) proteins, including PEX10, a C(3)HC(4) Zn RING finger peroxisomal membrane protein. Loss of function of PEX10 causes embryo lethality at the heart stage. We investigated the function of PEX10 with conditional sublethal mutants. Four T-DNA insertion lines expressing pex10 with a dysfunctional RING finger were created in an Arabidopsis WT background (DeltaZn plants). They could be normalized by growth in an atmosphere of high CO(2) partial pressure, indicating a defect in photorespiration. beta-Oxidation in mutant glyoxysomes was not affected. However, an abnormal accumulation of the photorespiratory metabolite glyoxylate, a lowered content of carotenoids and chlorophyll a and b, and a decreased quantum yield of photosystem II were detected under normal atmosphere, suggesting impaired leaf peroxisomes. Light and transmission electron microscopy demonstrated leaf peroxisomes of the DeltaZn plants to be more numerous, multilobed, clustered, and not appressed to the chloroplast envelope as in WT. We suggest that inactivation of the RING finger domain in PEX10 has eliminated protein interaction required for attachment of peroxisomes to chloroplasts and movement of metabolites between peroxisomes and chloroplasts.
- Published
- 2007
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43. Ricinosomes and endosperm transfer cell structure in programmed cell death of the nucellus during Ricinus seed development.
- Author
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Greenwood JS, Helm M, and Gietl C
- Subjects
- Cellular Senescence, Cysteine Endopeptidases metabolism, DNA Fragmentation, In Situ Nick-End Labeling, Organelles ultrastructure, Ricin metabolism, Apoptosis physiology, Organelles metabolism, Plant Proteins metabolism, Ricinus cytology, Ricinus physiology, Seeds chemistry, Seeds growth & development, Seeds ultrastructure
- Abstract
The ricinosome (precursor protease vesicle) is an organelle found exclusively in plant cells. Ricinosomes contain a 45-kDa pro-cysteine endopeptidase (CysEP) with a C-terminal KDEL endoplasmic reticulum retention signal. CysEP is a member of a unique group of papain-type cysteine peptidases found specifically in senescing and ricinosome-containing tissues. During seed development in the castor oil plant (Ricinus communis L.), the cells of the nucellus are killed as the major seed storage organ, the cellular endosperm, expands and begins to accumulate reserves. The destruction of the maternal seed tissues is a developmentally programmed cell death. Terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling revealed that nuclear DNA fragmentation occurs in the nucellar cells adjacent to the expanding endosperm. These cells exhibit ultrastructural features consistent with programmed cell death, including vesiculation of the cytosol, development of irregularly shaped nuclei, vacuolar collapse, and shrinkage of the cytoplasm. Ricinosomes containing the CysEP were identified in the nucellar cells by light and electron microscopy and immunocytochemistry. Both proCysEP and mature CysEP are present in protein extracts of the nucellar tissues during seed development. Upon collapse of the nucellar cells, the content of the ricinosomes is released into the cytoplasm, where the activated CysEP digests the remaining proteinaceous cellular debris. Digestion products of the nucellar cells are presumed taken up by the outermost cells of the endosperm, which have labyrinthine ingrowths of the outer walls typical of transfer cells.
- Published
- 2005
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44. Organelle and translocatable forms of glyoxysomal malate dehydrogenase. The effect of the N-terminal presequence.
- Author
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Cox B, Chit MM, Weaver T, Gietl C, Bailey J, Bell E, and Banaszak L
- Subjects
- Amino Acid Sequence, Calorimetry, Differential Scanning, Catalysis, Crystallography, X-Ray, Enzyme Stability, Malate Dehydrogenase chemistry, Malate Dehydrogenase isolation & purification, Molecular Sequence Data, Protein Conformation, Protein Transport, Sequence Homology, Amino Acid, Glyoxysomes enzymology, Malate Dehydrogenase metabolism
- Abstract
Many organelle enzymes coded for by nuclear genes have N-terminal sequences, which directs them into the organelle (precursor) and are removed upon import (mature). The experiments described below characterize the differences between the precursor and mature forms of watermelon glyoxysomal malate dehydrogenase. Using recombinant protein methods, the precursor (p-gMDH) and mature (gMDH) forms were purified to homogeneity using Ni2+-NTA affinity chromatography. Gel filtration and dynamic light scattering have shown both gMDH and p-gMDH to be dimers in solution with p-gMDH having a correspondingly higher molecular weight. p-gMDH also exhibited a smaller translational diffusion coefficient (D(t)) at temperatures between 4 and 32 degrees C resulting from the extra amino acids on the N-terminal. Differential scanning calorimetry described marked differences in the unfolding properties of the two proteins with p-gMDH showing additional temperature dependent transitions. In addition, some differences were found in the steady state kinetic constants and the pH dependence of the K(m) for oxaloacetate. Both the organelle-precursor and the mature form of this glyoxysomal enzyme were crystallized under identical conditions. The crystal structure of p-gMDH, the first structure of a cleavable and translocatable protein, was solved to a resolution of 2.55 A. GMDH is the first glyoxysomal MDH structure and was solved to a resolution of 2.50 A. A comparison of the two structures shows that there are few visible tertiary or quaternary structural differences between corresponding elements of p-gMDH, gMDH and other MDHs. Maps from both the mature and translocatable proteins lack significant electron density prior to G44. While no portion of the translocation sequences from either monomer in the biological dimer was visible, all of the other solution properties indicated measurable effects of the additional residues at the N-terminal.
- Published
- 2005
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45. The 2.0 A crystal structure and substrate specificity of the KDEL-tailed cysteine endopeptidase functioning in programmed cell death of Ricinus communis endosperm.
- Author
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Than ME, Helm M, Simpson DJ, Lottspeich F, Huber R, and Gietl C
- Subjects
- Amino Acid Sequence, Animals, Binding Sites, Caseins metabolism, Cattle, Cysteine Endopeptidases metabolism, Cysteine Proteinase Inhibitors chemistry, Fluorescent Dyes, Kinetics, Models, Molecular, Peptide Fragments chemistry, Peptide Fragments pharmacology, Plant Proteins, Substrate Specificity, Apoptosis, Crystallography, X-Ray, Cysteine Endopeptidases chemistry, Ricinus enzymology
- Abstract
In the senescing endosperm of germinating castor bean (Ricinus communis) a special organelle (the ricinosome) releases a papain-type cysteine endopeptidase (CysEP) during the final stages of cellular disintegration. Protein cleavage sites for the Ricinus CysEP were determined with fluorogenic peptides (Abz-Xaa-Arg-/-Gln-Gln-Tyr(NO2)-Asp). The highest kcat/Km values were obtained with neutral amino acid residues with large aliphatic and non-polar (Leu, Val, Ile, Met) or aromatic (Phe, Tyr, Trp) side-chains. A second series (Abz-Leu-Xaa-/Gln-Pro-Tyr(NO2)-Asp) was evaluated. Based on these results, the covalent binding inhibitor H-D-Val-Leu-Lys-chloromethylketone (CMK) was chosen as substrate analogue for replacement in the catalytic site. Unusually, CysEP cleaved beta-casein N and C-terminal to the amino acid proline. CysEP was crystallized, its structure was solved by molecular replacement at 2.0 A resolution and refined to a R-factor of 18.1% (Rfree=22.6%). The polypeptide chain folds as in papain into two domains divided by the active site cleft, an elongated surface depression harboring the active site. The non-primed specificity subsites of the proteinase are clearly defined by the H-D-Val-Leu-Lys-CMK-inhibitor covalently bound to the active site. The absence of the occluding loop, which blocks the active site of exopeptidases at the C-terminal side of the scissile bond, identifies CysEP as an endopeptidase. The more open pocket of the Ricinus CysEP correlates with the extended variety of substrate amino acid residues accommodated by this enzyme, including even proline at the P1 and P1' positions. This may allow the enzyme to attack a greater variety of proteins during programmed cell death.
- Published
- 2004
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46. AthPEX10, a nuclear gene essential for peroxisome and storage organelle formation during Arabidopsis embryogenesis.
- Author
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Schumann U, Wanner G, Veenhuis M, Schmid M, and Gietl C
- Subjects
- Amino Acid Sequence, Cell Membrane metabolism, Cytosol metabolism, DNA, Complementary metabolism, Endoplasmic Reticulum metabolism, Exons, Lipid Bilayers, Lipid Metabolism, Microscopy, Electron, Molecular Sequence Data, Mutation, Peroxins, Peroxisomes metabolism, Phospholipids metabolism, Plants, Genetically Modified, Protein Structure, Tertiary, Arabidopsis genetics, Arabidopsis physiology, Arabidopsis Proteins genetics, Arabidopsis Proteins physiology, Carrier Proteins genetics, Carrier Proteins physiology, Cell Nucleus metabolism, Membrane Transport Proteins, Organelles metabolism, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear physiology
- Abstract
In yeasts and mammals, PEX10 encodes an integral membrane protein with a C3HC4 RING finger motif in its C-terminal domain and is required for peroxisome biogenesis and matrix protein import. In humans, its dysfunction in peroxisome biogenesis leads to severe Zellweger Syndrome and infantile Refsum disease. Here we show that dysfunction of a homologous gene in Arabidopsis leads to lethality at the heart stage of embryogenesis, impairing the biogenesis of peroxisomes, lipid bodies, and protein bodies. In a T-DNA insertion mutant disrupting the fourth exon of the AthPEX10 gene, ultrastructural analyses fail to detect peroxisomes characteristic for wild-type embryogenesis. Storage triacyl glycerides are not assembled into lipid bodies (oil bodies; oleosomes) surrounded by the phospholipid-protein monolayer membrane. Instead, the dysfunctional monolayer membranes, which derive from the bilayer membrane of the endoplasmic reticulum, accumulate in the cytosol. Concomitantly the transfer of the storage proteins from their site of synthesis at the endoplasmic reticulum to the vacuoles is disturbed. The mutant can be rescued by transformation with wild-type AthPEX10 cDNA. Transformants of wild-type Hansenula polymorpha cells with the AthPEX10 cDNA did produce the encoded protein without targeting it to peroxisomes. Additionally, the cDNA could not complement a Hansenula pex10 mutant unable to form peroxisomes. The ultrastructural knockout phenotype of AthPEX10p suggests that this protein in Arabidopsis is essential for peroxisome, oleosome, and protein transport vesicle formation.
- Published
- 2003
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47. A soybean plastid-targeted NADH-malate dehydrogenase: cloning and expression analyses.
- Author
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Imsande J, Berkemeyer M, Scheibe R, Schumann U, Gietl C, and Palmer RG
- Abstract
A typical soybean (Glycine max) plant assimilates nitrogen rapidly both in active root nodules and in developing seeds and pods. Oxaloacetate and 2-ketoglutarate are major acceptors of ammonia during rapid nitrogen assimilation. Oxaloacetate can be derived from the tricarboxylic acid (TCA) cycle, and it also can be synthesized from phosphoenolpyruvate and carbon dioxide by phosphoenolpyruvate carboxylase. An active malate dehydrogenase is required to facilitate carbon flow from phosphoenolpyruvate to oxaloacetate. We report the cloning and sequence analyses of a complete and novel malate dehydrogenase gene in soybean. The derived amino acid sequence was highly similar to the nodule-enhanced malate dehydrogenases from Medicago sativa and Pisum sativum in terms of the transit peptide and the mature subunit (i.e., the functional enzyme). Furthermore, the mature subunit exhibited a very high homology to the plastid-localized NAD-dependent malate dehydrogenase from Arabidopsis thaliana, which has a completely different transit peptide. In addition, the soybean nodule-enhanced malate dehydrogenase was abundant in both immature soybean seeds and pods. Only trace amounts of the enzyme were found in leaves and nonnodulated roots. In vitro synthesized labeled precursor protein was imported into the stroma of spinach chloroplasts and processed to the mature subunit, which has a molecular mass of ∼34 kDa. We propose that this new malate dehydrogenase facilitates rapid nitrogen assimilation both in soybean root nodules and in developing soybean seeds, which are rich in protein. In addition, the complete coding region of a geranylgeranyl hydrogenase gene, which is essential for chlorophyll synthesis, was found immediately upstream from the new malate dehydrogenase gene.
- Published
- 2001
48. The ricinosomes of senescing plant tissue bud from the endoplasmic reticulum.
- Author
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Schmid M, Simpson DJ, Sarioglu H, Lottspeich F, and Gietl C
- Subjects
- Amino Acid Sequence, Apoptosis, Blotting, Western, Carrier Proteins analysis, Centrifugation, Density Gradient, Cyanogen Bromide metabolism, Cysteine Endopeptidases chemistry, Cysteine Endopeptidases metabolism, Cytoplasmic Vesicles chemistry, Cytoplasmic Vesicles enzymology, Cytoplasmic Vesicles ultrastructure, Endoplasmic Reticulum chemistry, Endoplasmic Reticulum enzymology, Endoplasmic Reticulum ultrastructure, Endoplasmic Reticulum Chaperone BiP, Enzyme Activation, Fluorescent Antibody Technique, Hydrogen-Ion Concentration, Mass Spectrometry, Microscopy, Electron, Molecular Chaperones analysis, Molecular Sequence Data, Molecular Weight, Oligopeptides metabolism, Peptide Fragments chemistry, Peptide Fragments metabolism, Protein Disulfide-Isomerases metabolism, Protein Precursors chemistry, Protein Precursors metabolism, Protein Processing, Post-Translational, Protein Sorting Signals, Ricinus ultrastructure, Vacuoles metabolism, Cytoplasmic Vesicles metabolism, Endoplasmic Reticulum metabolism, Heat-Shock Proteins, Plants, Toxic, Ricinus cytology, Ricinus growth & development
- Abstract
The ricinosome (synonym, precursor protease vesicle) is a novel organelle, found so far exclusively in plant cells. Electron microscopic studies suggest that it buds off from the endoplasmic reticulum in senescing tissues. Biochemical support for this unusual origin now comes from the composition of the purified organelle, which contains large amounts of a 45-kDa cysteine endoprotease precursor with a C-terminal KDEL motif and the endoplasmic reticulum lumen residents BiP (binding protein) and protein disulfide isomerase. Western blot analysis, peptide sequencing, and mass spectrometry demonstrate retention of KDEL in the protease proform. Acidification of isolated ricinosomes causes castor bean cysteine endopeptidase activation, with cleavage of the N-terminal propeptide and the C-terminal KDEL motif. We propose that ricinosomes accumulate during senescence by programmed cell death and are activated by release of protons from acidic vacuoles.
- Published
- 2001
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49. Programmed cell death in castor bean endosperm is associated with the accumulation and release of a cysteine endopeptidase from ricinosomes.
- Author
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Schmid M, Simpson D, and Gietl C
- Subjects
- Ricinus communis cytology, Ricinus communis genetics, Cell Nucleus, DNA Fragmentation, DNA, Plant, Germination, In Situ Hybridization, Organelles, Seeds anatomy & histology, Seeds growth & development, Apoptosis, Ricinus communis enzymology, Cysteine Endopeptidases metabolism, Plants, Toxic
- Abstract
The cells of the endosperm of castor bean seeds (Ricinus communis) undergo programmed cell death during germination, after their oil and protein reserves have been mobilized. Nuclear DNA fragmentation first was observed at day 3 in the endosperm cells immediately adjacent to the cotyledons and progressed across to the outermost cell layers by day 5. We also detected the accumulation of small organelles known as ricinosomes, by using an antibody against a cysteine endoprotease. By the time the nuclear DNA was susceptible to heavy label by terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling, the ricinosomes had released into the cytoplasm their content of cysteine endoprotease, which became activated because of the cleavage of its propeptide. The cysteine endoprotease is distinguished by a C-terminal KDEL sequence, although it is not retained in the lumen of the endoplasmic reticulum and is a marker for ricinosomes. Homologous proteases are found in the senescing tissues of other plants, including the petals of the daylily. Ricinosomes were identified in this tissue by electron microscopy and immunocytochemistry. It seems that ricinosomes are not unique to Ricinus and play an important role in the degradation of plant cell contents during programmed cell death.
- Published
- 1999
- Full Text
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50. The plant PTS1 receptor: similarities and differences to its human and yeast counterparts.
- Author
-
Wimmer C, Schmid M, Veenhuis M, and Gietl C
- Subjects
- Amino Acid Sequence, Base Sequence, Cloning, Molecular, Cotyledon metabolism, Fruit physiology, Fungal Proteins, Genes, Plant, Humans, Microbodies ultrastructure, Molecular Sequence Data, Peroxisome-Targeting Signal 1 Receptor, Pichia genetics, Pichia ultrastructure, Receptors, Cytoplasmic and Nuclear chemistry, Recombinant Proteins chemistry, Recombinant Proteins metabolism, Repetitive Sequences, Amino Acid, Sequence Alignment, Sequence Homology, Amino Acid, Fruit genetics, Microbodies physiology, Receptors, Cytoplasmic and Nuclear genetics, Receptors, Cytoplasmic and Nuclear metabolism
- Abstract
Two targeting signals, PTS1 and PTS2, mediate import of proteins into the peroxisomal matrix. We have cloned and sequenced the watermelon (Citrullus vulgaris) cDNA homologue to the PTS1 receptor gene (PEX5). Its gene product, CvPex5p, belongs to the family of tetratricopeptide repeat (TPR) containing proteins like the human and yeast counterparts, and exhibits 11 repeats of the sequence W-X2-(E/S)-(Y/F/Q) in its N-terminal half. According to fractionation studies the plant Pex5p is located mainly in the cytosolic fraction and therefore could function as a cycling receptor between the cytosol and glyoxysomes, as has been proposed for the Pex5p of human and some yeast peroxisomes. Transformation of the Hansenula polymorpha peroxisome deficient pex5 mutant with watermelon PEX5 resulted in restoration of peroxisome formation and the synthesis of additional membranes surrounding the peroxisomes. These structures are labeled in immunogold experiments using antibodies against the Hansenula polymorpha integral membrane protein Pex3p, confirming their peroxisomal nature. The plant Pex5p was localized by immunogold labelling mainly in the cytosol of the yeast, but also inside the newly formed peroxisomes. However, import of the PTS1 protein alcohol oxidase is only partially restored by CvPex5p.
- Published
- 1998
- Full Text
- View/download PDF
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