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Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane‐bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII‐mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site‐directed mutagenesis to generate a dominant‐negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER‐Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER‐to‐Golgi protein secretory pathway in green algae. [ABSTRACT FROM AUTHOR]

Proteins carrying a signal peptide and/or a transmembrane domain enter the intracellular secretory pathway at the endoplasmic reticulum (ER) and are transported to the Golgi apparatus via COPII vesicles or tubules. SAR1 initiates COPII coat assembly by recruiting other coat proteins to the ER membrane. Mammalian genomes encode two SAR1 paralogs, SAR1A and SAR1B. While these paralogs exhibit ~90% amino acid sequence identity, it is unknown whether they perform distinct or overlapping functions in vivo. We now report that genetic inactivation of Sar1a in mice results in lethality during midembryogenesis. We also confirm previous reports that complete deficiency of murine Sar1b results in perinatal lethality. In contrast, we demonstrate that deletion of Sar1b restricted to hepatocytes is compatible with survival, though resulting in hypocholesterolemia that can be rescued by adenovirus-mediated overexpression of either SAR1A or SAR1B. To further examine the in vivo function of these two paralogs, we genetically engineered mice with the Sar1a coding sequence replacing that of Sar1b at the endogenous Sar1b locus. Mice homozygous for this allele survive to adulthood and are phenotypically normal, demonstrating complete or near-complete overlap in function between the two SAR1 protein paralogs in mice. These data also suggest upregulation of SAR1A gene expression as a potential approach for the treatment of SAR1B deficiency (chylomicron retention disease) in humans. [ABSTRACT FROM AUTHOR]

Abstract Eukaryotic cells are highly compartmentalized, requiring elaborate transport mechanisms to facilitate the movement of proteins between membrane‐bound compartments. Most proteins synthesized in the endoplasmic reticulum (ER) are transported to the Golgi apparatus through COPII‐mediated vesicular trafficking. Sar1, a small GTPase that facilitates the formation of COPII vesicles, plays a critical role in the early steps of this protein secretory pathway. Sar1 was characterized in yeast, animals and plants, but no Sar1 homolog has been identified and functionally analyzed in algae. Here we identified a putative Sar1 homolog (CrSar1) in the model green alga Chlamydomonas reinhardtii through amino acid sequence similarity. We employed site‐directed mutagenesis to generate a dominant‐negative mutant of CrSar1 (CrSar1DN). Using protein secretion assays, we demonstrate the inhibitory effect of CrSar1DN on protein secretion. However, different from previously studied organisms, ectopic expression of CrSar1DN did not result in collapse of the ER‐Golgi interface in Chlamydomonas. Nonetheless, our data suggest a largely conserved role of CrSar1 in the ER‐to‐Golgi protein secretory pathway in green algae.

Nuclear Pore Complexes (NPCs) are embedded in the nuclear envelope (NE), regulating macromolecule transport and physically interacting with chromatin. The NE undergoes dramatic breakdown and reformation during plant cell division. In addition, this structure has a specific meiotic function, anchoring and positioning telomeres to facilitate the pairing of homologous chromosomes. To elucidate a possible function of the structural components of the NPCs in meiosis, we have characterized several Arabidopsis lines with mutations in genes encoding nucleoporins belonging to the outer ring complex. Plants defective for either SUPPRESSOR OF AUXIN RESISTANCE1 (SAR1, also called NUP160) or SAR3 (NUP96) present condensation abnormalities and SPO11-dependent chromosome fragmentation in a fraction of meiocytes, which is increased in the double mutant sar1 sar3. We also observed these meiotic defects in mutants deficient in the outer ring complex protein HOS1, but not in mutants affected in other components of this complex. Furthermore, our findings may suggest defects in the structure of NPCs in sar1 and a potential link between the meiotic role of this nucleoporin and a component of the RUBylation pathway. These results provide the first insights in plants into the role of nucleoporins in meiotic chromosome behavior. [ABSTRACT FROM AUTHOR]

Nuclear Pore Complexes (NPCs) are embedded in the nuclear envelope (NE), regulating macromolecule transport and physically interacting with chromatin. The NE undergoes dramatic breakdown and reformation during plant cell division. In addition, this structure has a specific meiotic function, anchoring and positioning telomeres to facilitate the pairing of homologous chromosomes. To elucidate a possible function of the structural components of the NPCs in meiosis, we have characterized several Arabidopsis lines with mutations in genes encoding nucleoporins belonging to the outer ring complex. Plants defective for either SUPPRESSOR OF AUXIN RESISTANCE1 (SAR1, also called NUP160) or SAR3 (NUP96) present condensation abnormalities and SPO11-dependent chromosome fragmentation in a fraction of meiocytes, which is increased in the double mutant sar1 sar3. We also observed these meiotic defects in mutants deficient in the outer ring complex protein HOS1, but not in mutants affected in other components of this complex. Furthermore, our findings may suggest defects in the structure of NPCs in sar1 and a potential link between the meiotic role of this nucleoporin and a component of the RUBylation pathway. These results provide the first insights in plants into the role of nucleoporins in meiotic chromosome behavior.

Transport of newly synthesized proteins from endoplasmic reticulum (ER) to Golgi is mediated by coat protein complex II (COPII). The assembly and disassembly of COPII vesicles is regulated by the molecular switch Sar1, which is a small GTPase and a component of COPII. Usually a small GTPase binds GDP (inactive form) or GTP (active form). Mammals have two Sar1 isoforms, Sar1a and Sar1b, that have approximately 90% sequence identity. Some experiments demonstrated that these two isoforms had distinct but overlapping functions. Here we found another instance of differing behavior: the alarmone ppGpp could bind to and inhibit the GTPase activity of human Sar1a but could not inhibit the GTPase activity of human Sar1b. The crystal structures of Sar1a⋅ppGpp and Sar1b⋅GDP have been determined. Superposition of the structures shows that ppGpp binds to the nucleotide‐binding pocket, its guanosine base, ribose ring and 5′‐diphosphate occupying nearly the same positions as for GDP. However, its 3′‐diphosphate points away from the active site and, hence, away from the surface of the protein. The overall structure of Sar1a⋅ppGpp is more similar to Sar1b⋅GDP than to Sar1b⋅GTP. We also find that the Asp140‐Arg138–water–ligand interaction net is important for the binding of ppGpp to Sar1a. This study provides further evidence showing that there are biochemical differences between the Sar1a and Sar1b isoforms of Sar1. [ABSTRACT FROM AUTHOR]

Sar1 is a small GTPase of the ARF family. Upon exchange of GDP for GTP, Sar1 associates with the endoplasmic reticulum (ER) membrane and recruits COPII components, orchestrating cargo concentration and membrane deformation. Many aspects of the role of Sar1 and regulation of its GTP cycle remain unclear, especially as complexity increases in higher organisms that secrete a wider range of cargoes. This review focusses on the regulation of GTP hydrolysis and its role in coat assembly, as well as the mechanism of Sar1‐induced membrane deformation and scission. Finally, we highlight the additional specialisation in higher eukaryotes and the outstanding questions on how Sar1 functions are orchestrated. [ABSTRACT FROM AUTHOR]

Key message: We identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. Higher plants accumlate large amounts of seed storage proteins (SSPs). However, mechanisms underlying SSP trafficking are largely unknown, especially the ER-Golgi anterograde process. Here, we showed that a rice glutelin precursor accumulation13 (gpa13) mutant exhibited floury endosperm and overaccumulated glutelin precursors, which phenocopied the reported RNAi-Sar1abc line. Molecular cloning revealed that the gpa13 allele encodes a mutated Sar1c (mSar1c) with a deletion of two conserved amino acids Pro134 and Try135. Knockdown or knockout of Sar1c alone caused no obvious phenotype, while overexpression of mSar1c resulted in seedling lethality similar to the gpa13 mutant. Transient expression experiment in tobacco combined with subcellular fractionation experiment in gpa13 demonstrated that the expression of mSar1c affects the subcellular distribution of all Sar1 isoforms and Sec23c. In addition, mSar1c failed to interact with COPII component Sec23. Conversely, mSar1c competed with Sar1a/b/d to interact with guanine nucleotide exchange factor Sec12. Together, we identified a dosage-dependent dominant negative form of Sar1c, which confirms the essential role of COPII system in mediating ER export of storage proteins in rice endosperm. [ABSTRACT FROM AUTHOR]

ABSTRACT Microsporidia, as unicellular eukaryotes, also have an endomembrane system for transporting proteins, which is essentially similar to those of other eukaryotes. In eukaryotes, coat protein complex II (COPII) consists of Sar1, Sec23, Sec24, Sec13, and Sec31 and mediates protein transport from the endoplasmic reticulum (ER) to the Golgi apparatus. Sar1 is the central player in the regulation of coat protein complex II vesicle formation in the endoplasmic reticulum. In this study, we successfully cloned the NbSar1, NbSec23-1, NbSec23-2, NbSec24-1, NbSec24-2, NbSec13, NbSec31-1, and NbSec31-2 genes and prepared NbSar1 polyclonal antibody. We found that NbSar1 was localized mainly in the perinuclear cytoplasm of Nosema bombycis by immunofluorescence analysis (IFA). Yeast two-hybrid assays demonstrated that NbSar1 interacts with NbSec23-2, NbSec23-2 interacts with NbSec24-1 or NbSec24-2, NbSec23-1 interacts with NbSec31, and NbSec31 interacts with NbSec13. Moreover, the silencing of NbSar1 by RNA interference resulted in the aberrant expression of NbSar1, NbSec23-1, NbSec24-1, NbSec24-2, NbSec13, NbSec31-1, and NbSec31-2 and significantly inhibited the proliferation of N. bombycis. Altogether, these findings indicated that the subunits of coat protein complex II work together to perform functions in the proliferation of N. bombycis and that NbSar1 may play a crucial role in coat protein complex II vesicle formation. IMPORTANCE As eukaryotes, microsporidia have retained the endomembrane system for transporting and sorting proteins throughout their evolution. Whether the microsporidia form coat protein complex II (COPII) vesicles to transport cargo proteins and whether they play other roles besides cargo transport are not fully explained at present. Our results showed that NbSar1, NbSec23-1/NbSec23-2, NbSec24-1/NbSec24-2, NbSec13, and NbSec31 might be assembled to form COPII in the ER of N. bombycis, and the functions of COPII are also closely related to the proliferation of N. bombycis, this may be a new target for the prevention of pébrine disease of the silkworm.

Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex Ⅱ (COPⅡ) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells. [ABSTRACT FROM AUTHOR]

Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes.

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions.

Gene silencing induced by hairpin RNA or virus infection expression is one of the major tools in genetics studies in plants. However, when dealing with essential genes, virus-induced gene silencing (VIGS) and transgenic expression of hairpin RNA could lead to plant death, while transient expression of hairpin RNA in leaves is often less competent in downregulating target gene mRNA levels. Here, we developed a transient double-stranded RNA (dsRNA) expression system assisted by a modified viral RNA-dependent RNA polymerase (RdRp) in plant leaves. We show that this system is more effective in inducing gene silencing than the intron-spliced hairpin RNA expression. Furthermore, by using this system, we tested the role of the early secretory pathway during infection of Soybean mosaic potyvirus (SMV). We found that key components of the coat protein complex II vesicles are required for the multiplication of SMV. Overall, this dsRNA-based gene silencing system is effective in downregulating plant gene expression and can be used to identify host genes involved in plant-virus interactions. [ABSTRACT FROM AUTHOR]

Lipid droplets (LDs) are intracellular organelles that are ubiquitous in many types of cells. The LD core consists of triacylglycerols (TGs) surrounded by a phospholipid monolayer and surface proteins such as perilipin 2 (PLIN2). Although TGs accumulate in the phospholipid bilayer of the endoplasmic reticulum (ER) and subsequently nascent LDs buds from ER, the mechanism by which LD proteins are transported to LD particles is not fully understood. Sar1 is a GTPase known as a regulator of coat protein complex Ⅱ (COPⅡ) vesicle budding, and its role in LD formation was investigated in this study. HuH7 human hepatoma cells were infected with adenoviral particles containing genes coding GFP fused with wild-type Sar1 (Sar1 WT) or a GTPase mutant form (Sar1 H79G). When HuH7 cells were treated with oleic acid, Sar1 WT formed a ring-like structure around the LDs. The transient expression of Sar1 did not significantly alter the levels of TG and PLIN2 in the cells. However, the localization of PLIN2 to the LDs decreased in the cells expressing Sar1 H79G. Furthermore, the effects of Sar1 on PLIN2 localization to the LDs were verified by the suppression of endogenous Sar1 using the short hairpin RNA technique. In conclusion, it was found that Sar1 has some roles in the intracellular distribution of PLIN2 to LDs in liver cells.

In eukaryotes, secretory proteins traffic from the endoplasmic reticulum (ER) to the Golgi apparatus via coat protein complex II (COPII) vesicles. Intriguingly, during nutrient starvation, the COPII machinery acts constructively as a membrane source for autophagosomes during autophagy to maintain cellular homeostasis by recycling intermediate metabolites. In higher plants, essential roles of autophagy have been implicated in plant development and stress responses. Nonetheless, the membrane sources of autophagosomes, especially the participation of the COPII machinery in the autophagic pathway and autophagosome biogenesis, remains elusive in plants. Here, we provided evidence in support of a novel role of a specific Sar1 homolog AtSar1d in plant autophagy in concert with a unique Rab1/Ypt1 homolog AtRabD2a. First, proteomic analysis of the plant ATG (autophagy-related gene) interactome uncovered the mechanistic connections between ATG machinery and specific COPII components including AtSar1d and Sec23s, while a dominant negative mutant of AtSar1d exhibited distinct inhibition on YFP-ATG8 vacuolar degradation upon autophagic induction. Second, a transfer DNA insertion mutant of AtSar1d displayed starvation-related phenotypes. Third, AtSar1d regulated autophagosome progression through specific recognition of ATG8e by a noncanonical motif. Fourth, we demonstrated that a plant-unique Rab1/Ypt1 homolog AtRabD2a coordinates with AtSar1d to function as the molecular switch in mediating the COPII functions in the autophagy pathway. AtRabD2a appears to be essential for bridging the specific AtSar1dpositive COPII vesicles to the autophagy initiation complex and therefore contributes to autophagosome formation in plants. Taken together, we identified a plant-specific nexus of AtSar1d-AtRabD2a in regulating autophagosome biogenesis. [ABSTRACT FROM AUTHOR]

Carbon tetrachloride (CCl4) causes hepatotoxicity in mammals, with its hepatocytic metabolism producing radicals that attack the intracellular membrane system and destabilize intracellular vesicle transport. Inhibition of intracellular transport causes lipid droplet retention and abnormal protein distribution. The intracellular transport of synthesized lipids and proteins from the endoplasmic reticulum (ER) to the Golgi apparatus is performed by coat complex II (COPII) vesicle transport, but how CCl4 inhibits COPII vesicle transport has not been elucidated. COPII vesicle formation on the ER membrane is initiated by the recruitment of Sar1 protein from the cytoplasm to the ER membrane, followed by that of the COPII coat constituent proteins (Sec23, Sec24, Sec13, and Sec31). In this study, we evaluated the effect of CCl4 on COPII vesicle formation using the RLC‐16 rat hepatocyte cell line. Our results showed that CCl4 suppressed ER‐Golgi transport in RLC‐16 cells. Using a reconstituted system of rat liver tissue‐derived cytoplasm and RLC‐16 cell‐derived ER membranes, CCl4 treatment inhibited the recruitment of Sar1 and Sec13 from the cytosolic fraction to ER membranes. CCl4‐induced changes in the ER membrane accordingly inhibited the accumulation of COPII vesicle‐coated constituent proteins on the ER membrane, as well as the formation of COPII vesicles, which suppressed lipid and protein transport between the ER and Golgi apparatus. Our data suggest that CCl4 inhibits ER‐Golgi intracellular transport by inhibiting COPII vesicle formation on the ER membrane in hepatocytes. [ABSTRACT FROM AUTHOR]

Secretion-associated and ras-related protein 1 (Sar1) is a small GTPase that plays an important role in the transport of protein coated with coat protein complex II vesicles. However, its alternative roles in the biological processes of Procambarus clarkii remain unclear. Here, a sar1 gene (named as Pc-sar1) with an open reading frame of 582 bp from P. clarkii was identified. Pc-sar1 was expressed in all examined tissues with highest expression levels in muscle, which was determined by real-time PCR and western blotting. After the induction of lipopolysaccharide (LPS) and polycytidylic acid (Poly I: C), the transcriptional levels of Pc-sar1 differed in hepatopancreas, gill, muscle and intestine. In contrast, the expression of Pc-sar1 was upregulated by 20-hydroxyecdysone in these four tissues. In addition, the RNA interference of Pc-sar1 significantly affected the expression levels of immune and hormone-related genes. These results indicate that Pc-sar1 is involved in the innate immune response and ecdysteroid signaling pathway. [ABSTRACT FROM AUTHOR]

Contrary to the opinion recently offered by Dimou et al., our previously published biochemical, subcellular and genetic data supported our contention that AN11127 corresponds to the A. nidulans gene encoding Sec12, which is the guanine nucleotide exchange factor (GEF) specific for SAR1. We add here additional bioinformatics evidence that fully disprove the otherwise negative evidence reported by Dimou et al., highlighting the dangers associated with the lax interpretation of genomic data. On the positive side, we establish guidelines for the identification of this key secretory gene in other species of Ascomycota and Basidiomycota, including species of medical and applied interest.

Cargo selection and export from the endoplasmic reticulum is mediated by the COPII coat machinery that includes the small GTPase Sar1 and the Sec23/24 and Sec13/31 complexes. We have analyzed the sequential events regulated by purified Sar1 and COPII coat complexes during synchronized export of cargo from the ER in vitro. We find that activation of Sar1 alone, in the absence of other cytosolic components, leads to the formation of ER-derived tubular domains that resemble ER transitional elements that initiate cargo selection. These Sar1-generated tubular domains were shown to be transient, functional intermediates in ER to Golgi transport in vitro. By following cargo export in live cells, we show that ER export in vivo is also characterized by the formation of dynamic tubular structures. Our results demonstrate an unanticipated and novel role for Sar1 in linking cargo selection with ER morphogenesis through the generation of transitional tubular ER export sites.

Abstract A plasma membrane a mino acid t ransporter B 0,+ (ATB0,+), encoded by the SLC6A14 gene, is specific for neutral and basic amino acids. It is up-regulated in several types of malignant cancers. Neurotransmitter transporters of the SLC6 family interact with specific SEC24 proteins of the COPII complex along their pathway from the endoplasmic reticulum (ER) to Golgi. This study focused on the possible role of SEC24 proteins in ATB0,+ trafficking. Rat ATB0,+ was expressed in HEK293 cells, its localization and trafficking were examined by Western blot, deglycosylation, immunofluorescence (co-localization with ER and trans -Golgi markers) and biotinylation. The expression of ATB0,+ at the plasma membrane was decreased by dominant negative mutants of SAR1, a GTPase, whose activity triggers the formation of the COPII complex. ATB0,+ co-precipitated with SEC24C (but not with the remaining isoforms A, B and D). This interaction was confirmed by immunocytochemistry and the proximity ligation assay. Co-localization of SEC24C with endogenous ATB0,+ was also observed in MCF-7 breast cancer cells. Contrary to the endogenous transporter, part of the overexpressed ATB0,+ is directed to proteolysis, a process significantly reversed by a proteasome inhibitor bortezomib. Co-transfection with a SEC24C dominant negative mutant attenuated ATB0,+ expression at the plasma membrane, due to proteolytic degradation. These results support a hypothesis that lysine at position +2 downstream of the ER export "RI" motif on the cargo protein is crucial for SEC24C binding and for further trafficking to the Golgi. Moreover, there is an equilibrium between ER export and degradation mechanisms in case of overexpressed transporter. Highlights • A major portion of the amino acid transporter B0,+ (SLC6A14) resides in the ER. • B0,+ requires the coatomer II (COPII) complex for its exit from the ER. • Within the cargo recognizing components of COPII, only SEC24C interacts with B0,+. • A fraction of the newly translated B0,+ is directed to proteasomal degradation. [ABSTRACT FROM AUTHOR]

A large number of proteins are synthesized de novo in the endoplasmic reticulum (ER). They are transported through the Golgi apparatus and then delivered to their proper destinations. The ER and the Golgi play a central role in protein processing and sorting and show dynamic features in their forms. Ras super family small GTPases mediate the protein transport through and between these organelles. The ER-localized GTPase, Sar1, facilitates the formation of COPII transport carriers at the ER exit sites (ERES) on the ER for the transport of cargo proteins from the ER to the Golgi. The Golgi-localized GTPase, Arf1, controls intra-Golgi, and Golgi-to-ER transport of cargo proteins by the formation of COPI carriers. Rab GTPases localized at the Golgi, which are responsible for fusion of membranes, are thought to establish the identities of compartments. Recent evidence suggests that these small GTPases regulate not only discrete sites for generation/fusion of transport carriers, but also membrane dynamics of the organelles where they locate to ensure the integrity of transport. Here we summarize the current understandings about the membrane traffic between these organelles and highlight the cutting-edge advances from super-resolution live imaging of budding yeast, Saccharomyces cerevisiae.

Our view of the secretory pathway has evolved from a morphological one to one that includes molecular mechanistic understanding of basic traffic components. These components include coat complexes involved in cargo sorting and budding and proteins that mediate targeting, tethering and fusion. The expanding repertoire of regulators that control basic traffic activities begins to paint a unified morphological‐molecular view of secretion. The emerging picture provides key insights into the coupling of secretion with physiology. This review examines aspects of morphological‐molecular relations that are derived from studies on traffic from the endoplasmic reticulum carried by the coat protein complex II. Biosynthetic traffic of proteins from the endoplasmic reticulum (ER) is mediated by coat protein complex II (COPII). This traffic step is utilized as a model to examine emerging links between the cellular organization of membranes at sorting sites and the molecular machineries that mediate traffic. The evolutionary expansion of proteins involved in membrane organization and COPII regulation at ER exit sites and the role of these proteins in coupling biosynthetic secretion with physiological demands is examined. [ABSTRACT FROM AUTHOR]

Most metazoan organisms have evolved a mildly acidified and calcium diminished sorting hub in the early secretory pathway commonly referred to as the Endoplasmic Reticulum‐Golgi intermediate compartment (ERGIC). These membranous vesicular‐tubular clusters are found tightly juxtaposed to ER subdomains that are competent for the production of COPII‐coated transport carriers. In contrast to many unicellular systems, metazoan COPII carriers largely transit just a few hundred nanometers to the ERGIC, prior to COPI‐dependent transport on to the cis‐Golgi. The mechanisms underlying formation and maintenance of ERGIC membranes are poorly defined. However, recent evidence suggests an important role for Trk‐fused gene (TFG) in regulating the integrity of the ER/ERGIC interface. Moreover, in the absence of cytoskeletal elements to scaffold tracks on which COPII carriers might move, TFG appears to promote anterograde cargo transport by locally tethering COPII carriers adjacent to ERGIC membranes. This action, regulated in part by the intrinsically disordered domain of TFG, provides sufficient time for COPII coat disassembly prior to heterotypic membrane fusion and cargo delivery to the ERGIC. [ABSTRACT FROM AUTHOR]

Transmembrane glycoproteins, synthesized at the endoplasmic reticulum (ER), generally reach the Golgi apparatus in COPII‐coated vesicles en route to the cell surface. Here, we show that the bona fide nonglycoprotein Nox5, a transmembrane superoxide‐producing NADPH oxidase, is transported to the cell surface in a manner resistant to co‐expression of Sar1 (H79G), a GTP‐fixed mutant of the small GTPase Sar1, which blocks COPII vesicle fission from the ER. In contrast, Sar1 (H79G) effectively inhibits ER‐to‐Golgi transport of glycoproteins including the Nox5‐related oxidase Nox2. The trafficking of Nox2, but not that of Nox5, is highly sensitive to over‐expression of syntaxin 5 (Stx5), a t‐SNARE required for COPII ER‐to‐Golgi transport. Thus, Nox2 and Nox5 mainly traffic via the Sar1/Stx5‐dependent and ‐independent pathways, respectively. Both participate in Nox1 trafficking, as Nox1 advances to the cell surface in two differentially N‐glycosylated forms, one complex and one high mannose, in a Sar1/Stx5‐dependent and ‐independent manner, respectively. Nox2 and Nox5 also can use both pathways: a glycosylation‐defective mutant Nox2 is weakly recruited to the plasma membrane in a less Sar1‐dependent manner; N‐glycosylated Nox5 mutants reach the cell surface in part as the complex form Sar1‐dependently, albeit mainly as the high‐mannose form in a Sar1‐independent manner. [ABSTRACT FROM AUTHOR]

Many questions remain about how the stacked structure of the Golgi is formed and maintained. In our previous study, we challenged this question using tobacco BY-2 cells and revealed that, upon Brefeldin A (BFA) treatment, previously undescribed small punctate structures containing a particular subset of cis-Golgi proteins are formed adjacent to the ER-exit sites and act as scaffolds for Golgi regeneration after BFA removal. In this study, we analyzed these structures further. The proteins that localize to these punctate structures originate from the cis-most cisternae. 3D time-lapse observations show that the trans-Golgi marker is transported through these structures during Golgi regeneration. These data indicate that the cis-most cisternae have a specialized region that receives cargo from the ER, which becomes obvious upon BFA treatment. Expression of a dominant mutant form of SAR1 does not affect the formation of the punctate structures. We propose to call these punctate structures the 'Golgi entry core compartment' (GECCO). They act as receivers for the rest of the Golgi materials and are formed independently of the COPII machinery. This article has an associated First Person interview with the first author of the paper. [ABSTRACT FROM AUTHOR]

Proteins synthesized within the endoplasmic reticulum (ER) are transported to the Golgi via coat protein complex II (COPII)-coated vesicles. The formation of COPII-coated vesicles is regulated by the GTPase cycle of Sar1. Activated Sar1 is recruited to ER membranes and forms a pre-budding complex with cargoes and the inner-coat complex. The outer-coat complex then stimulates Sar1 inactivation and completes vesicle formation. The mechanisms of forming transport carriers are well-conserved among species; however, in mammalian cells, several cargo molecules such as collagen, and chylomicrons are too large to be accommodated in conventional COPII-coated vesicles. Thus, special cargo-receptor complexes are required for their export from the ER. cTAGE5/TANGO1 complexes and their isoforms have been identified as cargo receptors for these macromolecules. Recent reports suggest that the cTAGE5/TANGO1 complex interacts with the GEF and the GAP of Sar1 and tightly regulates its GTPase cycle to accomplish large cargo secretion.

Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein–protein and protein–lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII). The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS), we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH). The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane. [ABSTRACT FROM AUTHOR]

The COPII coat and the small GTPase Sar1 mediate protein export from the endoplasmic reticulum (ER) via specialized domains known as the ER exit sites. The peripheral ER protein Sec16 has been proposed to organize ER exit sites. However, it remains unclear how these molecules drive COPII coat polymerization. Here, we characterized the spatiotemporal relationships between the Saccharomyces cerevisiae COPII components during their polymerization by performing fluorescence microscopy of an artificial planar membrane. We demonstrated that Sar1 dissociates from the membrane shortly after the COPII coat recruitment and Sar1 is then no longer required for the COPII coat to bind to the membrane. Furthermore, we found that Sec16 is incorporated within the COPII-cargo clusters and that this is dependent on the Sar1 GTPase cycle. These data show how Sar1 drives the polymerization of COPII coat and how Sec16 is spatially distributed during COPII coat polymerization. [ABSTRACT FROM AUTHOR]

Wntless (Wls) transports Wnt molecules for secretion; however, the cellular mechanism underlying the initial assembly of Wnt secretory vesicles is still not fully defined. Here, we performed proteomic and mutagenic analyses of mammalian Wls, and report a mechanism for formation of early Wnt secretory vesicles on ER membrane. Wls forms a complex with SEC12 (also known as PREB), an ER membrane-localized guanine nucleotide-exchange factor (GEF) activator of the SAR1 (the SAR1A isoform) small GTPase. Compared to palmitoylation-deficient Wnt molecules, binding of mature Wnt to Wls increases Wls-SEC12 interaction and promotes association of Wls with SAR1, the key activator of the COPII machinery. Incorporation of Wls into this exporting ER compartment is affected by Wnt ligand binding and SEC12 binding to Wls, as well as the structural integrity and, potentially, the folding of the cytosolic tail of Wls. In contrast, Wls-SEC12 binding is stable, with the interacting interface biochemically mapped to cytosolic segments of individual proteins. Mutant Wls that fails to communicate with the COPII machinery cannot effectively support Wnt secretion. These data suggest that formation of early Wnt secretory vesicles is carefully regulated to ensure proper export of functional ligands. [ABSTRACT FROM AUTHOR]

Small GTPases largely control membrane traffic, which is essential for the survival of all eukaryotes. Among the small GTP-binding proteins, ARF1 (ADP-ribosylation factor 1) and SAR1 (Secretion-Associated RAS super family 1) are commonly conserved among all eukaryotes with respect to both their functional and sequential characteristics. The ARF1 and SAR1 GTP-binding proteins are involved in the formation and budding of vesicles throughout plant endomembrane systems. ARF1 has been shown to play a critical role in COPI (Coat Protein Complex I)-mediated retrograde trafficking in eukaryotic systems, whereas SAR1 GTPases are involved in intracellular COPII-mediated protein trafficking from the ER to the Golgi apparatus. This review offers a summary of vesicular trafficking with an emphasis on the ARF1 and SAR1 expression patterns at early growth stages and in the de-etiolation process.

The Sar1 GTPase controls coat assembly on coat protein complex II (COPII)-coated vesicles, which mediate protein transport from the endoplasmic reticulum (ER) to the Golgi. The GTP-bound form of Sar1, activated by the ER-localized guanine nucleotide exchange factor (GEF) Sec12, associates with the ER membrane. GTP hydrolysis by Sar1, stimulated by the COPII-vesicle-localized GTPase-activating protein (GAP) Sec23, in turn causes Sar1 to dissociate from the membrane. Thus, Sar1 is cycled between active and inactive states, and on and off vesicle membranes, but its precise spatiotemporal regulation remains unknown. Here, we examined Sar1 localization on COPII-coated membranes in living Saccharomyces cerevisiae cells. Two-dimensional (2D) observation demonstrated that Sar1 showed modest accumulation around the ER exit sites (ERES) in a manner that was dependent on Sec16 function. Detailed three-dimensional (3D) observation further demonstrated that Sar1 localized at the rims of the COPII-coated membranes, but was excluded from the rest of the COPII membranes. Additionally, a GTP-locked form of Sar1 induced abnormally enlarged COPII-coated structures and covered the entirety of these structures. These results suggested that the reversible membrane association of Sar1 GTPase leads to its localization being restricted to the rims of COPII-coated membranes in vivo. [ABSTRACT FROM AUTHOR]

Membrane-interacting proteins are polyphilic polymers that engage in dynamic protein–protein and protein–lipid interactions while undergoing changes in conformation, orientation and binding interfaces. Predicting the sites of interactions between such polypeptides and phospholipid membranes is still a challenge. One example is the small eukaryotic GTPase Sar1, which functions in phospholipid bilayer remodeling and vesicle formation as part of the multimeric coat protein complex (COPII). The membrane interaction of Sar1 is strongly dependent on its N-terminal 23 amino acids. By monolayer adsorption experiments and infrared reflection-absorption spectroscopy (IRRAS), we elucidate the role of lipids in inducing the amphipathicity of this N-terminal stretch, which inserts into the monolayer as an amphipathic helix (AH). The AH inserting angle is determined and is consistent with the philicities and spatial distribution of the amino acid monomers. Using an advanced method of IRRAS data evaluation, the orientation of Sar1 with respect to the lipid layer prior to the recruitment of further COPII proteins is determined. The result indicates that only a slight reorientation of the membrane-bound Sar1 is needed to allow coat assembly. The time-course of the IRRAS analysis corroborates a role of slow GTP hydrolysis in Sar1 desorption from the membrane.

Secretory proteins traffic from endoplasmic reticulum (ER) to Golgi via the coat protein complex II (COPII) vesicle, which consists of five cytosolic components (Sar1, Sec23-24, and Sec13-31). In eukaryotes, COPII transport has diversified due to gene duplication, creating multiple COPII paralogs. Evidence has accumulated, revealing the functional heterogeneity of COPII paralogs in protein ER export. Sar1B, the small GTPase of COPII machinery, seems to be specialized for large cargo secretion in mammals. Arabidopsis contains five Sar1 and seven Sec23 homologs, and AtSar1a was previously shown to exhibit different effects on α-amylase secretion. However, mechanisms underlying the functional diversity of Sar1 paralogs remain unclear in higher organisms. Here, we show that the Arabidopsis Sar1 homolog AtSar1a exhibits distinct localization in plant cells. Transgenic Arabidopsis plants expressing dominant-negative AtSar1a exhibit distinct effects on ER cargo export. Mutagenesis analysis identified a single amino acid, Cys84, as being responsible for the functional diversity of AtSar1a. Structure homology modeling and interaction studies revealed that Cys84 is crucial for the specific interaction of AtSar1a with AtSec23a, a distinct Arabidopsis Sec23 homolog. Structure modeling and coimmunoprecipitation further identified a corresponding amino acid, Cys484, on AtSec23a as being essential for the specific pair formation. At the cellular level, the Cys484 mutation affects the distinct function of AtSec23a on vacuolar cargo trafficking. Additionally, dominant-negative AtSar1a affects the ER export of the transcription factor bZIP28 under ER stress. We have demonstrated a unique plant pair of COPII machinery function in ER export and the mechanism underlying the functional diversity of COPII paralogs in eukaryotes. [ABSTRACT FROM AUTHOR]

The Drosophila type I transmembrane protein Crumbs is an apical determinant required for the maintenance of apico-basal epithelial cell polarity. The level of Crumbs at the plasma membrane is crucial, but how it is regulated is poorly understood. In a genetic screen for regulators of Crumbs protein trafficking we identified Sar1, the core component of the coat protein complex II transport vesicles. sar1 mutant embryos show a reduced plasma membrane localization of Crumbs, a defect similar to that observed in haunted and ghost mutant embryos, which lack Sec23 and Sec24CD, respectively. By pulse-chase assays in Drosophila Schneider cells and analysis of protein transport kinetics based on Endoglycosidase H resistance we identified an RNKR motif in Crumbs, which contributes to efficient ER export. The motif identified fits the highly conserved di-basic RxKR motif and mediates interaction with Sar1. The RNKR motif is also required for plasma membrane delivery of transgene-encoded Crumbs in epithelial cells of Drosophila embryos. Our data are the first to show that a di-basic motif acts as a signal for ER exit of a type I plasma membrane protein in a metazoan organism. [ABSTRACT FROM AUTHOR]

The Golgi is a highly organized and dynamic organelle that receives and distributes material from and to the endoplasmic reticulum (ER) and the endocytic pathway. One open question about Golgi organization is whether it is solely based on ER-to-Golgi transport. Here, we analyzed the kinetics of Golgi breakdown in the absence of COPII-dependent ER export with high temporal and spatial resolution using quantitative fluorescence microscopy. We found that Golgi breakdown occurred in two phases. While Golgi enzymes continuously redistributed to the ER, we consistently observed extensive Golgi fragmentation at the beginning of the breakdown, followed by microtubule-dependent formation of a Golgi remnant structure (phase 1). Further Golgi disintegration occurred less uniformly (phase 2). Remarkably, cisternal Golgi morphology was lost early in phase 1 and Golgi fragments instead corresponded to variably sized vesicle clusters. These breakdown intermediates were devoid of COPI-dependent recycling material, but contained typical 'core' Golgi components. Furthermore, Golgi breakdown intermediates were able to disassemble and reassemble following cell division, indicating that they retained important regulatory capabilities. Taken together, these findings support the view that Golgi self-organization exists independently of ER-to-Golgi transport. [ABSTRACT FROM AUTHOR]

In the cytosol of plant cells vesicle transport occurs via secretory pathways among the endoplasmic reticulum network, Golgi bodies, secretory granules, endosome, and plasma membrane. Three systems transfer lipids, proteins and other important molecules through aqueous spaces to membrane-enclosed compartments, via vesicles that bud from donor membranes, being coated and uncoated before tethered and fused with acceptor membranes. In addition, molecular, biochemical and ultrastructural evidence indicates presence of a vesicle transport system in chloroplasts. Little is known about the protein components of this system. However, as chloroplasts harbor the photosynthetic apparatus that ultimately supports most organisms on the planet, close attention to their pathways is warranted. This may also reveal novel diversification and/or distinct solutions to the problems posed by the targeted intra-cellular trafficking of important molecules. To date two homologs to well-known yeast cytosolic vesicle transport proteins, CPSAR1 and CPRabA5e (CP, chloroplast localized), have been shown to have roles in chloroplast vesicle transport, both being GTPases. Bioinformatic data indicate that several homologs of cytosolic vesicle transport system components are putatively chloroplast-localized and in addition other proteins have been implicated to participate in chloroplast vesicle transport, including vesicle-inducing protein in plastids 1, thylakoid formation 1, snowy cotyledon 2/cotyledon chloroplast biogenesis factor, curvature thylakoid 1 proteins, and a dynamin like GTPase FZO-like protein. Several putative potential cargo proteins have also been identified, including building blocks of the photosynthetic apparatus. Here we discuss details of the largely unknown putative chloroplast vesicle transport system, focusing on GTPase-related components. [ABSTRACT FROM AUTHOR]

As shape transformations of membranes are vital for intracellular trafficking, it is crucial to understand both the mechanics and the biochemistry of these processes. The interplay of these two factors constitutes an experimental challenge, however, because biochemical experiments are not tailored to the investigation of mechanical processes, and biophysical studies using model membranes are not capable of emulating native biological complexity. Reconstituted liposome-based model systems have been widely used for investigating the formation of transport vesicles by the COPII complex that naturally occurs at the endoplasmic reticulum. Here we have revisited these model systems, to address the influence of lipid composition, GTP hydrolyzing conditions and mechanical perturbation on the experimental outcome. We observed that the lipid-dependence of COPII-induced membrane remodeling differs from that predicted based on the lipid-dependence of COPII membrane binding. Under GTP non-hydrolyzing conditions, a structured coat was seen while GTP-hydrolyzing conditions yielded uncoated membranes as well as membranes coated by a thick protein coat of rather unstructured appearance. Detailed up-to-date protocols for purifications of Saccharomyces cerevisiae COPII proteins and for reconstituted reactions using these proteins with giant liposomes are also provided. [ABSTRACT FROM AUTHOR]

Small GTPases largely control membrane traffic, which is essential for the survival of all eukaryotes. Among the small GTP-binding proteins, ARF1 (ADP-ribosylation factor 1) and SAR1 (Secretion-Associated RAS super family 1) are commonly conserved among all eukaryotes with respect to both their functional and sequential characteristics. The ARF1 and SAR1 GTP-binding proteins are involved in the formation and budding of vesicles throughout plant endomembrane systems. ARF1 has been shown to play a critical role in COPI (Coat Protein Complex I)-mediated retrograde trafficking in eukaryotic systems, whereas SAR1 GTPases are involved in intracellular COPII-mediated protein trafficking from the ER to the Golgi apparatus. This review offers a summary of vesicular trafficking with an emphasis on the ARF1 and SAR1 expression patterns at early growth stages and in the de-etiolation process. [ABSTRACT FROM AUTHOR]

The Sar1 GTPase coordinates the assembly of coat protein complex- II ( COPII) at specific sites of the endoplasmic reticulum ( ER). COPII is required for ER-to-Golgi transport, as it provides a structural and functional framework to ship out protein cargoes produced in the ER. To investigate the requirement of COPII-mediated transport in mammalian cells, we used small interfering RNA ( siRNA)-mediated depletion of Sar1A and Sar1B. We report that depletion of these two mammalian forms of Sar1 disrupts COPII assembly and the cells fail to organize transitional elements that coordinate classical ER-to-Golgi protein transfer. Under these conditions, minimal Golgi stacks are seen in proximity to juxtanuclear ER membranes that contain elements of the intermediate compartment, and from which these stacks coordinate biosynthetic transport of protein cargo, such as the vesicular stomatitis virus G protein and albumin. Here, transport of procollagen-I is inhibited. These data provide proof-of-principle for the contribution of alternative mechanisms that support biosynthetic trafficking in mammalian cells, providing evidence of a functional boundary associated with a bypass of COPII. [ABSTRACT FROM AUTHOR]

Transmembrane protein 132A (TMEM132A, KIAA1583) was first isolated as a novel gene that is enriched during the embryonic and postnatal stages of rat brain development and interacts with GRP78. However, the biological functions of TMEM132A are scarcely characterized because the protein does not contain any known structural domains. Using a cell-surface biotinylation assay and immunocytochemical staining, we found that TMEM132A is a transmembrane glycoprotein consisting of a large extracellular domain in the N-terminal region and a small cytosolic domain in the C-terminal region. Partial deletions of the intracellular domain of TMEM132A had little effect on its expression level and cell-surface localization in transfected HEK293 cells, whereas deletions of the extracellular domain hampered transport to the cell surface. The expression pattern of each N-terminal mutant was immunocytochemically confirmed in HeLa cells transfected with the same constructs. Treatment with tunicamycin, an inhibitor of protein glycosylation, led to the accumulation of the unglycosylated form of TMEM132A in inverse proportion to the glycosylated form; however, both forms were localized at the cell surface at almost equal rates. In contrast, GRP78 overexpression led to the accumulation of unglycosylated TMEM132A, which was not detected on the cell surface. Inhibition of ER-Golgi transport by treatment with brefeldin A or the overexpression of mutant Sar1 attenuated the amount of cell-surface localized TMEM132A in HEK293 cells. Treatment with reagents disrupting intracellular calcium rapidly down-regulated the amount of TMEM132A protein in Neuro2a cells without affecting the expression level of its mRNA. Taken together, our data show that the novel cell-surface localized glycoprotein, TMEM132A, is regulated by several factors, including GRP78, Sar1, and intracellular calcium, in a post-transcriptional manner. [ABSTRACT FROM AUTHOR]

Abstract: The sculpting of membranes into highly curved vesicles is central to intracellular cargo trafficking, yet the mechanical activities of trafficking proteins remain poorly understood. Using an optical trap based assay that measures in vitro membrane response to imposed deformations, we examined the behavior of the two human paralogs of Sar1, a key component of the COPII family of vesicle coat proteins. Like their yeast counterpart, the human Sar1 proteins can lower the mechanical rigidity of the membranes to which they bind. Unlike the yeast Sar1, the rigidity is not a monotonically decreasing function of concentration. At high concentrations, we find increased bending rigidity and decreased protein mobility. These features imply a model in which protein clustering governs membrane mechanical properties. [Copyright &y& Elsevier]

Membrane-bound transport carriers are used to transfer cargo between membranes of the secretory and the endocytic pathways. The generation of these carriers can be classified into three steps: segregation of cargo away from the residents of a donor compartment (cargo sorting), generation of membrane curvature commensurate with the size of the cargo (membrane budding or tubulation), and finally separation of the nascent carrier from the donor membrane by a scission or membrane fission event. This review summarizes advances in our understanding of some of the best-characterized proteins required for the membrane fission that separates a transport carrier from its progenitor compartment: the large GTPase dynamin, the small guanine nucleotide-binding (G) proteins of the Arf family, BAR (Bin-amphiphysin-Rvs) domain proteins, and protein kinase D. These proteins share their ability to insert into membranes and oligomerize to create the large curvatures; however, the overall process of fission that involves these proteins appears to be quite different. [ABSTRACT FROM AUTHOR]

ER-to-Golgi protein transport involves transport vesicles of which formation is initiated by assembly of Sar1. The assembly of Sar1 is suppressed by protein kinase inhibitor H89, suggesting that ER-to-Golgi transport is regulated progressively by H89 sensitive kinase. ER-resident G protein suppresses vesicle formation with inhibition of Sar1 assembly. This study examined whether these promotion and suppression of vesicle transport share the same signal pathway, by examining the effects of G protein activator mastoparan 7 (Mp-7) and H89 on Sar1 and Sec23 recruitment onto microsomes. In a cell-free system for Sar1 translocation assay, GTPγS addition induced the translocation of Sar1 onto microsomes. Mp-7 and H89 decreased the Sar1 translocation. Double treatment of Mp-7 and H89 strongly decreased Sar1 translocation. In single and double treatments, however, G protein inactivator pertussis toxin (IAP) partially restored the suppressive effect of Mp-7, but had not any effect on H89-induced effect. Then, the assembly of Sec23 onto the microsome was also increased by the addition of GTPγS. Sec23 translocation was decreased by Mp-7 and/or H89 treatment and recovered by IAP pretreatment except for H89 single treatment, similarly to Sar1 translocation in each treatment. Inhibitory effects of H89 and Mp-7on ER-to-Golgi vesicle transport by H89 or Mp-7 were also confirmed in a cell culture system by BFA-dispersion and BFA-reconstruction experiments. These findings indicate that promotion and suppression of ER-to-Golgi vesicle transport are modulated through separate signal pathways. [ABSTRACT FROM AUTHOR]

As part of a continuing effort to elucidate mechanisms that regulate the magnitude of ethylene signalling, an Arabidopsis mutant with an enhanced ethylene response was identified. Subsequent characterization of this loss-of-function mutant revealed severe hypocotyl shortening in the presence of saturating ethylene along with increased expression in leaves of a subset of ethylene-responsive genes. It was subsequently determined by map-based cloning that the mutant (sar1-7) represents a loss-of-function mutation in the previously described nucleoporin AtNUP160 (At1g33410, SAR1). In support of previously reported results, the sar1-7 mutant partially restored auxin responsiveness to roots of an rce1 loss-of-function mutant, indicating that AtNUP160/SAR1 is required for proper expression of factors responsible for the repression of auxin signalling. Analysis of arf7-1/sar1-7 and arf19-1/sar1-7 double mutants revealed that mutations affecting either ARF7 or ARF19 function almost fully blocked manifestation of the sar1-7-dependent ethylene hypersensitivity phenotype, suggesting that ARF7- and ARF19-mediated auxin signalling is responsible for regulating the magnitude of and/or competence for the ethylene response in Arabidopsis etiolated hypocotyls. Consistent with this, addition of auxin to ethylene-treated seedlings resulted in severe hypocotyl shortening, reminiscent of that seen for other eer (enhanced ethylene response) mutants, suggesting that auxin functions in part synergistically with ethylene to control hypocotyl elongation and other ethylene-dependent phenomena. [ABSTRACT FROM PUBLISHER]

Recently, mesencephalic astrocyte-derived neurotrophic factor (MANF) has been reported to prevent cell death under some pathophysiological conditions. MANF, also referred to as arginine rich, mutated in early stage of tumors (Armet), was identified as an endoplasmic reticulum (ER) stress-inducible factor. Using RT-PCR, we found two variants of MANF mRNA: wild type, which contains exon 1 (wt- MANF), and one lacking exon 1, which is presumably not secreted (ΔΝ- MANF) in Neuro2a cells. The latter has a putative translational start site upstream of the second exon in the mouse MANF gene. Comparing the expression of wt-MANF with that of ΔΝ-MANF, we found that the amount of intracellular ΔΝ-MANF was much lower than that of wt-MANF. Furthermore, ΔΝ-MANF was not detected in the culture medium after its transient transfection into Neuro2a cells. Deletion of several α-helices of mouse MANF decreased its intracellular stability and secretion. Secretion of wt-MANF was almost completely inhibited by either treatment with brefeldin A (BFA), which disrupts the Golgi apparatus structure, or overexpression of a dominant negative Sar1 (Sar1[H79G]), which is reported to impair COPII-mediated transport from the ER to the Golgi apparatus. In addition, the enforced expression of glucose-regulated protein 78 kDa (GRP78) attenuated the secretion of wt-MANF and led to its intracellular accumulation. MANF lacking the four C-terminal amino acids (ΔC-MANF) accumulated at low levels in the cells, but its intracellular level was increased by GRP78 overexpression. The amount of ΔC-MANF in the culture medium was partially down-regulated after co-transfection of GRP78. Substitution of the amino acids RTDL at the C-terminus of mouse MANF with KDEL, the canonical ER localization signal in GRP78, markedly decreased MANF secretion and its secretion was further attenuated by GRP78 overexpression. Taken together, our data show that the secretion of MANF is regulated via COPII-mediated transport and that its C-terminus could be responsible for its retention in the ER through GRP78. The alternate isotype, ΔΝ-MANF, may be less stable in cells than wt-MANF and may not be secreted extracellularly. [ABSTRACT FROM AUTHOR]

Abstract: In this study, we found that Cysteine-rich with EGF-like domains 2 (CRELD2), a novel endoplasmic reticulum stress-inducible protein, is not only localized in the ER-Golgi apparatus but also spontaneously secreted. Deletion of four C-terminal amino acids from mouse CRELD2 or addition of tag-peptides to its C-terminus dramatically enhanced CRELD2 secretion. Intra- and extra-cellular CRELD2 is differentially glycosylated and its spontaneous secretion was significantly prevented by overexpression of a dominant negative mutant Sar1 and treatment with brefeldin A. Overexpression of wild-type GRP78 remarkably enhanced the secretion of wild-type but not mutant CRELD2. Our results demonstrate both that CRELD2 is a novel secretory glycoprotein regulated by Sar1 and GRP78 and that the C-terminal of CRELD2 plays a crucial role in its secretion. [Copyright &y& Elsevier]

Abstract: ER-to-Golgi protein transport is carried out by transport vesicles which are formed at the ER-exit sites with recruitment of cytoplasmic coat proteins. Vesicle formation is initiated by assembly of the small G protein (Sar1) onto the ER membrane. Sar1 assembly onto the ER membrane is suppressed by protein kinase inhibitor H89, suggesting participation of H89-sensitive kinase in this process. The present study identified an effector of H89-sensitive kinase by LC–MS PMF analysis combined with 1D- and 2D-PAGE autoradiography, and examined the changes on the effector and Sar1 translocation induced by H89. H89 significantly suppressed the phosphorylation of 55kDa protein with dosage dependency, and phosphorylation of 55kDa, pI 5.5 protein spot in 2-D-autoradiography was drastically diminished by H89. LC–MS PMF analysis showed that the protein spot was β-tubulin. H89 significantly suppressed Sar1 translocation onto the ER. These findings indicate that β-tubulin is one of downstream effectors of H89-sensitive kinase, and that suppression of ER-coupled β-tubulin phosphorylation decreases Sar1 translocation onto the ER, suggesting that phosphorylation of β-tubulin regulates Sar1 translocation. [Copyright &y& Elsevier]