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PubMed1996

A v-SNARE implicated in intra-Golgi transport.

We report the identification of a putative v-SNARE (GOS-28), localized primarily to transport vesicles at the terminal rims of Golgi stacks. In vitro, GOS-28, A Golgi SNARE of 28 kD, is efficiently packaged into Golgi-derived vesicles, which are most likely COPI coated. Antibodies directed against GOS-28 block its ability to bind alpha-SNAP, partially inhibit transport from the cis to the medial cisternae, and do not inhibit budding of COP-coated vesicles, but do accumulate docked uncoated vesicles.

Amino Acid Sequence↗
PubMed1995

Immunocytochemical localization of beta-COP to the ER-Golgi boundary and the TGN.

Recent data strongly suggest that the coatomer (COP) complex is involved in membrane transport between the ER and Golgi complex. This vesicular coat has been implicated in ER to Golgi, in intra Golgi as well as in Golgi to ER traffic. In this study we present a detailed immunocytochemical analysis of the distribution of beta-COP in different tissue culture cells. Our results extend previous studies by showing, using electron microscopy, that beta-COP accumulates on vesicular profiles and buds in the intermediate compartment (IC) under conditions that block ER to Golgi transport (15 degrees C). Importantly, under these conditions beta-COP co-localizes on these structures with a passenger protein, the membrane glycoprotein of vesicular stomatis virus (ts-O45-G). Furthermore, quantitative immunofluorescence microscopy of cells with ts-045-G accumulated in the ER, IC and trans-Golgi network, shifted briefly to the permissive temperature, showed that beta-COP was associated with many of the putative transport intermediates containing the viral glycoprotein which is in transit between the ER/IC and the cis-Golgi. The simplest interpretation of these data is that COP-coated vesicles are involved in anterograde transport of ts-045-G from the IC to the Golgi complex. Since many putative COP vesicle lacked the G protein following release of the 15 degrees C block this pool could be involved in retrograde transport. We also show that beta-COP is present on the membranes of the trans-Golgi network. However, in contrast to the ER-Golgi boundary, we could find no convincing evidence that this pool of beta-COP is associated with buds or trans-Golgi network-derived transport vesicles.

Animals↗
PubMed1996

Identification of two types of beta-COP vesicles in the Golgi complex of rat spermatids.

In the present study, immunoelectron microscopy was used to characterize two types of coated vesicles at the trans Golgi reticulum (TGR) of rat early spermatids. Most of the coated vesicles and buds had a 18.4 +/- 0.5 nm thick electron-dense coat. The size of the vesicles without the coat was 102.6 +/- 3.6 nm. This coat reacted with antibodies against clathrin. Immunolabeling for clathrin was almost restricted to the trans Golgi area (86% of the total labeling in the Golgi complex). In addition, we found a homogeneous population of small vesicles and buds bearing a approximately 10 nm thick coat which reacted with antibodies against beta-COP. beta-COP-immunoreactive vesicles were detected at the cis side (32%), lateral rims (27%) and trans face (40%) of the Golgi complex. The diameters of beta-COP-immunoreactive vesicles at the TGR and cis Golgi side were 53.9 +/- 1.3 and 42.1 +/- 1.1 nm, respectively. Cis Golgi elements were identified by using antibodies against markers of the Golgi intermediate compartment p 58, p 53 and Rab 2. Some beta-COP labeling was also found at the acrosomal membrane and associated buds. These results indicate that the TGR of early spermatids contains COP-coated vesicles which are different to those found at the cis Golgi side.

Acrosome↗
PubMed1995

Uncoupled packaging of targeting and cargo molecules during transport vesicle budding from the endoplasmic reticulum.

Formation of vesicular intermediates in protein transport between the endoplasmic reticulum and the Golgi apparatus involves a mechanism that sorts and packages two classes of molecules into transport vesicles: targeting molecules, which are required for targeting and consumption of vesicular intermediates, and cargo proteins. In order to examine the importance of cargo in this packaging reaction, we developed an in vitro assay that quantifies vesicle formation based on segregation of targeting molecules. Here we document that endoplasmic reticulum devoid of cargo proteins is competent in the formation and release of targeting molecule-containing vesicles in a fashion indistinguishable from its normal counterpart. This observation implies that packaging of cargo proteins may be uncoupled from the recruitment of targeting molecules during vesicle budding from the endoplasmic reticulum. Using the same assay, we demonstrate that the packaging of targeting molecules into vesicles is not dependent on the lumenal chaperone, BiP (Kar2p).

COP-Coated Vesicles↗
PubMed1995

Yeast SEC16 gene encodes a multidomain vesicle coat protein that interacts with Sec23p.

Temperature-sensitive mutations in the SEC16 gene of Saccharomyces cerevisiae block budding of transport vesicles from the ER. SEC16 was cloned by complementation of the sec16-1 mutation and encodes a 240-kD protein located in the insoluble, particulate component of cell lysates. Sec16p is released from this particulate fraction by high salt, but not by nonionic detergents or urea. Some Sec16p is localized to the ER by immunofluorescence microscopy. Membrane-associated Sec16p is incorporated into transport vesicles derived from the ER that are formed in an in vitro vesicle budding reaction. Sec16p binds to Sec23p, a COPII vesicle coat protein, as shown by the two-hybrid interaction assay and affinity studies in cell extracts. These findings indicate that Sec16p associates with Sec23p as part of the transport vesicle coat structure. Genetic analysis of SEC16 identifies three functionally distinguishable domains. One domain is defined by the five temperature-sensitive mutations clustered in the middle of SEC16. Each of these mutations can be complemented by the central domain of SEC16 expressed alone. The stoichiometry of Sec16p is critical for secretory function since overexpression of Sec16p causes a lethal secretion defect. This lethal function maps to the NH2-terminus of the protein, defining a second functional domain. A separate function for the COOH-terminal domain of Sec16p is shown by its ability to bind Sec23p. Together, these results suggest that Sec16p engages in multiple protein-protein interactions both on the ER membrane and as part of the coat of a completed vesicle.

Alleles↗
PubMed1996

Amino acid permeases require COPII components and the ER resident membrane protein Shr3p for packaging into transport vesicles in vitro.

In S. cerevisiae lacking SHR3, amino acid permeases specifically accumulate in membranes of the endoplasmic reticulum (ER) and fail to be transported to the plasma membrane. We examined the requirements of transport of the permeases from the ER to the Golgi in vitro. Addition of soluble COPII components (Sec23/24p, Sec13/31p, and Sar1p) to yeast membrane preparations generated vesicles containing the general amino acid permease. Gap1p, and the histidine permease, Hip1p. Shr3p was required for the packaging of Gap1p and Hip1p but was not itself incorporated into transport vesicles. In contrast, the packaging of the plasma membrane ATPase, Pma1p, and the soluble yeast pheromone precursor, glycosylated pro alpha factor, was independent of Shr3p. In addition, we show that integral membrane and soluble cargo colocalize in transport vesicles, indicating that different types of cargo are not segregated at an early step in secretion. Our data suggest that specific ancillary proteins in the ER membrane recruit subsets of integral membrane protein cargo into COPII transport vesicles.

ATP-Binding Cassette Transporters↗
PubMed1998

COPII-cargo interactions direct protein sorting into ER-derived transport vesicles.

Vesicles coated with coat protein complex II (COPII) selectively transport molecules (cargo) and vesicle fusion proteins from the endoplasmic reticulum (ER) to the Golgi complex. We have investigated the role of coat proteins in cargo selection and recruitment. We isolated integral membrane and soluble cargo proteins destined for transport from the ER in complexes formed in the presence of Sar1 and Sec23/24, a subset of the COPII components, and GTP or GMP-PNP. Vesicle fusion proteins of the vSNARE family and Emp24, a member of a putative cargo carrier family, were also found in COPII complexes. The inclusion of amino-acid permease molecules into the complex depended on the presence of Shr3, a protein required for the permease to leave the ER. Resident ER proteins Sec61, BiP (Kar2) and Shr3 were not included in the complexes, indicating that the COPII components bound specifically to vesicle cargo. COPII-cargo complexes and putative cargo adaptor-cargo complexes were also isolated from COPII vesicles. Our results indicate that cargo packaging signals and soluble cargo adaptors are recognized by a recruitment complex comprising Sar1-GTP and Sec23/24.

Amino Acid Transport Systems↗
PubMed1993

Requirement for a GTPase-activating protein in vesicle budding from the endoplasmic reticulum.

The binding and hydrolysis of guanosine triphosphate (GTP) by the small GTP-binding protein Sar1p is required to form transport vesicles from the endoplasmic reticulum (ER) in Saccharomyces cerevisiae. Experiments revealed that an interaction between Sar1p and the Sec23p subunit of an oligomeric protein is also required for vesicle budding. The isolated Sec23p subunit and the oligomeric complex stimulated guanosine triphosphatase (GTPase) activity of Sar1p 10- to 15-fold but did not activate two other small GTP-binding proteins involved in vesicle traffic (Ypt1p and ARF). Activation of GTPase was inhibited by an antibody to Sec23p but not by an antibody that inhibits the budding activity of the other subunit of the Sec23p complex. Also, activation was thermolabile in pure samples of Sec23p that were isolated from two independent sec23 mutant strains. It appears that Sec23p represents a new class of GTPase-activating protein because its sequence shows no similarity to any known member of this family.

COP-Coated Vesicles↗
PubMed1996

Cloning and functional characterization of mammalian homologues of the COPII component Sec23.

We screened a human cDNA library with a probe derived from a partial SEC23 mouse homologue and isolated two different cDNA clones (hSec23A and hSec23B) encoding proteins of a predicted molecular mass of 85 kDa. hSec23Ap and hSec23Bp were 85% identical and shared 48% identity with the yeast Sec23p. Affinity-purified anti-hSec23A recognized a protein of approximately 85 kDa on immunoblots of human, mouse, and rat cell extracts but did not recognize yeast Sec23p. Cytosolic hSec23Ap migrated with an apparent molecular weight of 350 kDa on a gel filtration column, suggesting that it is part of a protein complex. By immunoelectron microscopy, hSec23Ap was found essentially in the ribosome-free transitional face of the endoplasmic reticulum (ER) and associated vesicles. hSec23Ap is a functional homologue of the yeast Sec23p as the hSec23A isoform complemented the temperature sensitivity of the Saccharomyces cerevisiae sec23-1 mutation at a restrictive temperature of 34 degrees C. RNase protection assays indicated that both hSec23 isoforms are coexpressed in various human tissues, although at a variable ratio. Our data demonstrate that hSec23Ap is the functional human counterpart of the yeast COPII component Sec23p and suggest that it plays a similar role in mammalian protein export from the ER. The exact function of hSec23Bp remains to be determined.

Animals↗
PubMed1993

Identification and differential expression of yeast SEC23-related gene (Msec23) in mouse tissues.

We have isolated a yeast SEC23-related clone (Msec23) from mouse fibroblast cDNA library. It has an open reading frame of 1721 bp (64% homologous to SEC23-2.3 kb) which can potentially encode a 64.7 kDa protein (61% homologous to 85.4 kDa product of SEC23). The deduced Msec23 protein (Msec23p) sequence contains three successive Ig-like domains at the N-terminus followed by amphipathic alpha-helical regions, suggesting the potential of Msec23p to interact with other protein components. Further, Msec23 is differentially expressed in mouse tissues with its high level in brain and fibroblasts.

Amino Acid Sequence↗
PubMed1995

COPII: a membrane coat that forms endoplasmic reticulum-derived vesicles.

Vesicle budding from the endoplasmic reticulum (ER) has been reconstituted with washed membranes and three soluble proteins: Sec13 complex, Sec23 complex and the small GTPase Sar1p. The proteins that drive this cell-free vesicle budding reaction form an approximately 10 nm thick electron dense coat on ER-derived vesicles. Although the overall mechanism of membrane budding driven by various cytoplasmic coats appears similar, the constituents of this new membrane coat are molecularly distinct from the non-clathrin coat (COP) involved in intra-Golgi transport and the clathrin-containing coats. The new vesicle coat has been termed COPII.

Biological Transport↗
PubMed1994

COPII: a membrane coat formed by Sec proteins that drive vesicle budding from the endoplasmic reticulum.

In vitro synthesis of endoplasmic reticulum-derived transport vesicles has been reconstituted with washed membranes and three soluble proteins (Sar1p, Sec13p complex, and Sec23p complex). Vesicle formation requires GTP but can be driven by nonhydrolyzable analogs such as GMP-PNP. However, GMP-PNP vesicles fail to target and fuse with the Golgi complex whereas GTP vesicles are functional. All the cytosolic proteins required for vesicle formation are retained on GMP-PNP vesicles, while Sar1p dissociates from GTP vesicles. Thin section electron microscopy of purified preparations reveals a uniform population of 60-65 nm vesicles with a 10 nm thick electron dense coat. The subunits of this novel coat complex are molecularly distinct from the constituents of the nonclathrin coatomer involved in intra-Golgi transport. Because the overall cycle of budding driven by these two types of coats appears mechanistically similar, we propose that the coat structures be called COPI and COPII.

Base Sequence↗
PubMed2024

Functional overlap between the mammalian Sar1a and Sar1b paralogs in vivo.

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.

Animals↗
PubMed1996

Interleaflet clear space is reduced in the membrane of COP I and COP II-coated buds/vesicles.

Intracellular transfers between membrane-bound compartments occur through vesicles that bud from a donor compartment to fuse subsequently with an acceptor membrane. We report that the membrane that delimits COP I or COP II-coated buds/vesicles from the endoplasmic reticulum and the Golgi complex has a thinner interleaflet clear space as compared with the surrounding, noncoated parental membrane. This change is compatible with a compositional change of the membrane bilayer during the budding process.

COP-Coated Vesicles↗
PubMed1997

COPII subunit interactions in the assembly of the vesicle coat.

In vitro analysis of COPII vesicle formation in the yeast Saccharomyces cerevisiae has demonstrated the requirement for three cytosolic factors: Sec31p-Sec13p, Sec23p-Sec24p, and Sar1p. Convergent evidence suggests that the peripheral endoplasmic reticulum (ER) membrane protein Sec16p also represents an important component of the vesicle assembly apparatus: SEC16 interacts genetically with all five COPII genes; Sec16p binds to Sec23p and Sec24p, is found on ER-derived transport vesicles, and is required in vitro for the efficient release of ER-derived vesicle cargo. In this report, we demonstrate an important functional interaction between Sec16p and Sec31p. First, we map onto Sec31p binding regions for Sec16p, Sec23p, Sec24p, and Sec13p. Second, we show that a truncation mutant of Sec31p specifically defective for Sec16p binding is unable to complement a sec31Delta mutant and cannot rescue the secretion defect of a temperature-sensitive sec31 mutant at nonpermissive temperatures. We propose that Sec16p organizes the assembly of a coat that is stabilized both by the interaction of Sec31p with Sec23p and Sec24p and by the interaction of these three components with Sec16p.

Binding Sites↗
PubMed1996

COPII coat subunit interactions: Sec24p and Sec23p bind to adjacent regions of Sec16p.

Formation of COPII-coated vesicles at the endoplasmic reticulum (ER) requires assembly onto the membrane of five cytosolic coat proteins, Sec23p, Sec24p, Sec13p, Sec31p, and Sar1p. A sixth vesicle coat component, Sec16p, is tightly associated with the ER membrane and has been proposed to act as a scaffold for membrane association of the soluble coat proteins. We previously showed that Sec23p binds to the C-terminal region of Sec16p. Here we use two-hybrid and coprecipitation assays to demonstrate that the essential COPII protein Sec24p binds to the central region of Sec16p. In vitro reconstitution of binding with purified recombinant proteins demonstrates that the interaction of Sec24p with the central domain of Sec16p does not depend on the presence of Sec23p. However, Sec23p facilitates binding of Sec24p to Sec16p, and the three proteins can form a ternary complex in vitro. Truncations of Sec24p demonstrate that the N-terminal and C-terminal regions of Sec24p display different binding specificities. The C terminus binds to the central domain of Sec16p, whereas the N terminus of Sec24p binds to both the central domain of Sec16p and to Sec23p. These findings define binding to Sec16p as a new function for Sec24p and support the idea that Sec16p organizes assembly of the COPII coat.

Binding Sites↗