GPI anchor attachment is required for Gas1p transport from the endoplasmic reticulum in COP II vesicles.

Inositol starvation of auxotrophic yeast interrupts glycolipid biosynthesis and prevents lipid modification of a normally glycosyl phosphatidylinositol (GPI)-linked protein, Gas1p. The unanchored Gas1p precursor undergoes progressive modification in the endoplasmic reticulum (ER), but is not modified by Golgi-specific glycosylation. Starvation-induced defects in anchor assembly and protein processing are rapid, and occur without altered maturation of other proteins. Cells remain competent to manufacture anchor components and to process Gas1p efficiently once inositol is restored. Newly synthesized Gas1p is packaged into vesicles formed in vitro from perforated yeast spheroplasts incubated with either yeast cytosol or the purified Sec proteins (COP II) required for vesicle budding from the ER. In vitro synthesized vesicles produced by inositol-starved membranes do not contain detectable Gas1p. These studies demonstrate that COP II components fulfill the soluble protein requirements for packaging a GPI-anchored protein into ER-derived transport vesicles. However, GPI anchor attachment is required for this packaging to occur.     

Jagunal is required for reorganizing the endoplasmic reticulum during Drosophila oogenesis

Vesicular traffic in the Drosophila melanogaster oocyte occurs actively during vitellogenesis. Although endocytosis in the oocyte has been well characterized, exocytic vesicular traffic is less well understood. We show that the oocyte endoplasmic reticulum (ER) becomes concentrated into subcortical clusters during vitellogenesis. This ER reorganization requires Jagunal, which is an evolutionarily conserved ER membrane protein. Loss of Jagunal reduces vesicular traffic to the oocyte lateral membrane, but does not affect posterior polarized vesicular traffic, suggesting a role for Jagunal in facilitating vesicular traffic in the subcortex. Reduced membrane traffic caused by loss of Jagunal affects oocyte and bristle growth. We propose that ER reorganization is an important mechanism used by cells to prepare for an increased demand for membrane traffic, and Jagunal facilitates this process through ER clustering.     

cFLIP downregulation is an early event required for endoplasmic reticulum stress-induced apoptosis in tumor cells

Protein misfolding or unfolding and the resulting endoplasmic reticulum (ER) stress frequently occur in highly proliferative tumors. How tumor cells escape cell death by apoptosis after chronic ER stress remains poorly understood. We have investigated in both two-dimensional (2D) cultures and multicellular tumor spheroids (MCTSs) the role of caspase-8 inhibitor cFLIP as a regulator of the balance between apoptosis and survival in colon cancer cells undergoing ER stress. We report that downregulation of cFLIP proteins levels is an early event upon treatment of 2D cultures of colon cancer cells with ER stress inducers, preceding TNF-related apoptosis-inducing ligand receptor 2 (TRAIL-R2) upregulation, caspase-8 activation, and apoptosis. Maintaining high cFLIP levels during ER stress by ectopic expression of cFLIP markedly inhibits ER stress-induced caspase-8 activation and apoptosis. Conversely, cFLIP knockdown by RNA interference significantly accelerates caspase-8 activation and apoptosis upon ER stress. Despite activation of the proapoptotic PERK branch of the unfolded protein response (UPR) and upregulation of TRAIL-R2, MCTSs are markedly more resistant to ER stress than 2D cultures of tumor cells. Resistance of MCTSs to ER stress-induced apoptosis correlates with sustained cFLIP_L expression. Interestingly, resistance to ER stress-induced apoptosis is abolished in MCTSs generated from cFLIP_L knockdown tumor cells. Overall, our results suggest that controlling cFLIP levels in tumors is an adaptive strategy to prevent tumor cell’s demise in the unfavorable conditions of the tumor microenvironment.Subject terms: Cancer microenvironment, Apoptosis     

The carboxyl-terminal valine is required for transport of glycoprotein CD8 alpha from the endoplasmic reticulum to the intermediate compartment

There is evidence that a carboxyl-terminal valine residue is an anterograde transport signal for type I transmembrane proteins. Removal of the signal would either delay glycosylation in the Golgi complex of proteins destined to recycle to the endoplasmic reticulum or determine accumulation in the endoplasmic reticulum of newly synthesized proteins destined for the plasma membrane. We used the human CD8 alpha glycoprotein to investigate the role of the carboxyl-terminal valine in the exocytic pathway. Using immunofluorescence light microscopy, metabolic labeling, and cell fractionation, we demonstrate that removal of the carboxyl-terminal valine residue delays transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment. Removal of the residue did not affect the other steps of the exocytic pathway or the folding/dimerization and glycosylation processes. Therefore, it is likely that this signal plays a role in the transport of CD8 alpha from the endoplasmic reticulum to the intermediate compartment either before or during the formation of the transport vesicles that drive the exit the protein from the endoplasmic reticulum.     

Human cytomegalovirus specifically controls the levels of the endoplasmic reticulum chaperone BiP/GRP78, which is required for virion assembly

The endoplasmic reticulum (ER) chaperone BiP/GRP78 regulates ER function and the unfolded protein response (UPR). Human cytomegalovirus infection of human fibroblasts induces the UPR but modifies it to benefit viral replication. BiP/GRP78 protein levels are tightly regulated during infection, rising after 36 h postinfection (hpi), peaking at 60 hpi, and decreasing thereafter. To determine the effects of this regulation on viral replication, BiP/GRP78 was depleted using the SubAB subtilase cytotoxin, which rapidly and specifically cleaves BiP/GRP78. Toxin treatment of infected cells for 12-h periods beginning at 36, 48, 60, and 84 hpi caused complete loss of BiP but had little effect on viral protein synthesis. However, progeny virion formation was significantly inhibited, suggesting that BiP/GRP78 is important for virion formation. Electron microscopic analysis showed that infected cells were resistant to the toxin and showed none of the cytotoxic effects seen in uninfected cells. However, all viral activity in the cytoplasm ceased, with nucleocapsids remaining in the nucleus or concentrated in the cytoplasmic space just outside of the outer nuclear membrane. These data suggest that one effect of the controlled expression of BiP/GRP78 in infected cells is to aid in cytoplasmic virion assembly and egress.     

Ethanolaminephosphate side chain added to glycosylphosphatidylinositol (GPI) anchor by mcd4p is required for ceramide remodeling and forward transport of GPI proteins from endoplasmic reticulum to Golgi

Glycosylphosphatidylinositol (GPI) anchors of mammals as well as yeast contain ethanolaminephosphate side chains on the alpha1-4- and the alpha1-6-linked mannoses of the anchor core structure (protein-CO-NH-(CH(2))(2)-PO(4)-6Manalpha1-2Manalpha1-6Manalpha1-4GlcNH(2)-inositol-PO(4)-lipid). In yeast, the ethanolaminephosphate on the alpha1-4-linked mannose is added during the biosynthesis of the GPI lipid by Mcd4p. MCD4 is essential because Gpi10p, the mannosyltransferase adding the subsequent alpha1-2-linked mannose, requires substrates with an ethanolaminephosphate on the alpha1-4-linked mannose. The Gpi10p ortholog of Trypanosoma brucei has no such requirement. Here we show that the overexpression of this ortholog rescues mcd4Delta cells. Phenotypic analysis of the rescued mcd4Delta cells leads to the conclusion that the ethanolaminephosphate on the alpha1-4-linked mannose, beyond being an essential determinant for Gpi10p, is necessary for an efficient recognition of GPI lipids and GPI proteins by the GPI transamidase for the efficient transport of GPI-anchored proteins from the endoplasmic reticulum to Golgi and for the physiological incorporation of ceramides into GPI anchors by lipid remodeling. Furthermore, mcd4Delta cells have a marked defect in axial bud site selection, whereas this process is normal in gpi7Delta and gpi1. This also suggests that axial bud site selection specifically depends on the presence of the ethanolaminephosphate on the alpha1-4-linked mannose.     

Transmembrane lipid transfer is crucial for providing neutral lipids during very low density lipoprotein assembly in endoplasmic reticulum

Very low density lipoprotein (VLDL), a large particle containing apolipoprotein B (apoB) and large amounts of neutral lipids, is formed in the luminal space within the endoplasmic reticulum (ER) of hepatic cells. The assembly mechanism of VLDL particles is a tightly regulated process where apoB, associated with an insufficient amount of lipids, is selectively degraded intracellularly. In this study we found that treatment of HuH-7 human hepatoma cells with verapamil inhibited secretion of apoB-containing lipoprotein particles through increasing degradation of apoB. Addition of N-acetylleucyl-leucyl-norleucinal, an inhibitor of proteasome and other cysteinyl proteases that are responsible for apoB degradation, restored apoB recovery from verapamil-treated cells. De novo synthesis of lipids from [14C]acetate was increased in the presence of verapamil, suggesting that verapamil decreases lipid availability for apoB thus leading to the secretion of apoB-containing lipoprotein. We prepared cytosolic fractions from cells preincubated with [14C]acetate and used as a donor of radioactive lipids. When this cytosolic fraction was incubated with microsomes isolated separately, radioactive triglyceride (TG) accumulated in the luminal space of the microsomes. The transfer of radioactive TG from the cytosolic fraction to the microsomal lumen was inhibited in the presence of verapamil, suggesting that there is a verapamil-sensitive mechanism for TG transfer across ER membranes that is involved in formation of apoB-containing lipoprotein particles in ER. Verapamil showed no inhibitory effect on microsomal TG transfer protein, a well known lipid transfer protein in ER. We propose from these results that there is novel machinery for transmembrane movement of neutral lipids, which is involved in providing TG for apoB during VLDL assembly in ER.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

A microsomal GTPase is required for glycopeptide export from the mammalian endoplasmic reticulum

Bidirectional transport of proteins via the Sec61p translocon across the endoplasmic reticulum (ER) membrane is a recognized component of the ER quality control machinery. Following translocation and engagement by the luminal quality control system, misfolded and unassembled proteins are exported from the ER lumen back to the cytosol for degradation by the proteasome. Additionally, other ER contents, including oligosaccharides, oligopeptides, and glycopeptides, are efficiently exported from mammalian and yeast systems, indicating that bidirectional transport across ER membranes is a general eukaryotic phenomenon. Glycopeptide and protein export from the ER in in vitro systems is both ATP- and cytosol-dependent. Using a well established system to study glycopeptide export and conventional liquid chromatography, we isolated a single polypeptide species of 23 kDa from rat liver cytosol that was capable of fully supporting glycopeptide export from rat microsomes in the presence of an ATP-regenerating system. The protein was identified by mass spectrometric sequence analysis as guanylate kinase (GK), a housekeeping enzyme critical in the regulation of cellular GTP levels. We confirmed the ability of GK to substitute for complete cytosol by reconstitution of glycopeptide export from rat liver microsomes using highly purified recombinant GK from Saccharomyces cerevisiae. Most significantly, we found that the GK (and hence the cytosolic component) requirement was fully bypassed by low micromolar concentrations of GDP or GTP. Similarly, export was inhibited by non-hydrolyzable analogues of GDP and GTP, indicating a requirement for GTP hydrolysis. Membrane integrity was fully maintained under assay conditions, as no ER luminal proteins were released. Competence for glycopeptide export was abolished by very mild protease treatment of microsomes, indicating the presence of an essential protein on the cytosolic face of the ER membrane. These data demonstrate that export of glycopeptide export is controlled by a microsomal GTPase and is independent of cytosolic protein factors.     

Yeast GTB1 encodes a subunit of glucosidase II required for glycoprotein processing in the endoplasmic reticulum

Glucosidase II is essential for sequential removal of two glucose residues from N-linked glycans during glycoprotein biogenesis in the endoplasmic reticulum. The enzyme is a heterodimer whose alpha-subunit contains the glycosyl hydrolase active site. The function of the beta-subunit has yet to be defined, but mutations in the human gene have been linked to an autosomal dominant form of polycystic liver disease. Here we report the identification and characterization of a Saccharomyces cerevisiae gene, GTB1, encoding a polypeptide with 21% sequence similarity to the beta-subunit of human glucosidase II. The Gtb1 protein was shown to be a soluble glycoprotein (96-102 kDa) localized to the endoplasmic reticulum lumen where it was present in a complex together with the yeast alpha-subunit homologue Gls2p. Surprisingly, we found that Deltagtb1 mutant cells were specifically defective in the processing of monoglucosylated glycans. Thus, although Gls2p is sufficient for cleavage of the penultimate glucose residue, Gtb1p is essential for cleavage of the final glucose. Our data demonstrate that Gtb1p is required for normal glycoprotein biogenesis and reveal that the final two glucose-trimming steps in N-glycan processing are mechanistically distinct.     

Dynein light intermediate chain 1 is required for progress through the spindle assembly checkpoint

The spindle assembly checkpoint monitors microtubule attachment to kinetochores and tension across sister kinetochores to ensure accurate division of chromosomes between daughter cells. Cytoplasmic dynein functions in the checkpoint, apparently by moving critical checkpoint components off kinetochores. The dynein subunit required for this function is unknown. Here we show that human cells depleted of dynein light intermediate chain 1 (LIC1) delay in metaphase with increased interkinetochore distances; dynein remains intact, localised and functional. The checkpoint proteins Mad1/2 and Zw10 localise to kinetochores under full tension, whereas BubR1 is diminished at kinetochores. Metaphase delay and increased interkinetochore distances are suppressed by depletion of Mad1, Mad2 or BubR1 or by re‐expression of wtLIC1 or a Cdk1 site phosphomimetic LIC1 mutant, but not Cdk1‐phosphorylation‐deficient LIC1. When the checkpoint is activated by microtubule depolymerisation, Mad1/2 and BubR1 localise to kinetochores. We conclude that a Cdk1 phosphorylated form of LIC1 is required to remove Mad1/2 and Zw10 but not BubR1 from kinetochores during spindle assembly checkpoint silencing.     

The tyrosine phosphatase PRL-1 localizes to the endoplasmic reticulum and the mitotic spindle and is required for normal mitosis

PRL-1 is one of three closely related protein-tyrosine phosphatases, which are characterized by C-terminal farnesylation. Recent reports suggest that they are involved in the regulation of cell proliferation and transformation. However, their biological function has not yet been determined. Here we show that PRL-1 mRNA is overexpressed in a number of human tumor cell lines, including HeLa cells. Using immunofluorescence we studied the subcellular localization of endogenous PRL-1, and our results demonstrate that PRL-1 exhibits cell cycle-dependent localization; in non-mitotic HeLa cells, PRL-1 is localized to the endoplasmic reticulum in a farnesylation-dependent manner. In mitotic cells PRL-1 relocalizes to the centrosomes and the spindle apparatus, proximal to the centrosomes, in a farnesylation-independent manner. Conditional expression of a catalytic domain mutant in HeLa cells results in a delay in the progression of cells through mitosis but has no effect on other phases of the cell cycle. Further, expression of a farnesylation site PRL-1 mutant results in mitotic defects, characterized by chromosomal bridges in anaphase and lagging chromosomes, without affecting spindle checkpoint function. Together, these results suggest that PRL-1 function is regulated in a cell cycle-dependent manner and implicate PRL-1 in regulating progression through mitosis, possibly by modulating spindle dynamics.     

Vacuole membrane protein 1 is an endoplasmic reticulum protein required for organelle biogenesis, protein secretion, and development

Vacuole membrane protein 1 (Vmp1) is membrane protein of unknown molecular function that has been associated with pancreatitis and cancer. The social amoeba Dictyostelium discoideum has a vmp1-related gene that we identified previously in a functional genomic study. Loss-of-function of this gene leads to a severe phenotype that compromises Dictyostelium growth and development. The expression of mammalian Vmp1 in a vmp1(-) Dictyostelium mutant complemented the phenotype, suggesting a functional conservation of the protein among evolutionarily distant species and highlights Dictyostelium as a valid experimental system to address the function of this gene. Dictyostelium Vmp1 is an endoplasmic reticulum protein necessary for the integrity of this organelle. Cells deficient in Vmp1 display pleiotropic defects in the secretory pathway and organelle biogenesis. The contractile vacuole, which is necessary to survive under hypoosmotic conditions, is not functional in the mutant. The structure of the Golgi apparatus, the function of the endocytic pathway and conventional protein secretion are also affected in these cells. Transmission electron microscopy of vmp1(-) cells showed the accumulation of autophagic features that suggests a role of Vmp1 in macroautophagy. In addition to these defects observed at the vegetative stage, the onset of multicellular development and early developmental gene expression are also compromised.     

Viral protein targeting to the cortical endoplasmic reticulum is required for cell-cell spreading in plants

Many plant RNA viruses use their nonstructural proteins to target and move through the cortical endoplasmic reticulum (ER) tubules within the plant intercellular junction for cell-to-cell spreading. Most of these proteins, including the triple-gene-block 3 protein (TGBp3) of Potexvirus, are ER membrane proteins. We previously showed that TGBp3 of the Bamboo mosaic potexvirus partitions into tubular subdomains of the ER in both yeast and plants, but the mechanism and physiological significance of this localization is unclear. Here, we demonstrate that a sorting signal present in TGBp3 is necessary and sufficient for its oligomerization and for targeting integral membrane proteins into puncta within curved ER tubules. Mutations in the TGBp3 sorting signal impair viral spread, and plants infected with viruses harboring these mutants were either asymptomatic or had reduced symptoms. Thus, we propose that Potexvirus use the sorting signal in TGBp3 to target infectious viral derivatives to cortical ER tubules for transmission through the intercellular junctions in plants.     

Molecular machinery required for protein transport from the endoplasmic reticulum to the Golgi complex

The cellular machinery responsible for conveying proteins between the endoplasmic reticulum and the Golgi is being investigated using genetics and biochemistry. A role for vesicles in mediating protein traffic between the ER and the Golgi has been established by characterizing yeast mutants defective in this process, and by using recently developed cell-free assays that measure ER to Golgi transport. These tools have also allowed the identification of several proteins crucial to intracellular protein trafficking. The characterization and possible functions of several GTP-binding proteins, peripheral membrane proteins, and an integral membrane protein during ER to Golgi transport are discussed here.     

Protein kinase Ctheta is required for autophagy in response to stress in the endoplasmic reticulum

Autophagy is an evolutionally conserved process for the bulk degradation of cytoplasmic proteins and organelles. Recent observations indicate that autophagy is induced in response to cellular insults that result in the accumulation of misfolded proteins in the lumen of the endoplasmic reticulum (ER). However, the signaling mechanisms that activate autophagy under these conditions are not understood. Here, we report that ER stress-induced autophagy requires the activation of protein kinase C (PKC), a member of the novel-type PKC family. Induction of ER stress by treatment with either thapsigargin or tunicamycin activated autophagy in immortalized hepatocytes as monitored by the conversion LC3-I to LC3-II, clustering of LC3 into dot-like cytoplasmic structures, and electron microscopic detection of autophagosomes. Pharmacological inhibition of PKC or small interfering RNA-mediated knockdown of PKC prevented the autophagic response to ER stress. Treatment with ER stressors induced PKC phosphorylation within the activation loop and localization of phospho-PKC to LC3-containing dot structures in the cytoplasm. However, signaling through the known unfolded protein response sensors was not required for PKC activation. PKC activation and stress-induced autophagy were blocked by chelation of intracellular Ca(2+) with BAPTA-AM. PKC was not activated or required for autophagy in response to amino acid starvation. These observations indicate that Ca(2+)-dependent PKC activation is specifically required for autophagy in response to ER stress but not in response to amino acid starvation.     

An energy-dependent maturation step is required for release of the cystic fibrosis transmembrane conductance regulator from early endoplasmic reticulum biosynthetic machinery

Polytopic proteins are synthesized in the endoplasmic reticulum (ER) by ribosomes docked at the Sec61 translocation channel. It is generally assumed that, upon termination of translation, polypeptides are spontaneously released into the ER membrane where final stages of folding and assembly are completed. Here we investigate early interactions between the ribosome-translocon complex and cystic fibrosis transmembrane conductance regulator (CFTR), a multidomain ABC transporter, and demonstrate that this is not always the case. Using in vitro and Xenopus oocyte expression systems we show that, during and immediately following synthesis, nascent CFTR polypeptides associate with large, heterogeneous, and dynamic protein complexes. Partial-length precursors were quantitatively isolated in a non-covalent, puromycin-sensitive complex (>3,500 kDa) that contained the Sec61 ER translocation machinery and the cytosolic chaperone Hsc70. Following the completion of synthesis, CFTR was gradually released into a smaller (600-800 kDa) ATP-sensitive complex. Surprisingly, release of full-length CFTR from the ribosome and translocon was significantly delayed after translation was completed. Moreover, this step required both nucleotide triphosphates and cytosol. Release of control proteins varied depending on their size and domain complexity. These studies thus identify a novel energy-dependent step early in the CFTR maturation pathway that is required to disengage nascent CFTR from ER biosynthetic machinery. We propose that, contrary to current models, the final stage of membrane integration is a regulated process that can be influenced by the state of nascent chain folding, and we speculate that this step is influenced by the complex multidomain structure of CFTR.     

The essential endoplasmic reticulum chaperone Rot1 is required for protein N- and O-glycosylation in yeast

Rot1 is an essential yeast protein originally shown to be implicated in such diverse processes such as β-1,6-glucan synthesis, actin cytoskeleton dynamics or lysis of autophagic bodies. More recently also a role as a molecular chaperone has been discovered. Here, we report that Rot1 interacts in a synthetic manner with Ost3, one of the nine subunits of the oligosaccharyltransferase (OST) complex, the key enzyme of N-glycosylation. The deletion of OST3 in the rot1-1 mutant causes a temperature sensitive phenotype as well as sensitivity toward compounds interfering with cell wall biogenesis such as Calcofluor White, caffeine, Congo Red and hygromycin B, whereas the deletion of OST6, a functional homolog of OST3, has no effect. OST activity in vitro determined in membranes from rot1-1ost3Δ cells was found to be decreased to 45% compared with wild-type membranes, and model glycoproteins of N-glycosylation, like carboxypeptidase Y, Gas1 or dipeptidyl aminopeptidase B, displayed an underglycosylation pattern. By affinity chromatography, a physical interaction between Rot1 and Ost3 was demonstrated. Moreover, Rot1 was found to be involved also in the O-mannosylation process, as the glycosylation of distinct glycoproteins of this type were affected as well. Altogether, the data extend the role of Rot1 as a chaperone required to ensure proper glycosylation.     

Cytosolic Sec13p complex is required for vesicle formation from the endoplasmic reticulum in vitro

The SEC13 gene of Saccharomyces cerevisiae is required in vesicle biogenesis at a step before or concurrent with the release of transport vesicles from the ER membrane. SEC13 encodes a 33-kD protein with sequence homology to a series of conserved internal repeat motifs found in beta subunits of heterotrimeric G proteins. The product of this gene, Sec13p, is a cytosolic protein peripherally associated with membranes. We developed a cell-free Sec13p-dependent vesicle formation reaction. Sec13p-depleted membranes and cytosol fractions were generated by urea treatment of membranes and affinity depletion of a Sec13p-dihydrofolate reductase fusion protein, respectively. These fractions were unable to support vesicle formation from the ER unless cytosol containing Sec13p was added. Cytosolic Sec13p fractionated by gel filtration as a large complex of about 700 kD. Fractions containing the Sec13p complex restored activity to the Sec13p- dependent vesicle formation reaction. Expression of SEC13 on a multicopy plasmid resulted in overproduction of a monomeric form of Sec13p, suggesting that another member of the complex becomes limiting when Sec13p is overproduced. Overproduced, monomeric Sec13p was inactive in the Sec13p- dependent vesicle formation assay.