Little is known about the integral membrane proteins that participate in the early secretory pathway of mammalian cells. The complementary DNA encoding a 28-kilodalton protein (p28) of the cis-Golgi was cloned and sequenced. The protein was predicted to contain a central coiled-coil domain with a carboxyl-terminal membrane anchor. An in vitro assay for endoplasmic reticulum-Golgi transport was used to show that p28 participates in the docking and fusion stage of this transport event. Biochemical studies established that p28 is a core component of the Golgi SNAP receptor (SNARE) complex.

The role of COPII components in endoplasmic reticulum (ER)-Golgi transport, first identified in the yeastSaccharomyces cerevisiae, has yet to be fully characterized in higher eukaryotes. A human cDNA whose predicted amino acid sequence showed 70% similarity to the yeast Sec13p has previously been cloned. Antibodies raised against the human SEC13 protein (mSEC13) recognized a cellular protein of 35 kDa in both the soluble and membrane fractions. Like the yeast Sec13p, mSEC13 exist in the cytosol in both monomeric and highermolecular-weight forms. Immunofluorescence microscopy localized mSEC13 to the characteristic spotty ERGolgi intermediate compartment (ERGIC) in cells of all species examined, where it colocalized well with the KDEL receptor, an ERGIC marker, at 15؇C. Immunoelectron microscopy also localized mSEC13 to membrane structures close to the Golgi apparatus. mSEC13 is essential for ER-to-Golgi transport, since both the His 6 -tagged mSEC13 recombinant protein and the affinity-purified mSEC13 antibody inhibited the transport of restrictive temperature-arrested vesicular stomatitis virus G protein from the ER to the Golgi apparatus in a semi-intact cell assay. Moreover, cytosol immunodepleted of mSEC13 could no longer support ER-Golgi transport. Transport could be restored in a dose-dependent manner by a cytosol fraction enriched in the high-molecular-weight mSEC13 complex but not by a fraction enriched in either monomeric mSEC13 or recombinant mSEC13. As a putative component of the mammalian COPII complex, mSEC13 showed partially overlapping but mostly different properties in terms of localization, membrane recruitment, and dynamics compared to that of ␤-COP, a component of the COPI complex.

Yeast Bet1p participates in vesicular transport from the endoplasmic reticulum to the Golgi apparatus and functions as a soluble N-ethylmaleimide–sensitive factor attachment protein receptor (SNARE) associated with ER-derived vesicles. A mammalian protein (rbet1) homologous to Bet1p was recently identified, and it was concluded that rbet1 is associated with the Golgi apparatus based on the subcellular localization of transiently expressed epitope-tagged rbet1. In the present study using rabbit antibodies raised against the cytoplasmic domain of rbet1, we found that the majority of rbet1 is not associated with the Golgi apparatus as marked by the Golgi mannosidase II in normal rat kidney cells. Rather, rbet1 is predominantly associated with vesicular spotty structures that concentrate in the peri-Golgi region but are also present throughout the cytoplasm. These structures colocalize with the KDEL receptor and ERGIC-53, which are known to be enriched in the intermediate compartment. When the Golgi apparatus is fragmented by nocodazole treatment, a significant portion of rbet1 is not colocalized with structures marked by Golgi mannosidase II or the KDEL receptor. Association of rbet1 in cytoplasmic spotty structures is apparently not altered by preincubation of cells at 15°C. However, upon warming up from 15 to 37°C, rbet1 concentrates into the peri-Golgi region. Furthermore, rbet1 colocalizes with vesicular stomatitis virus G-protein en route from the ER to the Golgi. Antibodies against rbet1 inhibit in vitro transport of G-protein from the ER to the Golgi apparatus in a dose-dependent manner. This inhibition can be neutralized by preincubation of antibodies with recombinant rbet1. EGTA is known to inhibit ER-Golgi transport at a stage after vesicle docking but before the actual fusion event. Antibodies against rbet1 inhibit ER-Golgi transport only when they are added before the EGTA-sensitive stage. These results suggest that rbet1 may be involved in the docking process of ER- derived vesicles with the cis-Golgi membrane.

Abstract. The small GTPase Rabl is required for vesicular traffic from the ER to the cis-Golgi compartment, and for transport between the cis and medial compartments of the Golgi stack. In the present study, we examine the role of guanine nucleotide dissociation inhibitor (GDI) in regulating the function of Rabl in the transport of vesicular stomatitis virus glycoprotein (VSV-G) in vitro. Incubation in the presence of excess GDI rapidly (h/2 < 30 s) extracted Rabl from membranes, inhibiting vesicle budding from the ER and sequential transport between the cis-, medial-, and transGolgi cisternae. These results demonstrate a direct role for GDI in the recycling of Rab proteins. Analysis of rat liver cytosol by gel filtration revealed that a major pool of Rabl fractionates with a molecular mass of ,,o80 kD in the form of a GDI-Rabl complex. When the GDI-Rabl complex was depleted from cytosol by use of a Rabl-specific antibody, VSV-G failed to exit the ER. However, supplementation of depleted cytosol with a GDI-Rabl complex prepared in vitro from recombinant forms of Rabl and GDI efficiently restored export from the ER, and transport through the Golgi stack. These results provide evidence that a cytosolic GDI-Rabl complex is required for the formation of non-clathrin-coated vesicles mediating transport through the secretory pathway.

Abstract. Using a novel in vitro assay which allows us to distinguish vesicle budding from subsequent targeting and fusion steps, we provide the first biological evidence that B-COP, a component of non-clathrincoated vesicles believed to mediate intraGolgi transport, is essential for transport of protein from the ER to the cis-Golgi compartment. Incubation in the presence of B-COP specific antibodies and Fab fragments prevents the exit of vesicular stomatitis virus glycoprotein (VSV-G) from the ER. These results demonstrate that fl-COP is required for the assembly of coat complexes mediating vesicle budding. Fractionation of rat liver cytosol revealed that a major biologically active form of B-COP was found in a high molecular pool (>1,000 kD) distinct from coatomer and which promoted efficient vesicle budding from the ER. Surprisingly, rablB could be quantitatively coprecipitated with this/3-COP containing complex and was also essential for function. We suggest that/3-COP functions in an early step during vesicle formation and that rablB may be recruited as a component of a precoat complex which participates in the export of protein from the ER via vesicular carders.

In eukaryotic cells, the Golgi apparatus receives newly synthesized proteins from the endoplasmic reticulum (ER) and delivers them after covalent modification to their destination in the cell. These proteins move from the inside (cis) face to the plasma-membrane side (trans) of the Golgi, through a stack of cisternae, towards the trans-Golgi network (TGN), but very little is known about how proteins are moved through the Golgi compartments. In a model known as the maturation model, no special transport process was considered necessary, with protein movement along the Golgi being achieved by maturation of the cisternae. Alternatively, proteins could be transported by vesicles or membrane tubules. Although little is known about membrane-tubule-mediated transport, the molecular mechanism for vesicle-mediated transport is quite well understood, occurring through docking of SNAREs on the vesicle with those on the target membrane. We have now identified a protein of relative molecular mass 27K which is associated with the Golgi apparatus. The cytoplasmic domain of this protein or antibodies raised against it quantitatively inhibit transport in vitro from the ER to the trans-Golgi/TGN, acting at a stage between the cis/medial- and the trans-Golgi/TGN. This protein, which behaves like a SNARE and has been named GS27 (for Golgi SNARE of 27K), is identical to membrin, a protein implicated earlier in ER-to-Golgi transport. Our results suggest that protein movement from medial- to the trans-Golgi/TGN depends on SNARE-mediated vesicular transport.

ER to the Golgi apparatus. In this system, antibodies against Bet3 inhibit transport in a dose-dependent manner, and cytosol that is immunodepleted of Bet3 is also defective in this transport. This defect can be rescued by supplementing the Bet3-depleted cytosol with recombinant GST-Bet3. We also show that Bet3 acts after COPII but before Rab1, α-SNAP and the EGTA-sensitive stage during ER-Golgi transport. Gel filtration analysis demonstrates that Bet3 exists in two distinct pools in the cytosol, the highmolecular-weight pool may represent the TRAPP complex, whereas the other probably represents the monomeric Bet3.

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