Cargo is selectively exported from the ER in COPII vesicles. To analyze the role of COPII in selective transport from the ER, we have purified components of the mammalian COPII complex from rat liver cytosol and then analyzed their role in cargo selection and ER export. The purified mammalian Sec23–24 complex is composed of an 85-kD (Sec23) protein and a 120-kD (Sec24) protein. Although the Sec23–24 complex or the monomeric Sec23 subunit were found to be the minimal cytosolic components recruited to membranes after the activation of Sar1, the addition of the mammalian Sec13–31 complex is required to complete budding. To define possible protein interactions between cargo and coat components, we recruited either glutathione-S-transferase (GST)–tagged Sar1 or GST– Sec23 to ER microsomes. Subsequently, we solubilized and reisolated the tagged subunits using glutathione-Sepharose beads to probe for interactions with cargo. We find that activated Sar1 in combination with either Sec23 or the Sec23–24 complex is necessary and sufficient to recover with high efficiency the type 1 transmembrane cargo protein vesicular stomatitis virus glycoprotein in a detergent-soluble prebudding protein complex that excludes ER resident proteins. Supplementing these minimal cargo recruitment conditions with the mammalian Sec13–31 complex leads to export of the selected cargo into COPII vesicles. The ability of cargo to interact with a partial COPII coat demonstrates that these proteins initiate cargo sorting on the ER membrane before budding and establishes the role of GTPase-dependent coat recruitment in cargo selection.

Abstract. ER to Golgi transport requires the function of two distinct vesicle coat complexes, termed COPI (coatomer) and COPII, whose assembly is regulated by the small GTPases ADP-ribosylation factor 1 (ARF1) and Sarl, respectively. To address their individual roles in transport, we have developed a new assay using mammalian microsomes that reconstitute the formation of ER-derived vesicular carriers. Vesicles released from the ER were found to contain the cargo molecule vesicular stomatitis virus glycoprotein (VSV-G) and p58, an endogenous protein that continuously recycles between the ER and pre-Golgi intermediates. Cargo was efficiently sorted from resident ER proteins during vesicle formation in vitro. Export of VSV-G and p58 were found to be exclusively mediated by COPII. Subsequent movement of ER-derived carriers to the Golgi stack was blocked by a trans-dominant ARF1 mutant restricted to the GDP-bound state, which is known to prevent COPI recruitment. To establish the initial site of coatomer assembly after export from the ER, we immunoisolated the vesicular intermediates and tested their ability to recruit COPI. Vesicles bound coatomer in a physiological fashion requiring an ARFl-guanine nucleotide exchange activity. These results suggest that coat exchange is an early event preceding the targeting of ER-derived vesicles to pre-Golgi intermediates.

Members of the yeast p24 family, including Emp24p and Erv25p, form a heteromeric complex required for the efficient transport of selected proteins from the endoplasmic reticulum (ER) to the Golgi apparatus. The specific functions and sites of action of this complex are unknown. We show that Emp24p is directly required for efficient packaging of a lumenal cargo protein, Gas1p, into ER-derived vesicles. Emp24p and Erv25p can be directly cross-linked to Gas1p in ER-derived vesicles. Gap1p, which was not affected by emp24 mutation, was not cross-linked. These results suggest that the Emp24 complex acts as a cargo receptor in vesicle biogenesis from the ER.

A 125-kDa glycoprotein exposed on the surface of Saccharomyces cerevisiae cells belongs to a class of eucaryotic membrane proteins anchored to the lipid bilayer by covalent linkage to an inositol-containing glycophospholipid. We have cloned the gene (GAS)) encoding the 125-kDa protein (Gaslp) and found that the function of Gaslp is not essential for cell viability. The nucleotide sequence of GAS) predicts a 60-kDa polypeptide with a cleavable N-terminal signal sequence, potential sites for N-and 0-linked glycosylation, and a C-terminal hydrophobic domain. Determination of the anchor attachment site revealed that the C-terminal hydrophobic domain of Gaslp is removed during anchor addition. However, this domain is essential for addition of the glycophospholipid anchor, since a truncated form of the protein failed to become attached to the membrane. Anchor addition was also abolished by a point mutation affecting the hydrophobic character of the C-terminal sequence. We conclude that glycophospholipid anchoring of Gaslp depends on the integrity of the C-terminal hydrophobic domain that is removed during anchor attachment.A number of eucaryotic membrane proteins are anchored to the lipid bilayer by a covalently linked glycosyl phosphatidylinositol (GPI) moiety (reviewed in references 20, 26, and 46). This particular mode of membrane attachment occurs in a wide variety of eucaryotic organisms. The modified proteins fall into diverse functional groups, including hydrolytic enzymes, cell adhesion molecules, protozoan coat proteins, and numerous cell surface antigens of unknown function.The complete structure of the GPI moiety has been determined for two forms of the variant surface glycoprotein of Trypanosoma brucei (25, 58) and the mammalian cell surface antigen . GPI anchors from these distantly related organisms share a common core structure, consisting of a phosphatidylinositol molecule linked to a linear tetrasaccharide composed of one nonacetylated glucosaminyl and three mannosyl residues. At its nonreducing end, the glycan is attached via a phosphodiester to ethanolamine, which is amide linked to the a-carboxyl group of the C-terminal amino acid of the mature protein.GPI-anchored proteins are commonly synthesized with a cleavable N-terminal signal sequence and a C-terminal domain composed predominantly of hydrophobic amino acids. This particular feature seems to be important in the mechanism of anchor addition. In all cases studied so far, addition of the GPI anchor involves the removal of 17 to 31 residues from the C terminus of a larger precursor (7,15,22,27,29,32,35,36,47,51,53,60,62,65,68). Since processing rapidly follows protein synthesis (2, 18, 24), it is believed that the GPI moiety is preassembled and transferred en bloc to the protein in the endoplasmic reticulum.Several lines of evidence suggest that a signal for GPI anchor attachment resides in the C-terminal domain of the proteins. However, the C-terminal sequences of GPI-anchored proteins do not exhibit any recognizable homology. Although the stru...

Abstract. Rabl is a small GTPase regulating vesicular traffic between early compartments of the secretory pathway. To explore the role of rabl we have analyzed the function of a mutant (rabla[S25N]) containing a substitution which perturbs Mg 2+ coordination and reduces the affinity for GTE resulting in a form which is likely to be restricted to the GDP-bound state. The rabla(S25N) mutant led to a marked reduction in protein export from the ER in vivo and in vitro, indicating that a guanine nucleotide exchange protein (GEP) is critical for the recruitment of rabl during vesicle budding. The mutant protein required posttranslational isoprenylation for inhibition and behaved as a competitive inhibitor of wild-type rabl function. Both rabla and rablb (92% identity) were able to antagonize the inhibitory activity of the rabla(S25N) mutant, suggesting that these two isoforms are functionally interchangeable. The rabl mutant also inhibited transport between Golgi compartments and resulted in an apparent loss of the Golgi apparatus, suggesting that Golgi integrity is coupled to rabl function in vesicular traffic.

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Abstract. Members of the rab/YPTI/SEC4 gene family of small molecular weight GTPases play key roles in the regulation of vesicular traffic between compartments of the exocyfic pathway. Using immunoelectron microscopy, we demonstrate that a dominant negative rabla mutant, rabla(N124I), defective for guanine nucleotide binding in vitro, leads to the accumulation of vesicular stomatitis virus glycoprotein (VSV-G) in numerous pre-cis-Golgi vesicles and vesicular-tubular clusters containing rabl and B-COP, a subunit of the coatomer complex. Similar to previous observations . Cell. 76:841-852), VSV-G was concentrated nearly 5-1G-fold in vesicular carriers that aocumulate in the presence of the rabla(N124I) mutant. VSV-G containing vesicles and vesiculartubular clusters were also found to accumulate in the presence of a rabla effector domain peptide mimetic that inhibits endoplasmic reticulum to Golgi transport, as well as in the absence of Ca 2+. These results suggest that the combined action of a Ca2+-dependent prorein and conformational changes associated with the GTPase cycle of rabl are essential for a late targeting/fusion step controlling the delivery of vesicles to Golgi compartments.

Abstract. The Golgi apparatus is a dynamic organdie whose structure is sensitive to vesicular traffic and to cell cycle control. We have examined the potential role for mbla, a GTPase previously associated with ER to Golgi and intra-Golgi transport, 'in the formation and maintenance of Golgi structure. Bacterially expressed, recombinant rabla protein was mieroinjected into rat embryonic fibroblasts, followed by analysis of Golgi morphology by fluorescence and electron microscopy. Three recombinant proteins were tested: wild-type rab, mutant rabla(S25N), a constitutively GDP-botmd form