Distinct coated vesicles labeled for p 200 bud from trans - Golgi network membranes ( vesicle trafficking / Golgi / vesicle coat ) NAVNEET NARULA

Golgi-associated cytoplasmic proteins, such as the coatomer protein complex, are required for vesicle budding and trafficking. We have previously described a cytoplasmic phosphoprotein, p200, which binds dynamically and specifically to Golgi membranes. The p200 protein is dissociated from Golgi membranes in the presence ofbrefeldin A and it is induced to bind to Golgi membranes by activation of guanine nucleotide binding proteins (G proteins) with guanosine 5'-[y-thio]triphosphate or aluminum fluoride. To establish the role of p200 in vesicle budding, we localized membrane-bound p200 in intact cells and on isolated Golgi membranes. We show that p200 is preferentially associated with vesicles in the trans-Golgi network (TGN). Activation of G proteins induced budding and accumulation of small, coated vesicles from Golgi membranes and p200 was localized on the cytoplasmic surface of some of these vesicles. Using immunogold labeling we further demonstrate that p200 and 13-COP are localized on different populations of Golgi-derived vesicles. These data establish that p200 is involved in the budding and coating of a class of Golgi vesicles that are likely to be derived from the TGN. The data also show that there are distinct populations of non-clathrin-coated vesicles budded from Golgi membranes, and vesicles labeled for either 18-COP or p200 may represent transport vesicles for separate steps of protein transport. The transport of proteins through the secretory pathway is believed to occur via a series of carrier vesicles (1), although the vesicle populations mediating specific stages of transport have not been fully characterized. Progress in identifying the transport vesicles and their associated proteins has been achieved by cell-free assays, which were first used to investigate intra-Golgi transport and have since been used to study other steps of transport (reviewed in refs. 2 and 3). From these studies, a variety of cytoplasmic proteins and regulatory guanine nucleotide binding proteins (G proteins) have been described, which bind to membranes and form the coats of budding vesicles. The coatomer, a complex of COP proteins, including the 110 kDa (3-COP protein (4, 5) and the ADPribosylation factor (ARF) (6), are responsible for coating small non-clathrin-coated vesicles derived from Golgi membranes. Clathrin vesicles, which are coated with specific sets of adaptor proteins and clathrin also bud from Golgi membranes under similar conditions (7). Other complexes of cytoplasmic proteins, such as the COP II complex (8), are found on vesicles involved in rough endoplasmic reticulum (RER)-Golgi transport in yeast. Distinct cytoplasmic proteins also define populations of exocytic (9) and transcytotic (10) vesicles. Both monomeric and heterotrimeric G proteins have been implicated in regulating the binding of coat proteins to Golgi membranes to initiate the coating and budding of different vesicles (11-15). (3-COP, ARF, and y-adaptin are recruited onto Golgi membranes, followed by accumulation of coated vesicles in the presence of the nonhydrolyzable nucleotide guanosine 5'-[y-thio]triphosphate (GTP[-yS]) or by activation of heterotrimeric G proteins with aluminum fluoride (AlF,) (7, 16). We have described another cytosolic protein, p200, which also binds selectively and reversibly to Golgi membranes (17). Binding of p200 to Golgi membranes is induced by activation of G proteins with GTP[-yS], AlF,, or mastoparan, particularly through the action of the heterotrimeric G protein subunit Gai-3 (12). The properties of p200 demonstrated to date, including (i) its distribution on Golgi membranes and in the cytoplasm (17), (ii) its dissociation from membranes in the presence of brefeldin A (BFA) (17), and (iii) the induction of membrane binding by GTP analogs or activators of heterotrimeric G proteins (12), are consistent with a role for p200 in vesicle budding. The present study was undertaken to provide definitive evidence for the association of p200 with Golgi vesicles. In this report, we present data on the localization of membrane-bound p200 within the Golgi and on coated vesicles generated from the Golgi complex. In addition, we compared the distributions of p200 and (3-COP on Golgi vesicles, providing evidence for heterogeneity and subcompartment specificity among potential transport vesicles. MATERIALS AND METHODS Incubation of Isolated Golgi Membranes. Golgi membranes and cytosol fractions were prepared from rat liver (18) and in vitro incubations were carried out as described (12) to induce budding of Golgi vesicles. Golgi membranes (100 ,ug of protein) were incubated with cytosol (500',g of protein) in 30 mM Hepes/20 mM KCl/5 mM magnesium acetate/20 mM Tris HCl, pH 7.4, buffer containing 100 ,uM GTP[yS] for 30 min at 37°C; membranes were pelleted, fixed, and cryosectioned for immunogold labeling as described below. Immunocytochemistry. NRK cells grown on coverslips were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and incubated with specific primary antibodies followed by fluorescein isothiocyanate-labeled conjugates for immunofluorescence staining (17). For ultrathin cryosectioning and immunogold labeling, NRK cells grown on filters (Transwell; Corning/Costar) were fixed in 4% paraformaldehyde with 5% sucrose, cryoprotected by infiltration with 2.3 M sucrose, and snap frozen in liquid nitrogen, and then 60-nm frozen sections were cut on a Reichert' FC4D ultracryomicrotome and mounted on coated nickel grids. Sections were then sequentially incubated with specific antibodies followed by protein A conjugated to 5-, 8-, or 15-nm gold particles. Abbreviations: BFA, brefeldin A; AlF, aluminum fluoride; M6PR, mannose 6-phosphate receptor; TGN, trans-Golgi network; G protein, guanine nucleotide binding protein; ARF, ADP-ribosylation factor; RER, rough endoplasmic reticulum; GTP[yS], guanosine 5'-[,ythio]triphosphate. *To whom reprint requests should be addressed at: Centre for Molecular and Cellular Biology, University of Queensland, Brisbane 4072, QLD, Australia. 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