A Rabl Mutant Affecting Guanine Nueleotide Exchange Promotes Disassembly of the Golgi Apparatus

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 (Nuoffer, C., H. W. Davidson, J. Matteson, J. Meinkoth, and W. E. Balch, 1994. J. Cell Biol. 125: 225-237), and mutant rabla(N124I) defective in guanine nucleotide binding. Microinjection of wildtype rabla protein or a variety of negative controls (injection buffer alone or activated ras protein) did not affect the appearance of the Golgi, as visualized by immunofluorescence of a-mannosidase II (Man 1I), used as a Golgi marker. In contrast, mieroinjection of the mutant forms promoted the disassembly of the Golgi stacks into dispersed vesicular structures visualized by immunofluorescence. When S25N-injected cells were analyzed by EM after immunoperoxidase labeling, Man II was found in isolated ministacks and large vesicular elements that were often surrounded by numerous smaller unlabeled vesicles resembling carrier vesicles. Golgi disassembly caused by rabia mutants differs from BFA-induced disruption, since B-COP remains membrane associated, and Man II does not redistribute to the ER. BFA can still cause these residual Golgi elements to fuse and disperse, albeit at a slower rate. Moreover, BFA recovery is incomplete in the presence of rabl mutants or GTPTS. We conclude that GTP exchange and hydrolysis by GTPases, specifically rabla, are required to form and maintain normal Golgi stacks. The similarity of Golgi disassembly seen with rabla mutants to that occurring during mitosis, may point to a molecular basis involving rabla for fragmentation of the Golgi apparatus during cell division. T H~ Golgi apparatus is an exceptionally dynamic organelle which at any one time contains abundant newly synthesized membrane and secretory proteins en route to the cell surface and to other cellular organelles (23). In most cells, the Golgi apparatus resembles a stack of flattened discs that form an extensive intercalated network. This organization is partially dismantled in cells which are treated with microtubule-depolymerizing agents such as nocodozol, colchicine, or vinblastine (33) and is disassembled to vesicular elements when cells enter mitosis (60). Rapid disassembly of the Golgi apparatus is also seen after cells are exposed to the lipophilic fungal toxin, brefeldin A Dr. W'tlson's present address is Dept. of Pathology, University of New Mexico School of Medicine, Albuquerque, NM 87131. Address all correspondence to Dr. M. (3. Farquhar, Division of Cellular and Molecular Medicine, 0651, University of California at San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0651. (BFA).t In many cell types, BFA causes the fusion of socalled "homotypic" membranes which is assumed to be due to a loss of vesicular coat proteins (32, 64). The latter include the coatomer protein/3-COP implicated in ER to Golgi as well as intra-Golgi trafficking (21) and 7-adaptin and clathrin (47), involved in traffic from the trans-Golgi network to endosomes and/or lysosomes. It is now recognized that the rab and ARF family of GTPases regulate intracellular trafficking through both the exocytic and endocytic pathways. In particular, the rab family of proteins has now grown to over 25 members (39). A combination of genetic, biochemical, and immunolocalization evidence suggests that these small GTPases mediate 1. Abbreviations used in this paper: BFA, brefeldin A; GEP, guanine nucleotide ezchanom¢ protein; Man II, a-mannosidase IT; NRK, normal rat kidney; RT, room temperature; VSV-G, vesicular stomatitus virus glycoprotein. © The Rockefeller University Press, 0021-95251941051557115 $2.00 The Journal of CeU Biology, Volume 125, Number 3, May 1994 557-571 557 on July 7, 2017 jcb.rress.org D ow nladed fom different aspects of vesicle budding, targeting, and/or fusion at specific sites in the cell's complex ~highway" system (4, 26, 46). Analysis of the function of members of the rab family has benefited from the extensive mutational studies of cellular ras which identified point mutations that limit the ability of mutated ms protein tO assume different conformations brought about by exchange of GTP for GDP, or those that lower the intrinsic rate of hydrolysis of GTP which returns a protein to a GDP-bound state (6). Rab proteins are proposed to confer directionality and/or specificity to transport vesicle function through their role as molecular switches to control the vectorial interaction of components involved in budding, targeting, and fusion. The necessity for cycling of rab and other GTI'ases involved in vesicular traffic is underscored by the sensitivity of many vesicular transport steps to the introduction of nonhydrolyzable analogues of GTP into cell-free or permeabilized cell transport assays (8, 41). In particular, mutational analysis of closely related rabla and rablb proteins (14, 45) and ARF1 (13) strongly implicate them in control of trafficking events from the ER to the Golgi apparatus and between early Golgi compartments (14, 42, 45). To understand the role of the Golgi complex in the secretory pathway it is important to define the relationships between its structure and vesicular traffic. In this study we explore the possibility that rabia protein is not only actively involved in anterograde vesicular traffic, but also is critical for maintaining the structural integrity of the Golgi apparatus. This work follows the observations that BFA may inhibit the guanine nucleotide exchange protein that is critical for recruitment and/or maintenance of ARF on Golgi membranes (17, 28), and that GTP~,S (which targets all guanine nucleotide binding proteins) and A1F4(believed to target heterotrimeric G proteins [31]) interfere with Golgi apparatus disassembly after exposure to BFA (18, 34, 53). To avoid the use of these general reagents, we take a more direct molecular approach using mutants of the rabl protein defective in guanine nucleotide exchange to explore the specific role of rabl in this process. We find that microinjection of selected mutant rabl proteins directly into living cells triggers the complete disassembly of the Golgi stack without a BFA-like collapse into the ER. Thus we observed that extensive vesicle budding occurs in the presence of rabl mutants with an apparent absence of downstream fusion. Nevertheless, distinctive Golgi elements persist, suggesting they represent a possible template for assembly of the Golgi stack. Our findings indicate that normal function of rabl in control of vesicular traffic is essential for the integrity of the Golgi stack. Furthermore, multiple interactive GTPases are critical for this process since rabl mutants markedly inhibit the disassembly of the Golgi apparatus by BFA. The relationship of these results to the molecular basis for fragmentation of the Golgi apparatus during mitosis is discussed. Materials and Methods