The Small GTPbinding Protein Rholp Is Localized on the Golgi Apparatus and Pbst-Golgi Vesicles in Saccharomyces cerevisiae

In Saccharomyces cerevisiae the ras-related protein Rholp is essentially the only target for ADPribosylation by exoenzyme C3 of Clostridium botulinum. Using C3 to detect Rholp in subcellular fractions, Rholp was found primarily in the 10,000 g pellet (P2) containing large organelles ; small amounts also were detected in the 100,000 g pellet (P3), and cytosol . When P2 organelles were separated in sucrose density gradients Rholp comigrated with the Kex-2 activity, a late Golgi marker. Rholp distribution was shifted from P2 to P3 in several mutants that accumulate post-Golgi vesicles . Rholp comigrated with GPROTEINs are regulatory proteins that bind GTP and exhibit slow GTP hydrolysis activity. The two strucrural states of G-Proteins GTP bound or GDP bound, are finely regulated in response to specific signals . GTP binding results in structural changes that lead to transmission of a signal, while GTP hydrolysis returns the complex to the GDP bound form, the resting state ofG-proteins . By this mechanism G-proteins are thought to serve as molecular switches in many regulatory pathways. G-proteins can be divided into four classes. These are (a) the trimeric G-proreins, responding to extracellular signals, (b) certain soluble components of the protein synthetic machinery involved in translational accuracy, (c) proteins related to the ADP-ribosylation factor ARF, and (d) those related to the ras oncoprotein . The superfamily ofras-related proteins (Chardin, 1988) is composed of at least three subfamilies called Ras, Rho, and Ypt/Sec 4 (called rab in mammalian cells), the last one being the most diverse group of the three . Approximately 30% of the amino acids are identical among members of different subfamilies, and the GTP binding site comprises most ofthe conserved residues (De Vos et al ., 1988 ; Pai et al ., 1989) . M. McCaffrey's present address is Unité de Génétique Somatique (URA CNRS 361), Institut Pasteur, 25 Rue Dr. Roux, 75724 Paris Cedex 15, France . P. Madaules present address is Unitéde Recombinaison et Expression Génétique, Institut Pasteur, 28 Rue Dr. Roux, 75724 Paris Cedex 15, France. © The Rockefeller University Press, 0021-9525/91/10/309/11 $2 .00 The Journal of Cell Biology, Volume 115, Number 2, October 1991309-319 post-Golgi transport vesicles during fractionation of P3 organelles from wild-type or sec6 cells. Vesicles containing Rholp were of the same size but different density than those bearing Sec4p, a ras-related protein located both on post-Golgi vesicles and the plasma membrane . Immunofluorescence microscopy detected Rholp as a punctate pattern, with signal concentrated towards the cell periphery and in the bud . Thus, in S. cerevisiae Rholp resides primarily in the Golgi apparatus, and also in vesicles that are likely to be early post-Golgi vesicles . Another important region of the ras-related proteins is the carboxy-terminal domain known to undergo posttranslation modifications essential for attachment of the protein to a membrane (Willumsen et al ., 1984 ; Clarke et al ., 1988 ; Lowy and Willumsen, 1989 ; Hancock et al ., 1989) . ras-related proteins must serve fundamental roles within cells, because most members of this large family are extremely conserved in evolution . For instance, the yeast proteins Yptlp, Raslp, and Rholp all have counterparts in mammalian cells that are approximately 70% identical . The specific functions of ras-related proteins are largely unknown. However, members of the ras subfamily are involved in control of cell growth, and in the yeast Saccharomyces cerevisiae this function is achieved by stimulation of adenylate cyclase (Kataoka et al ., 1984 ; Broek et al ., 1985) . Increasing evidence indicates that members of the ypt family are involved in several steps of intracellular transport (Balch, 1989) . At least three rho proteins exist in human cells, rhoA, rhoB, and rhoC, which are more than 90 % identical to each other (Madaule and Axel, 1985 ; Yeramian et al ., 1987; Chardin et al ., 1988) . S. cerevisiae also contains rho genes ; RHO] is an essential gene that codes for Rholp, the single yeast counterpart of the human rhoA, rhoB and rhoC proteins, RHO2 codes for a nonessential protein 53 % identical to Rholp (Madaule et al ., 1987), and CDC42 codes for an essential protein 41 % identical to Rholp (Johnson and Prin309 on A ril 9, 2017 D ow nladed fom Published October 15, 1991