THE SMALL GTP-BINDING PROTEIN RHO1P IS LOCALIZED ON THE GOLGI-APPARATUS AND POST-GOLGI VESICLES IN SACCHAROMYCES-CEREVISIAE

In Saccharomyces cerevisiae the ras-related protein Rholp is essentially the only target for ADP-ribosylation 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 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.