Low levels of Ypt protein prenylation cause vesicle polarization defects and thermosensitive growth that can be suppressed by genes involved in cell wall maintenance.

Abstract

The Rab/Ypt small G proteins are essential for intracellular vesicle trafficking in mammals and yeast. The vesicle-docking process requires that Ypt proteins are located in the vesicle membrane. C-terminal geranylgeranyl anchors mediate the membrane attachment of these proteins. The Rab escort protein (REP) is essential for the recognition of Rab/Ypt small G proteins by geranylgeranyltransferase II (GGTase II) and for their delivery to acceptor membranes. What effect an alteration in the levels of prenylated Rab/Ypt proteins has on vesicle transport or other cellular processes is so far unknown. Here, we report the characterization of a yeast REP mutant, mrs6-2, in which reduced prenylation of Ypt proteins occurs even at the permissive temperature. A shift to the restrictive temperature does not alter exponential growth during the first 3 h. The amount of Sec4p, but not Ypt1p, bound to vesicle membranes is reduced 2.5 h after the shift compared with wild-type or mrs6-2 cells incubated at 25 degrees C. In addition, vesicles fail to be polarized towards the bud and small budded binucleate cells accumulate at this time point. Growth in 1 M sorbitol or overexpression of MLC1, encoding a myosin light chain able to bind the unconventional type V myosin Myo2, or of genes involved in cell wall maintenance, such as SLG1, GFA1 and LRE1, suppresses mrs6-2 thermosensitivity. Our data suggest that, at least at high temperature, a critical minimal level of Ypt protein prenylation is required for maintaining vesicle polarization.