Plasma Membrane Localization of Ras Requires Class C Vps Proteins and Functional Mitochondria in Saccharomyces cerevisiae

Abstract

Ras proteins are synthesized as cytosolic precursors, but then undergo posttranslational lipid addition, membrane association, and subcellular targeting to the plasma membrane. Although the enzymes responsible for farnesyl and palmitoyl lipid addition have been described, the mechanism by which these modifications contribute to the subcellular localization of Ras is not known. Following addition of the farnesyl group, Ras associates with the endoplasmic reticulum (ER), where palmitoylation occurs in Saccharomyces cerevisiae. The subsequent translocation of Ras from the ER to the plasma membrane does not require the classical secretory pathway or a functional Golgi apparatus. Vesicular and nonvesicular transport pathways for Ras proteins have been proposed, but the pathway is not known. Here we describe a genetic screen designed to identify mutants defective in Ras trafficking in S. cerevisiae. The screen implicates, for the first time, the class C VPS complex in Ras trafficking. Vps proteins are best characterized for their role in endosome and vacuole membrane fusion. However, the role of the class C Vps complex in Ras trafficking is distinct from its role in endosome and vacuole vesicle fusion, as a mitochondrial involvement was uncovered. Disruption of class C VPS genes results in mitochondrial defects and an accumulation of Ras proteins on mitochondrial membranes. Ras also fractionates with mitochondria in wild-type cells, where it is detected on the outer mitochondrial membrane by virtue of its sensitivity to protease treatment. These results point to a previously uncharacterized role of mitochondria in the subcellular trafficking of Ras proteins.

Nuclear translocation of Gln3 in response to nutrient signals requires Golgi-to-endosome trafficking in Saccharomyces cerevisiae

“…However, expression of the retrograde response gene CIT2 was induced in pep3 cells, even in nitrogen-rich medium, confirming earlier observations that vps class C mutants show mitochondrial defects (Fig. 1B) (16).…”

Whi2p links nutritional sensing to actin-dependent Ras-cAMP-PKA regulation and apoptosis in yeast

“…7). Evidence to support a role for vesicle trafficking in the regulation of Ras2p comes from a previous study that convincingly demonstrated that the disruption of the Class C VPS complex leads to mitochondrial localisation of Ras2p and respiratory defects (Wang and Deschenes, 2006). In addition we have recently demonstrated that mutations in the class C VPS machinery also result in the accumulation of ROS, formation of actin aggregates and failure to degrade Ras2p during the diauxic shift in a manner similar to that observed for Δwhi2 cells (data not shown).…”

Ras proteins: paradigms for compartmentalised and isoform-specific signalling

“…In yeast and adipocytes, palmitoylated Ras isoforms can also utilise a Golgi independent pathway to reach the plasma membrane [49,50]. Intriguingly, this pathway in yeast requires class C vps proteins that are classically associated with regulating endosome fusion [51]. The steady state distribution of the isoforms varies; whilst all Ras isoforms exhibit plasma membrane localisation, the extent of endomembranous labelling varies amongst isoforms and between cell types.…”