Ric1p and the Ypt6p GTPase Function in a Common Pathway Required for Localization ofTrans-Golgi Network Membrane Proteins

Abstract

In Saccharomyces cerevisiae, clathrin is necessary for localization of trans-Golgi network (TGN) membrane proteins, a process that involves cycling of TGN proteins between the TGN and endosomes. To characterize further TGN protein localization, we applied a screen for mutations that cause severe growth defects in combination with a temperature-sensitive clathrin heavy chain. This screen yielded a mutant allele of RIC1. Cells carrying a deletion of RIC1 (ric1⌬) mislocalize TGN membrane proteins Kex2p and Vps10p to the vacuole. Delivery to the vacuole occurs in ric1⌬ cells also harboring end3⌬ to block endocytosis, indicative of a defect in retrieval to the TGN rather than sorting to endosomes. SYS1, originally discovered as a multicopy suppressor of defects caused by the absence of the Rab GTPase YPT6, was identified as a multicopy suppressor of ric1⌬. Further comparison of ric1⌬ and ypt6⌬ cells demonstrated identical phenotypes. Multicopy plasmids expressing v-SNAREs Gos1p or Ykt6p, but not other v-and t-SNAREs, partially suppressed phenotypes of ric1⌬ and ypt6⌬ cells. SLY1-20, a dominant activator of the cis-Golgi network t-SNARE Sed5p, also functioned as a multicopy suppressor. Because Gos1p and Ykt6p interact with Sed5p, these results raise the possibility that TGN membrane protein localization requires Ric1p-and Ypt6p-dependent retrieval to the cis-Golgi network. INTRODUCTIONLocalization of proteins to appropriate membrane organelles is crucial for the functional compartmentalization of eukaryotic cells. For proteins that function in organelles of the secretory and endocytic pathways, localization requires not only targeting to the proper destination but mechanisms to maintain residence despite extensive membrane and protein flux through each organelle. Continued residence can be achieved through retention mechanisms that restrict incorporation into transport vesicles departing from an organelle and/or retrieval mechanisms that carry out vesicle-mediated return from distal sites in the pathway (Pelham and Munro, 1993;Rothman and Wieland, 1996).The Golgi apparatus in the yeast Saccharomyces cerevisiae, like its mammalian counterpart, is organized into dynamic, functionally distinct subcompartments that pose additional challenges for protein localization. Though not arranged into the cisternal stacks characteristic of the mammalian cell Golgi apparatus, yeast Golgi subcompartments can be considered functionally analogous to the mammalian cis-Golgi network (CGN), medial Golgi, and trans-Golgi (TGN) network (Graham and Emr, 1991;Preuss et al., 1992). The CGN serves as the site where endoplasmic reticulum-derived transport carriers dock and fuse and where mannose residues are first added to the core oligosaccharides of glycoproteins (Gaynor et al., 1994;Graham and Emr, 1991). Accordingly, this compartment is enriched for the t-SNARE Sed5p involved in the fusion of ER transport carriers and in the ␣-1,6 mannosyltransferase Och1p (Gaynor et al., 1994;Hardwick and Pelham, 1992). The medial Golgi compartment carries ...

Biochemical and Genetic Evidence for the Involvement of Yeast Ypt6-GTPase in Protein Retrieval to Different Golgi Compartments

“…Previous studies (7,9,12) had shown that overexpression of either Sys proteins, Ypt1p, Gos1p, or Ykt6p suppressed temperature-sensitive growth defects of ypt6 null mutants. In the present study we could show that high intracellular levels of Sys1p and of Ypt1p efficiently rescued also the ypt6-2 mutant from growth inhibition at 37°C (data not shown).…”

“…In the yeast Saccharomyces cerevisiae, this GTPase family has 11 members of which only those that act in forward transport through the secretory pathway (Ypt1p, Sec4p, and the pair of apparently redundant Ypt31/ Ypt32-GTPases) are essential for cell viability (3). Of the nonessential ones, Ypt6p, the homologue of the mammalian Rab6 GTPases, has been implicated in having a role in the retrieval of proteins from endosomes to the trans-Golgi network (7)(8)(9)(10) or in anterograde (11,12) and retrograde (9) Golgi transport.…”

“…Such mutants were found to be growthinhibited at elevated temperature, to partially mis-sort proteins in the trans-Golgi network already at permissive temperature, and to moderately interfere with protein secretion at nonpermissive conditions (7,11). Temperature sensitivity of ypt6 deletion mutants allowed us to isolate multicopy suppressors of which one, SYS1 (7), could also rescue another protein mis-sorting mutant, ric⌬ (9), later shown to be defective in the nucleotide exchange factor for Ypt6p (13). Cells lacking Ypt6p accumulate transport vesicles (8) of which at least a fraction may be derived from endosomes and destined to fuse with late Golgi membranes (10).…”

“…On the other hand, temperature-sensitive growth of ypt6 deletion mutants can be overcome efficiently by raising the intracellular level of Ypt1p (12), the GTPase required for ER 1 -to-Golgi (14) and early Golgi transport (15). Furthermore, partial suppression of growth defects of ypt6 and ric1 null mutants by high expression of a variety of genes that are known to act in either forward or retrograde Golgi transport have led to the assumption that Ypt6p might participate in regulating more than one transport step (9). This would in fact mirror the situation in mammalian cells where the homologue of yeast Ypt6p, Rab6, initially thought to function only in intra-Golgi transport (16), is also required for recycling of proteins between endosomes and the TGN (17).…”

Mutants defective in secretory/vacuolar pathways in the EUROFAN collection of yeast disruptants

“…YPT6 was recently reported to be involved in the retrieval pathway from either the PVC (Bensen et al, 2001) or early endosomes (Siniossoglou et al, 2000) back to the Golgi apparatus. SYS1 encodes a protein with four potential transmembrane segments that has not yet been localized.…”