Structural and Functional Analysis of a Novel Coiled-Coil Protein Involved in Ypt6 GTPase-regulated Protein Transport in Yeast

Abstract

The yeast transport GTPase Ypt6p is dispensable for cell growth and secretion, but its lack results in temperature sensitivity and missorting of vacuolar carboxypeptidase Y. We previously identified four yeast genes (SYS1, 2, 3, and 5) that on high expression suppressed these phenotypic alterations. SYS3 encodes a 105-kDa protein with a predicted high ␣-helical content. It is related to a variety of mammalian Golgi-associated proteins and to the yeast Uso1p, an essential protein involved in docking of endoplasmic reticulum-derived vesicles to the cis-Golgi. Like Uso1p, Sys3p is predominatly cytosolic. According to gel chromatographic, two-hybrid, and chemical cross-linking analyses, Sys3p forms dimers and larger protein complexes. Its loss of function results in partial missorting of carboxypeptidase Y. Double disruptions of SYS3 and YPT6 lead to a significant growth inhibition of the mutant cells, to a massive accumulation of 40-to 50-nm vesicles, to an aggravation of vacuolar protein missorting, and to a defect in ␣-pheromone processing apparently attributable to a perturbation of protease Kex2p cycling between the Golgi and a post-Golgi compartment. The results of this study suggest that Sys3p, like Ypt6p, acts in vesicular transport (presumably at a vesicledocking stage) between an endosomal compartment and the most distal Golgi compartment.

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

“…Although both deletions by themselves, or ypt6-2 alone, allowed cell growth at 25, 30, and 35°C, the double mutants did not grow at 35°C and were completely (ypt6-2/vps35⌬) or severely (ypt6-2/tlg2⌬) growth-inhibited at 30°C. As the syntaxin Tlg2p and the Vps35 protein are components of the machinery of endosome-to-Golgi trafficking, these results are in line with the conclusions drawn from previous studies (7)(8)(9)(10)13) indicating that Ypt6p has a function also in recycling between endosomes and the TGN.…”

“…Therefore, previous studies (7,8,11,12) on the functional role of Ypt6p were performed with ypt6 deletion or truncation mutants which are, however, sensitive to growth at elevated temperature. Since mutants lacking a given protein are sometimes able to adapt to such a situation by activating a by-path for example, we sought to investigate Ypt6p function in conditional mutants.…”

“…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.…”

“…The divergent views on Ypt6p function were based on the results of the initial studies (7,8,11,12) using ypt6 deletion or truncation mutants. 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).…”