Essential roles of class E Vps proteins for sorting into multivesicular bodies in Schizosaccharomyces pombe

Tomoko Iwaki,Masayuki Onishi,Masaru Ikeuchi,Yasuhisa Fukui,Reiko Sugiura,Yuko Giga-Hama,Ayako Kita,Kaoru Takegawa

doi:10.1099/mic.0.2007/006072-0

Abstract

The multivesicular body (MVB) sorting pathway is required for a number of biological processes, including downregulation of cell-surface proteins and protein sorting into the vacuolar lumen. The function of this pathway requires endosomal sorting complexes required for transport (ESCRT) composed of class E vacuolar protein sorting (Vps) proteins in Saccharomyces cerevisiae, many of which are conserved in Schizosaccharomyces pombe. Of these, sst4/vps27 (homologous to VPS27) and sst6 (similar to VPS23) have been identified as suppressors of sterility in ste12Δ (sst), although their functions have not been uncovered to date. In this report, these two sst genes are shown to be required for vacuolar sorting of carboxypeptidase Y (CPY) and an MVB marker, the ubiquitin–GFP–carboxypeptidase S (Ub–GFP–CPS) fusion protein, despite the lack of the ubiquitin E2 variant domain in Sst6p. Disruption mutants of a variety of other class E vps homologues also had defects in sorting of CPY and Ub–GFP–CPS. Sch. pombe has a mammalian AMSH homologue, sst2. Phenotypic analyses suggested that Sst2p is a class E Vps protein. Taken together, these results suggest that sorting into multivesicular bodies is dependent on class E Vps proteins, including Sst2p, in Sch. pombe.

Highlights

Genetic selections in Saccharomyces cerevisiae have resulted in the isolation of a large number of vps mutants defective in the delivery of proteins to lysosome-like vacuoles
The ubiquitin-interacting motif (UIM) domain was determined based on sequences identified in the subunit of the 26S proteasome that interacts with polyubiquitin (Young et al, 1998)
We found that sorting defects in the sst4/vps27D and sst6D Sch. pombe mutants are relatively modest; 30 % or less of carboxypeptidase Y (CPY) was not processed to the mature form (Fig. 2)

Summary

Introduction

Genetic selections in Saccharomyces cerevisiae have resulted in the isolation of a large number of vps mutants defective in the delivery of proteins to lysosome-like vacuoles. These define more than 60 complementation groups and have been categorized into six classes (A–F) with respect to their morphology, vacuolar protein sorting and acidification defects (Raymond et al, 1992; Banta et al, 1988). Class E vps mutants fail to transport newly synthesized hydrolases

Methods

Results

Conclusion