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

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

Atf1 Is a Target of the Mitogen-activated Protein Kinase Pmk1 and Regulates Cell Integrity in Fission Yeast

In fission yeast, knockout of the calcineurin gene resulted in hypersensitivity to Cl−, and the overexpression of pmp1+ encoding a dual-specificity phosphatase for Pmk1 mitogen-activated protein kinase (MAPK) or the knockout of the components of the Pmk1 pathway complemented the Cl− hypersensitivity of calcineurin deletion. Here, we showed that the overexpression of ptc1+ and ptc3+, both encoding type 2C protein phosphatase (PP2C), previously known to inactivate the Wis1–Spc1–Atf1 stress-activated MAPK signaling pathway, suppressed the Cl− hypersensitivity of calcineurin deletion. We also demonstrated that the mRNA levels of these two PP2Cs and pyp2+, another negative regulator of Spc1, are dependent on Pmk1. Notably, the deletion of Atf1, but not that of Spc1, displayed hypersensitivity to the cell wall-damaging agents and also suppressed the Cl− hypersensitivity of calcineurin deletion, both of which are characteristic phenotypes shared by the mutation of the components of the Pmk1 MAPK pathway. Moreover, micafungin treatment induced Pmk1 hyperactivation that resulted in Atf1 hyperphosphorylation. Together, our results suggest that PP2C is involved in a negative feedback loop of the Pmk1 signaling, and results also demonstrate that Atf1 is a key component of the cell integrity signaling downstream of Pmk1 MAPK