E3 ubiquitin ligase Pub1 is implicated in endocytosis of a GPI-anchored protein Ecm33 in fission yeast.

We previously identified three glycosylphosphatidylinositol (GPI)-anchored proteins including Ecm33, as multicopy suppressors of the phenotypes of a mutant allele of cis4(+) that encodes a zinc transporter in fission yeast. Here, we further identified two multicopy suppressor genes, ubi1 (+) and ubc4 (+), encoding ubiquitin-ribosomal fusion protein and ubiquitin conjugating enzyme E2, respectively. In addition, Ubi1 or Ubc4 overexpression failed to suppress the phenotypes of the double deletion of cis4 (+) and pub1 (+) gene, which encodes a HECT-type ubiquitin ligase E3. During exponential phase GFP-Ecm33 localized at the growing cell tips of the cell surface and the medial region in wild-type cells. Notably, during the post-exponential and stationary phase, GFP-Ecm33 in wild-type cells was internalized and mostly localized to the Golgi/endosomes, but it was still stably localized at the cell surface in Δpub1 cells. The Δpub1 cells showed osomoremedial phenotypes to various drugs indicating their defects in cell wall integrity. Altogether, our findings reveal a novel role for Pub1 in endocytosis of Ecm33 and regulation of cell wall integrity in fission yeast.