N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe
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In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.
KeywordsSchizosaccharomyces pombe ERAD Carboxypeptidase Y Protein degradation
This work was partly supported by the Project for Development of a Technological Infrastructure for Industrial Bioprocesses on R & D of New Industrial Science and Technology Frontiers by the Ministry of Economy, Trade & Industry, as supported by the New Energy and Industrial Technology Development Organization.
- Bordallo J, Plemper RK, Finger A, Wolf DH (1998) Der3p/Hrd1p is required for endoplasmic reticulum-associated degradation of misfolded lumenal and integral membrane proteins. Mol Biol Cell 9:209–222Google Scholar
- Broker M, Ragg H, Karges HE (1987) Expression of human antithrombin III in Saccharomyces cerevisiae and Schizosaccharomyces pombe. Biochim Bophys Acta 908:203–213Google Scholar
- Fernandez F, Jannatipour M, Hellman U, Rokeach LA, Parodi AJ (1996) A new stress protein: synthesis of Schizosaccharomyces pombe UDP-GIc:glycoprotein glucosyltransferase mRNA is induced by stress conditions but the enzyme is not essential for cell viability. EMBO J 15:705–713Google Scholar
- Giga-Hama Y, Kumagai H (1999) Expression system for foreign genes using the fission yeast Schizosaccharomyces pombe. Biotechnol Appl Biochem 30:235–244Google Scholar
- Hirao K, Natsuka Y, Tamura T, Wada I, Morito D, Natsuka S, Romero P, Sleno B, Tremblay LO, Herscovics A, Nagata K, Hosokawa N (2006) EDEM3, a soluble EDEM homolog, enhances glycoprotein endoplasmic reticulum-associated degradation and mannose trimming. J Biol Chem 281:9650–9658CrossRefGoogle Scholar
- Huyer G, Piluek WF, Fansler Z, Kreft SG, Hochstrasser M, Brodsky JL, Michaelis S (2004) Distinct machinery is required in Saccharomyces cerevisiae for the endoplasmic reticulum-associated degradation of a multispanning membrane protein and a soluble luminal protein. J Biol Chem 279:38369–38378CrossRefGoogle Scholar
- Knop M, Finger A, Braun T, Hellmuth K, Wolf DH (1996a) Der1, a novel protein specifically required for endoplasmic reticulum degradation in yeast. EMBO J 15:753–763Google Scholar
- Nakamura T, Nakamura-Kubo M, Hirata A, Shimoda C (2001) The Schizosaccharomyces pombe spo3+ gene is required for assembly of the forespore membrane and genetically interacts with psy1(+)-encoding syntaxin-like protein. Mol Biol Cell 12:3955–3972Google Scholar
- Sagt CM, Muller WH, van der Heide L, Boonstra J, Verkleij AJ, Verrips CT (2002) Impaired cutinase secretion in Saccharomyces cerevisiae induces irregular endoplasmic reticulum (ER) membrane proliferation, oxidative stress, and ER-associated degradation. Appl Environ Microbiol 68:2155–2160CrossRefGoogle Scholar
- Tabuchi M, Iwaihara O, Ohtani Y, Ohuchi N, Sakurai J, Morita T, Iwahara S, Takegawa K (1997) Vacuolar protein sorting in fission yeast: cloning, biosynthesis, transport, and processing of carboxypeptidase Y from Schizosaccharomyces pombe. J Bacteriol 179:4179–4189Google Scholar