Impaired Proteasome Function Rescues Thermosensitivity of Yeast Cells Lacking the Coatomer Subunit ε-COP*
- From the ‡Research and Education Center for Genetic Information, Nara Institute of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101 and §CREST, Japan Science and Technology Corporation, Tokyo 101-0062, Japan
Abstract
Formation of COPI-coated transport vesicles requires a cytosolic protein complex consisting of seven subunits: α-, β-, β′-, γ-, δ-, ε- and ζ-COP, collectively designated coatomer. The yeast Saccharomyces cerevisiaegene encoding the ε-COP subunit is known asSEC28/ANU2. anu2 null mutant cells (anu2Δ) are temperature-sensitive, and α-COP is rapidly degraded in these cells when they are shifted to the restrictive temperature. We isolated extragenic suppressors that rescue the temperature-sensitive growth defect of anu2Δ cells. Genetic analysis revealed that one of the suppressors is allelic toPRE8 (PRS4), which encodes a 20 S proteasome subunit. In the presence of a proteasome inhibitor, MG132,anu2Δ cells did not cease growth even at the restrictive temperature. Furthermore, MG132 inhibited the rapid decrease of α-COP levels in anu2Δ cells shifted to the restrictive temperature. However, secretion of certain proteins by these cells was impaired even in the presence of MG132. In conclusion, impairment of proteasome-dependent proteolysis rescued some, but not all, temperature-sensitive defects of anu2Δ cells. These results are discussed in terms of evidence that ε-COP plays a critical role in maintaining the structural integrity of α-COP.
Footnotes
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↵* This work was supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan, the Sasakawa Scientific Research Grant from the Japan Science Society, and Sapporo Bioscience Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵¶ To whom correspondence should be addressed: Research and Education Center for Genetic Information, Nara Inst. of Science and Technology, 8916-5 Takayama, Ikoma, Nara 630-0101, Japan. Tel.: 81-743-72-5640; Fax: 81-743-72-5649; E-mail: kkouno@bs.aist-nara.ac.jp.
- Abbreviations:
- COP(s)
-
coat protein(s)
- ER
-
endoplasmic reticulum
- CPY
-
carboxypeptidase Y
- NLS
-
nuclear localization sequence
- PAGE
-
polyacrylamide gel electrophoresis
- Leu-β-gal
-
leucine-β-galactosidase
- NLS-β-gal
-
SV40 T antigen NLS-tagged β-galactosidase
- SP
-
secretory protein
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- Received December 28, 1999.
- The American Society for Biochemistry and Molecular Biology, Inc.
