QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 30 November 2004
doi: 10.1242/jcs.01575

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Funakoshi, M. Articles by Kobayashi, H. Search for Related Content PubMed PubMed Citation Articles by Funakoshi, M. Articles by Kobayashi, H. Social Bookmarking                         What's this?

Sem1, the yeast ortholog of a human BRCA2-binding protein, is a component of the proteasome regulatory particle that enhances proteasome stability

Minoru Funakoshi^1,3,*, Xia Li^2,*, Irina Velichutina^2,*, Mark Hochstrasser² and Hideki Kobayashi^1,3,

¹ Department of Molecular Biology, Graduate School of Medical Sciences, Kyushu University, Maidashi 3-1-1, Higashi-ku, Fukuoka 812-8582, Japan
² Yale University, Department of Molecular Biophysics and Biochemistry, 266 Whitney Avenue, New Haven, CT 06520-8114, USA
³ CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

View larger version (34K): [in a new window]   Fig. 1. Identification of sem1 mutants in Saccharomyces cerevisiae. (A) Isolation of sem1 mutants. Two sem1 mutants were identified as suppressors of Dsk2-mediated lethality. Both the sem1 point mutants and sem1 were temperature-sensitive for growth. (B) Mutation sites of sem1 mutant alleles. Mutated sites (sem1-60 and sem1-64) are indicated in italics (60W and 64W). (C) Loss of Sem1 protein in sem1 mutants. Sem1 protein was detected in cell extracts by immunoblotting with anti-Sem1 antibody (top); Cdc28 was detected (bottom) as a loading control.

View larger version (53K): [in a new window]   Fig. 2. Mutation in SEM1 causes defects in proteasome function. (A) Genetic interactions between sem1 and proteasome mutations. Synthetic lethality of sem1 with rpn1-821 at 34°C (top) and with pre2-75 at 34°C (bottom) is shown. (B) Suppression of Dsk2-mediated growth arrest by disruption of PRE9. pGAL1-DSK2 was introduced into wild-type, pre9 or congenic sem1 cells. The disruption of PRE9, SEM1 or both genes did not affect the interaction of Dsk2 with the 26S proteasome (data not shown). (C) Accumulation of polyubiquitinated proteins in sem1 mutants. Cell extracts were subjected to immunoblot analysis with anti-polyubiquitin antibody (top) or with anti-Cdc28 antibody (bottom) as a loading control. (D) Defective protein degradation in sem1 cells. The rapidly degraded model substrate Leu-ß-gal and the stable Ala-ß-gal control proteins were expressed as ubiquitin fusions from galactose-induced genes. Transcription was blocked with glucose (0 minute), and proteins were followed for 80 minutes by anti-ß-gal immunoblotting.

View larger version (41K): [in a new window]   Fig. 5. The sem1 rpn10 double mutant has severe defects in RP lid integrity and DNA damage resistance. (A) Synthetic lethal interactions between sem1 and rpn10 at 36°C. (B) Disruption of the lid and dissociation of the lid from the base in sem1 rpn10 cells. Superose 6 fractions were subjected to immunoblot analysis with antibodies to the indicated proteins. (C) CP activity of the same column fractions used in B. (D) Growth defects of mutants exposed to canavanine, UV irradiation or hydroxyurea (HU). Cells were spotted onto plates in fivefold serial dilutions and were subjected to the indicated treatments at 30°C.

View larger version (36K): [in a new window]   Fig. 3. Sem1 is a component of the RP lid. (A) Sem1 is tightly associated with the RP. Endogenous PRE1 (CP) or RPT1 (RP) genes were replaced with alleles that expressed the corresponding proteins with Flag-His6 tags. Extracts from yeast expressing Flag-His6-Pre1 or Flag-His6-Rpt1 were precipitated with anti-Flag antibody, followed by immunoblotting with antibodies to the indicated antigens. Inputs (lanes 1-6) represent 15% (for -Sem1) or 45% (for -Rpt1, -20S and -Flag) of the amounts used for precipitation (lanes 7-12). (B) Co-elution of Sem1 in proteasome-containing gel-filtration-column fractions. Anti-Sem1 (top), anti-Rpt6 (middle), and anti-CP subunit (MCP231) plus anti-HA antibodies (bottom) were used for immunoblotting. Size standards are shown at the bottom. (C) Sem1 is tightly associated with the lid. The lid and base were salt eluted from RPs immobilized with protein-A tags on either the base (lanes 1-4) or the lid (lanes 5-8), followed by immunoblotting with antibodies as indicated. Proteins run on the gel were bound to the IgG resin prior to batch salt washes (lanes 1, 5); eluted with 0.3 M NaCl (lanes 2, 6); eluted with 1 M NaCl (lanes 3, 7); or remained after salt washes (lanes 4, 8). Positions of molecular weight standards are indicated on the left.

View larger version (49K): [in a new window]   Fig. 4. Sem1 contributes to RP stability. (A) Immunoprecipitation analysis of proteasomes. Flag-His6-Pre1 was expressed in YPH499 or sem1 cells. Extracts were prepared after cells were incubated at 37°C for 6 hours, and were immunoblotted with the indicated antibodies (lanes 1-3). In lanes 4-6, anti-Flag immunoprecipitation was followed by immunoblotting. Inputs (lanes 1-3) were 1/3 to 1/2 of the amounts used in lanes 4-6. (B) Proteasome stability in sem1 cells. Cell extracts at 30°C were resolved by nondenaturing PAGE either directly (lanes 1, 2) or following affinity purification of Flag-Pre1-containing proteasomes (lanes 3-6). CP-containing species were detected by an in-gel peptidase assay using the Suc-LLVY-AMC fluorogenic substrate and UV illumination of the gel. Extracts from cells grown at 37°C showed a similar pattern. (C) Glycerol gradient analysis of sem1 proteasomes. Glycerol gradient fractions were evaluated for the proteasomes by immunoblot analysis with antibodies against the indicated proteins (top) and by CP peptidase activity assays (bottom). The anti-CP antibody was MCP231, which recognizes multiple subunits. Asterisk indicates cross-reacting protein.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter