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A proteasome cap subunit required for spindle pole body duplication in yeast.

McDonald HB, Byers B - J. Cell Biol. (1997)

Bottom Line: Biol.EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants.We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Washington, Seattle 98195, USA.

ABSTRACT
Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810-32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1-green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.

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GFP–Pcs1p expression and localization. (A) Western  blot analysis of whole-cell extracts probed with anti-GFP antiserum. (Lane 1) Wx257-5c (PCS1). (Lane 2) YHM10.1.54 (GFP– PCS1). The presumptive GFP–Pcs1p fusion is indicated by the  arrow, and size markers are in kD. (B and C) Fluorescence microscopy of YHM10.1.54 cells. (B) GFP (FITC channel). (1) Unbudded cell; (2) small-budded cell; (3 and 4) large-budded cells.  (Asterisk) Less typical (∼5% of cells) appearance of GFP signal  that is not coincident with nuclear DNA staining. (C) DNA  (DAPI). Bar, 5 μm.
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Figure 10: GFP–Pcs1p expression and localization. (A) Western blot analysis of whole-cell extracts probed with anti-GFP antiserum. (Lane 1) Wx257-5c (PCS1). (Lane 2) YHM10.1.54 (GFP– PCS1). The presumptive GFP–Pcs1p fusion is indicated by the arrow, and size markers are in kD. (B and C) Fluorescence microscopy of YHM10.1.54 cells. (B) GFP (FITC channel). (1) Unbudded cell; (2) small-budded cell; (3 and 4) large-budded cells. (Asterisk) Less typical (∼5% of cells) appearance of GFP signal that is not coincident with nuclear DNA staining. (C) DNA (DAPI). Bar, 5 μm.

Mentions: The GFP–Pcs1p fusion is predicted to have a molecular mass of 76 kD. To confirm the presence of this product in strain YHM10.1.54, equivalent amounts of whole-cell extracts from strains YHM10.1.54 and Wx257-5c (wild-type control) were analyzed by Western blot (Fig. 10 A). AntiGFP antiserum recognized a band of approximately the correct size in YHM10.1.54 extract (Fig. 10 A, lane 2) but not in the wild-type control (lane 1).


A proteasome cap subunit required for spindle pole body duplication in yeast.

McDonald HB, Byers B - J. Cell Biol. (1997)

GFP–Pcs1p expression and localization. (A) Western  blot analysis of whole-cell extracts probed with anti-GFP antiserum. (Lane 1) Wx257-5c (PCS1). (Lane 2) YHM10.1.54 (GFP– PCS1). The presumptive GFP–Pcs1p fusion is indicated by the  arrow, and size markers are in kD. (B and C) Fluorescence microscopy of YHM10.1.54 cells. (B) GFP (FITC channel). (1) Unbudded cell; (2) small-budded cell; (3 and 4) large-budded cells.  (Asterisk) Less typical (∼5% of cells) appearance of GFP signal  that is not coincident with nuclear DNA staining. (C) DNA  (DAPI). Bar, 5 μm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139890&req=5

Figure 10: GFP–Pcs1p expression and localization. (A) Western blot analysis of whole-cell extracts probed with anti-GFP antiserum. (Lane 1) Wx257-5c (PCS1). (Lane 2) YHM10.1.54 (GFP– PCS1). The presumptive GFP–Pcs1p fusion is indicated by the arrow, and size markers are in kD. (B and C) Fluorescence microscopy of YHM10.1.54 cells. (B) GFP (FITC channel). (1) Unbudded cell; (2) small-budded cell; (3 and 4) large-budded cells. (Asterisk) Less typical (∼5% of cells) appearance of GFP signal that is not coincident with nuclear DNA staining. (C) DNA (DAPI). Bar, 5 μm.
Mentions: The GFP–Pcs1p fusion is predicted to have a molecular mass of 76 kD. To confirm the presence of this product in strain YHM10.1.54, equivalent amounts of whole-cell extracts from strains YHM10.1.54 and Wx257-5c (wild-type control) were analyzed by Western blot (Fig. 10 A). AntiGFP antiserum recognized a band of approximately the correct size in YHM10.1.54 extract (Fig. 10 A, lane 2) but not in the wild-type control (lane 1).

Bottom Line: Biol.EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants.We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, University of Washington, Seattle 98195, USA.

ABSTRACT
Proteasome-mediated protein degradation is a key regulatory mechanism in a diversity of complex processes, including the control of cell cycle progression. The selection of substrates for degradation clearly depends on the specificity of ubiquitination mechanisms, but further regulation may occur within the proteasomal 19S cap complexes, which attach to the ends of the 20S proteolytic core and are thought to control entry of substrates into the core. We have characterized a gene from Saccharomyces cerevisiae that displays extensive sequence similarity to members of a family of ATPases that are components of the 19S complex, including human subunit p42 and S. cerevisiae SUG1/CIM3 and CIM5 products. This gene, termed PCS1 (for proteasomal cap subunit), is identical to the recently described SUG2 gene (Russell, S.J., U.G. Sathyanarayana, and S.A. Johnston. 1996. J. Biol. Chem. 271:32810-32817). We have shown that PCS1 function is essential for viability. A temperature-sensitive pcs1 strain arrests principally in the second cycle after transfer to the restrictive temperature, blocking as large-budded cells with a G2 content of unsegregated DNA. EM reveals that each arrested pcs1 cell has failed to duplicate its spindle pole body (SPB), which becomes enlarged as in other monopolar mutants. Additionally, we have shown localization of a functional Pcs1-green fluorescent protein fusion to the nucleus throughout the cell cycle. We hypothesize that Pcs1p plays a role in the degradation of certain potentially nuclear component(s) in a manner that specifically is required for SPB duplication.

Show MeSH
Related in: MedlinePlus