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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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Characterization of  pcs1td. (A) Growth at different temperatures of yeast  strains containing PCS1 or  pcs1td. Each panel represents  duplicate patches of the following yeast strains: (a)  Wx257-5c (PCS1), (b)  YHM11.2 (pcs1td), (c)  YHM13.1 (PCS1, ubr1Δ:: HIS3), and (d) YHM12.1  (pcs1td, ubr1Δ::HIS3). (B)  Percentages of unbudded,  small-budded, and large-budded cells in asynchronous  PCS1 and pcs1td strains before and after shift to 37°C.  (C and D) Percentages of cell  types in synchronous PCS1  and pcs1td strains. A shift  from 23° to 37°C occurred at  time 0.
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Figure 4: Characterization of pcs1td. (A) Growth at different temperatures of yeast strains containing PCS1 or pcs1td. Each panel represents duplicate patches of the following yeast strains: (a) Wx257-5c (PCS1), (b) YHM11.2 (pcs1td), (c) YHM13.1 (PCS1, ubr1Δ:: HIS3), and (d) YHM12.1 (pcs1td, ubr1Δ::HIS3). (B) Percentages of unbudded, small-budded, and large-budded cells in asynchronous PCS1 and pcs1td strains before and after shift to 37°C. (C and D) Percentages of cell types in synchronous PCS1 and pcs1td strains. A shift from 23° to 37°C occurred at time 0.

Mentions: To obtain a conditional allele that could be used for more detailed phenotypic analysis, we constructed a haploid strain, designated YHM11.2, in which the chromosomal PCS1 gene was replaced with an allele (pcs1td) that encodes a degron–Pcs1p fusion (see Materials and Methods). The degron unit is a heat-inducible degradation signal (21) that at high temperature (37°C) is degraded, together with sequences fused downstream of it, by the N-end rule pathway of protein degradation (3). Strain YHM11.2 was viable at 23° and 30°C (Fig. 4 A, patch b), but was inviable at 36° and 37°C. Strain Wx257-5c, the wild-type strain from which YHM11.2 is derived, was viable at all temperatures tested (Fig. 4 A, patch a). Thus, pcs1td is a temperaturesensitive allele of PCS1. We also confirmed that the pcs1td mutation is recessive, as expected for an allele designed to encode a protein that is degraded rapidly: pcs1td/PCS1 diploid cells grew normally at 37°C (data not shown), and the mutant phenotype caused by pcs1td can be complemented by pHM49 (see above).


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

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

Characterization of  pcs1td. (A) Growth at different temperatures of yeast  strains containing PCS1 or  pcs1td. Each panel represents  duplicate patches of the following yeast strains: (a)  Wx257-5c (PCS1), (b)  YHM11.2 (pcs1td), (c)  YHM13.1 (PCS1, ubr1Δ:: HIS3), and (d) YHM12.1  (pcs1td, ubr1Δ::HIS3). (B)  Percentages of unbudded,  small-budded, and large-budded cells in asynchronous  PCS1 and pcs1td strains before and after shift to 37°C.  (C and D) Percentages of cell  types in synchronous PCS1  and pcs1td strains. A shift  from 23° to 37°C occurred at  time 0.
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Figure 4: Characterization of pcs1td. (A) Growth at different temperatures of yeast strains containing PCS1 or pcs1td. Each panel represents duplicate patches of the following yeast strains: (a) Wx257-5c (PCS1), (b) YHM11.2 (pcs1td), (c) YHM13.1 (PCS1, ubr1Δ:: HIS3), and (d) YHM12.1 (pcs1td, ubr1Δ::HIS3). (B) Percentages of unbudded, small-budded, and large-budded cells in asynchronous PCS1 and pcs1td strains before and after shift to 37°C. (C and D) Percentages of cell types in synchronous PCS1 and pcs1td strains. A shift from 23° to 37°C occurred at time 0.
Mentions: To obtain a conditional allele that could be used for more detailed phenotypic analysis, we constructed a haploid strain, designated YHM11.2, in which the chromosomal PCS1 gene was replaced with an allele (pcs1td) that encodes a degron–Pcs1p fusion (see Materials and Methods). The degron unit is a heat-inducible degradation signal (21) that at high temperature (37°C) is degraded, together with sequences fused downstream of it, by the N-end rule pathway of protein degradation (3). Strain YHM11.2 was viable at 23° and 30°C (Fig. 4 A, patch b), but was inviable at 36° and 37°C. Strain Wx257-5c, the wild-type strain from which YHM11.2 is derived, was viable at all temperatures tested (Fig. 4 A, patch a). Thus, pcs1td is a temperaturesensitive allele of PCS1. We also confirmed that the pcs1td mutation is recessive, as expected for an allele designed to encode a protein that is degraded rapidly: pcs1td/PCS1 diploid cells grew normally at 37°C (data not shown), and the mutant phenotype caused by pcs1td can be complemented by pHM49 (see above).

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