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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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Spindle morphology in (A) YHM11.2 (pcs1td), (B) CMY763 (cim3-1), and (C) CMY765 (cim5-1) strains at 37°C. (Bars) Percentages of large-budded cells with the indicated spindle forms.
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Figure 8: Spindle morphology in (A) YHM11.2 (pcs1td), (B) CMY763 (cim3-1), and (C) CMY765 (cim5-1) strains at 37°C. (Bars) Percentages of large-budded cells with the indicated spindle forms.

Mentions: Given our finding that the pcs1td cells arrest more quickly if held in stationary phase before a shift to the restrictive temperature, we also examined the microtubule arrays of cells treated in this manner at various times after the temperature shift. Since mutations in the closely related SUG1/CIM3 and CIM5 genes have previously been reported to cause cells to arrest with short spindles (31), we also wished to determine whether the phenotypes of pcs1td, cim3-1, and cim5-1 mutants were indeed distinct when the same conditions were used for the experiment. Accordingly, strains YHM11.2 (pcs1td), CMY763 (cim3-1), and CMY765 (cim5-1) (each congenic with the S288C background) were held at stationary phase for 3 d at 23°C, and then diluted into fresh media at 37°C. At the time of the temperature shift, no abnormal spindles were observed in any of the strains. Fig. 8 shows the percentage of largebudded cells containing short spindles, long spindles, or no spindles in each of the strains at different times after the temperature shift. While most large-budded pcs1td cells were observed to contain no spindles at 4 and 6 h after the shift, most large-budded cim3-1 and cim5-1 cells contained short spindles at these time points. Incidentally, we also observed cytoplasmic microtubule arrays in the cim3-1 cells containing short spindles that were broader and more elongated than those seen in wild-type or cim5-1 cells; this characteristic of cim3-1 is also apparent in the figure from the original paper describing these mutants (31). More strikingly, these data demonstrate that loss of PCS1 function causes an arrest that differs distinctly from that caused by loss of CIM3 or CIM5 function.


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

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

Spindle morphology in (A) YHM11.2 (pcs1td), (B) CMY763 (cim3-1), and (C) CMY765 (cim5-1) strains at 37°C. (Bars) Percentages of large-budded cells with the indicated spindle forms.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2139890&req=5

Figure 8: Spindle morphology in (A) YHM11.2 (pcs1td), (B) CMY763 (cim3-1), and (C) CMY765 (cim5-1) strains at 37°C. (Bars) Percentages of large-budded cells with the indicated spindle forms.
Mentions: Given our finding that the pcs1td cells arrest more quickly if held in stationary phase before a shift to the restrictive temperature, we also examined the microtubule arrays of cells treated in this manner at various times after the temperature shift. Since mutations in the closely related SUG1/CIM3 and CIM5 genes have previously been reported to cause cells to arrest with short spindles (31), we also wished to determine whether the phenotypes of pcs1td, cim3-1, and cim5-1 mutants were indeed distinct when the same conditions were used for the experiment. Accordingly, strains YHM11.2 (pcs1td), CMY763 (cim3-1), and CMY765 (cim5-1) (each congenic with the S288C background) were held at stationary phase for 3 d at 23°C, and then diluted into fresh media at 37°C. At the time of the temperature shift, no abnormal spindles were observed in any of the strains. Fig. 8 shows the percentage of largebudded cells containing short spindles, long spindles, or no spindles in each of the strains at different times after the temperature shift. While most large-budded pcs1td cells were observed to contain no spindles at 4 and 6 h after the shift, most large-budded cim3-1 and cim5-1 cells contained short spindles at these time points. Incidentally, we also observed cytoplasmic microtubule arrays in the cim3-1 cells containing short spindles that were broader and more elongated than those seen in wild-type or cim5-1 cells; this characteristic of cim3-1 is also apparent in the figure from the original paper describing these mutants (31). More strikingly, these data demonstrate that loss of PCS1 function causes an arrest that differs distinctly from that caused by loss of CIM3 or CIM5 function.

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