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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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Electron micrographs of the YHM11.2 (pcs1td) mutant after transfer to 37°C. A normal mitotic spindle was seen in 16% of the  cells analyzed, as shown in A. In this cell, the other SPB was located in a different section (not shown). The arrow indicates the halfbridge structure adjacent to the SPB. Pairs of images (B, B′ and C, C′) represent adjacent serial sections through the poles of monopolar  spindles, as were seen in 84% of arrested cells. Bar, 0.5 μm.
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Figure 9: Electron micrographs of the YHM11.2 (pcs1td) mutant after transfer to 37°C. A normal mitotic spindle was seen in 16% of the cells analyzed, as shown in A. In this cell, the other SPB was located in a different section (not shown). The arrow indicates the halfbridge structure adjacent to the SPB. Pairs of images (B, B′ and C, C′) represent adjacent serial sections through the poles of monopolar spindles, as were seen in 84% of arrested cells. Bar, 0.5 μm.

Mentions: To learn more about how loss of PCS1 function perturbs spindle formation, we used serial section EM to examine cells from strain YHM11.2 (pcs1td) held at 37°C for 9 h. We found that the majority of cells (21/25 examined) had failed to duplicate their SPBs and therefore possessed only a monopolar spindle. The remaining cells (4/25) had duplicated their SPBs and each contained a bipolar spindle. Fig. 9 A shows one pole of a bipolar spindle from one of these latter cells. The SPB seen here is indistinguishable from that of wild type: its half-bridge (arrow), the darkly staining segment of the nuclear envelope immediately adjacent to the SPB, has the same appearance as that which is normally seen in wild-type cells (7). Fig. 9, B, B′ and C, C′, show (in adjacent sections) the single SPBs from two cells containing monopolar spindles. These SPBs are larger than normal, as has been observed for other mutants that fail to duplicate their SPBs (e.g., 8, 101). Normal half-bridge structures were not seen in any of the cells (n = 21) containing unduplicated SPBs; instead, most cells examined contained an unusual type of structure in one of the adjacent sections in the series. This structure, shown in Fig. 9, B′ and C′, appears to represent an aberrant half-bridge that is unusually curved in profile and is wrapped partly around the SPB.


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

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

Electron micrographs of the YHM11.2 (pcs1td) mutant after transfer to 37°C. A normal mitotic spindle was seen in 16% of the  cells analyzed, as shown in A. In this cell, the other SPB was located in a different section (not shown). The arrow indicates the halfbridge structure adjacent to the SPB. Pairs of images (B, B′ and C, C′) represent adjacent serial sections through the poles of monopolar  spindles, as were seen in 84% of arrested cells. Bar, 0.5 μm.
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Related In: Results  -  Collection

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Figure 9: Electron micrographs of the YHM11.2 (pcs1td) mutant after transfer to 37°C. A normal mitotic spindle was seen in 16% of the cells analyzed, as shown in A. In this cell, the other SPB was located in a different section (not shown). The arrow indicates the halfbridge structure adjacent to the SPB. Pairs of images (B, B′ and C, C′) represent adjacent serial sections through the poles of monopolar spindles, as were seen in 84% of arrested cells. Bar, 0.5 μm.
Mentions: To learn more about how loss of PCS1 function perturbs spindle formation, we used serial section EM to examine cells from strain YHM11.2 (pcs1td) held at 37°C for 9 h. We found that the majority of cells (21/25 examined) had failed to duplicate their SPBs and therefore possessed only a monopolar spindle. The remaining cells (4/25) had duplicated their SPBs and each contained a bipolar spindle. Fig. 9 A shows one pole of a bipolar spindle from one of these latter cells. The SPB seen here is indistinguishable from that of wild type: its half-bridge (arrow), the darkly staining segment of the nuclear envelope immediately adjacent to the SPB, has the same appearance as that which is normally seen in wild-type cells (7). Fig. 9, B, B′ and C, C′, show (in adjacent sections) the single SPBs from two cells containing monopolar spindles. These SPBs are larger than normal, as has been observed for other mutants that fail to duplicate their SPBs (e.g., 8, 101). Normal half-bridge structures were not seen in any of the cells (n = 21) containing unduplicated SPBs; instead, most cells examined contained an unusual type of structure in one of the adjacent sections in the series. This structure, shown in Fig. 9, B′ and C′, appears to represent an aberrant half-bridge that is unusually curved in profile and is wrapped partly around the SPB.

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