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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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Sen3 protein localization. (A) MHY851 cells were  stained with anti-HA antibody to visualize the Sen3 protein (FITC).  (B) DNA (DAPI). Bar, 5 μm.
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Figure 11: Sen3 protein localization. (A) MHY851 cells were stained with anti-HA antibody to visualize the Sen3 protein (FITC). (B) DNA (DAPI). Bar, 5 μm.

Mentions: To gain a better understanding of how Pcs1p distribution compares with that of other proteasome subunits, we examined the localization of a different 19S subunit. The Sen3 protein from S. cerevisiae is a component of the proteasome cap, unrelated in sequence to the ATPase subunits (16). To study the localization of this protein, we used MHY851 cells, which express a functional Sen3 protein tagged with three copies of the HA epitope (16). We used anti-HA antibodies to visualize Sen3 protein distribution in these cells and found that it also colocalized with the nuclear DNA throughout the cell cycle (Fig. 11, A and B). No staining was observed in cells expressing untagged Sen3 protein (strain MHY849; data not shown). Similarly, the Sug1/Cim3 and Cim5 proteins have been localized primarily to the nucleus using affinity-purified antibodies, although more significant cytoplasmic staining was observed for Cim5 than for Cim3 (Mann, C., personal communication). Thus, a number of different proteasome cap subunits appear to localize largely within the nucleus.


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

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

Sen3 protein localization. (A) MHY851 cells were  stained with anti-HA antibody to visualize the Sen3 protein (FITC).  (B) DNA (DAPI). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2139890&req=5

Figure 11: Sen3 protein localization. (A) MHY851 cells were stained with anti-HA antibody to visualize the Sen3 protein (FITC). (B) DNA (DAPI). Bar, 5 μm.
Mentions: To gain a better understanding of how Pcs1p distribution compares with that of other proteasome subunits, we examined the localization of a different 19S subunit. The Sen3 protein from S. cerevisiae is a component of the proteasome cap, unrelated in sequence to the ATPase subunits (16). To study the localization of this protein, we used MHY851 cells, which express a functional Sen3 protein tagged with three copies of the HA epitope (16). We used anti-HA antibodies to visualize Sen3 protein distribution in these cells and found that it also colocalized with the nuclear DNA throughout the cell cycle (Fig. 11, A and B). No staining was observed in cells expressing untagged Sen3 protein (strain MHY849; data not shown). Similarly, the Sug1/Cim3 and Cim5 proteins have been localized primarily to the nucleus using affinity-purified antibodies, although more significant cytoplasmic staining was observed for Cim5 than for Cim3 (Mann, C., personal communication). Thus, a number of different proteasome cap subunits appear to localize largely within the nucleus.

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