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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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DNA localization in linked large-budded pcs1td cells at  37°C. (A) Nomarski image of cells. The numbers designate the  presumed order of budding, as explained in the text. (B) DNA  (DAPI). (C) Microtubules (FITC). Bar, 5 μm.
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Figure 7: DNA localization in linked large-budded pcs1td cells at 37°C. (A) Nomarski image of cells. The numbers designate the presumed order of budding, as explained in the text. (B) DNA (DAPI). (C) Microtubules (FITC). Bar, 5 μm.

Mentions: The attachment between pairs of large-budded cells seen after transfer of a pcs1td culture to the restrictive temperature (as described earlier) provided us with a method of identifying mother and daughter cells unambiguously. Fig. 7 A shows cells in this configuration. Since the cells had been arrested with α-factor before the shift to high temperature, the original mother is identifiable as the schmooshaped cell 1. This cell presumably had budded once to produce cell 2 and a second time to produce bud 3. Cell 2 had also budded to produce bud 4. In Fig. 7 B, it can be seen that the unsegregated DNA lies largely within the buds (3 and 4) that are the products of the second division. Cytoplasmic microtubule staining is evident in bud 4 (Fig. 7 C); faint microtubule staining was also observed in bud 3 in a different focal plane. Although we could not distinguish the mother cell and bud unambiguously among all cells examined in this manner, the DNA localized to the mother ∼50% of the time and bud 50% of the time when both could be identified (data not shown). A mutant that is specifically defective in spindle pole body duplication, ndc1-1, has previously been shown to display random localization of the single functional spindle pole and the bulk of the DNA between the mother cell and the bud (93, 97). Similarly, certain mutations that cause cells to arrest with a bipolar spindle also display such random localization of DNA (72).


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

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

DNA localization in linked large-budded pcs1td cells at  37°C. (A) Nomarski image of cells. The numbers designate the  presumed order of budding, as explained in the text. (B) DNA  (DAPI). (C) Microtubules (FITC). Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: DNA localization in linked large-budded pcs1td cells at 37°C. (A) Nomarski image of cells. The numbers designate the presumed order of budding, as explained in the text. (B) DNA (DAPI). (C) Microtubules (FITC). Bar, 5 μm.
Mentions: The attachment between pairs of large-budded cells seen after transfer of a pcs1td culture to the restrictive temperature (as described earlier) provided us with a method of identifying mother and daughter cells unambiguously. Fig. 7 A shows cells in this configuration. Since the cells had been arrested with α-factor before the shift to high temperature, the original mother is identifiable as the schmooshaped cell 1. This cell presumably had budded once to produce cell 2 and a second time to produce bud 3. Cell 2 had also budded to produce bud 4. In Fig. 7 B, it can be seen that the unsegregated DNA lies largely within the buds (3 and 4) that are the products of the second division. Cytoplasmic microtubule staining is evident in bud 4 (Fig. 7 C); faint microtubule staining was also observed in bud 3 in a different focal plane. Although we could not distinguish the mother cell and bud unambiguously among all cells examined in this manner, the DNA localized to the mother ∼50% of the time and bud 50% of the time when both could be identified (data not shown). A mutant that is specifically defective in spindle pole body duplication, ndc1-1, has previously been shown to display random localization of the single functional spindle pole and the bulk of the DNA between the mother cell and the bud (93, 97). Similarly, certain mutations that cause cells to arrest with a bipolar spindle also display such random localization of DNA (72).

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