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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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Related in: MedlinePlus

Flow cytometric analysis of pcs1td. (A) Wx257-5c (PCS1,  open curve) compared with YHM11.2 (pcs1td, shaded curve) at  23°C. (B) PCS1 (open curve) compared with pcs1td (shaded curve)  at 37°C. The left peak represents G1 cells and the right peak represents G2/M cells. In these histograms, the x-axis indicates relative DNA content measured by propidium iodide fluorescence,  and the y-axis indicates the relative number of cells. Each sample  represents 15,000 cells.
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Figure 5: Flow cytometric analysis of pcs1td. (A) Wx257-5c (PCS1, open curve) compared with YHM11.2 (pcs1td, shaded curve) at 23°C. (B) PCS1 (open curve) compared with pcs1td (shaded curve) at 37°C. The left peak represents G1 cells and the right peak represents G2/M cells. In these histograms, the x-axis indicates relative DNA content measured by propidium iodide fluorescence, and the y-axis indicates the relative number of cells. Each sample represents 15,000 cells.

Mentions: Flow cytometry experiments indicate that pcs1td cells arrest with a 2C (G2) content of DNA. Fig. 5 A shows the cytometry profiles of asynchronous PCS1 and pcs1td cultures at 23°C, where it can be seen that about equal proportions of pcs1td cells had apparent 1C and 2C DNA contents. After 9 h at 37°C, however, 87% of the pcs1td cells had accumulated in a state with a 2C content of DNA, whereas a higher proportion of PCS1 cells had a 1C content of DNA at 37°C than at 23°C (Fig. 5 B). These findings demonstrate that cells lacking PCS1 function undergo arrest in the division cycle after DNA synthesis, and that this function therefore must be required for progression from G2 into mitosis.


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

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

Flow cytometric analysis of pcs1td. (A) Wx257-5c (PCS1,  open curve) compared with YHM11.2 (pcs1td, shaded curve) at  23°C. (B) PCS1 (open curve) compared with pcs1td (shaded curve)  at 37°C. The left peak represents G1 cells and the right peak represents G2/M cells. In these histograms, the x-axis indicates relative DNA content measured by propidium iodide fluorescence,  and the y-axis indicates the relative number of cells. Each sample  represents 15,000 cells.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 5: Flow cytometric analysis of pcs1td. (A) Wx257-5c (PCS1, open curve) compared with YHM11.2 (pcs1td, shaded curve) at 23°C. (B) PCS1 (open curve) compared with pcs1td (shaded curve) at 37°C. The left peak represents G1 cells and the right peak represents G2/M cells. In these histograms, the x-axis indicates relative DNA content measured by propidium iodide fluorescence, and the y-axis indicates the relative number of cells. Each sample represents 15,000 cells.
Mentions: Flow cytometry experiments indicate that pcs1td cells arrest with a 2C (G2) content of DNA. Fig. 5 A shows the cytometry profiles of asynchronous PCS1 and pcs1td cultures at 23°C, where it can be seen that about equal proportions of pcs1td cells had apparent 1C and 2C DNA contents. After 9 h at 37°C, however, 87% of the pcs1td cells had accumulated in a state with a 2C content of DNA, whereas a higher proportion of PCS1 cells had a 1C content of DNA at 37°C than at 23°C (Fig. 5 B). These findings demonstrate that cells lacking PCS1 function undergo arrest in the division cycle after DNA synthesis, and that this function therefore must be required for progression from G2 into mitosis.

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