Limits...
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.

Show MeSH

Related in: MedlinePlus

Cytological analysis of pcs1td cells using immunofluorescence microscopy. (Left) DNA staining (DAPI); (right) microtubule staining (FITC). (A) Wx257-5c (PCS1) at 37°C. (B–E)  YHM11.2 (pcs1td) at 37°C. Intranuclear spindles appear to be absent in the mutant cells. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2139890&req=5

Figure 6: Cytological analysis of pcs1td cells using immunofluorescence microscopy. (Left) DNA staining (DAPI); (right) microtubule staining (FITC). (A) Wx257-5c (PCS1) at 37°C. (B–E) YHM11.2 (pcs1td) at 37°C. Intranuclear spindles appear to be absent in the mutant cells. Bar, 5 μm.

Mentions: To gain a better understanding of how a lack of PCS1 function perturbs mitosis, we visualized DNA and microtubules using fluorescence microscopy in pcs1td cells that had accumulated in the terminal arrest described by flow cytometry (see above). After 9 h at 37°C, the DNA remained unsegregated and was localized in a region immediately adjacent to the bud neck in ∼80% of these cells (Fig. 6, B–E). Nuclei occupying this position in PCS1 cells grown at 37°C were shown by tubulin antibody staining to contain a short intranuclear spindle (Fig. 6 A), as is characteristic of yeast cells in G2 or mitotic metaphase. However, the arrested pcs1td cells did not contain discrete spindles and generally had fewer detectable intranuclear microtubules. The most common phenotype is shown in Fig. 6 B, in which a single bundle of cytoplasmic microtubules extended away from the nucleus and through the bud neck. Additionally, some cells contained a relatively bright focus of microtubule staining at the nuclear periphery, as well as cytoplasmic microtubules emanating away from this focus (Fig. 6 C). Less frequently, we observed cells in which microtubules appeared to lie along the surface of the nucleus (Fig. 6 D), and other cells in which two bundles of cytoplasmic microtubules extended away from the nucleus in an antennae-like array (Fig. 6 E). (Significant numbers of cells lacking spindle structures were not observed at earlier time points in this experiment, presumably because the first cycle proceeded normally, albeit slowly.) These findings suggest that cells deprived of PCS1 function fail to form a mitotic spindle while retaining the ability to assemble cytoplasmic microtubule arrays.


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

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

Cytological analysis of pcs1td cells using immunofluorescence microscopy. (Left) DNA staining (DAPI); (right) microtubule staining (FITC). (A) Wx257-5c (PCS1) at 37°C. (B–E)  YHM11.2 (pcs1td) at 37°C. Intranuclear spindles appear to be absent in the mutant cells. Bar, 5 μm.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 6: Cytological analysis of pcs1td cells using immunofluorescence microscopy. (Left) DNA staining (DAPI); (right) microtubule staining (FITC). (A) Wx257-5c (PCS1) at 37°C. (B–E) YHM11.2 (pcs1td) at 37°C. Intranuclear spindles appear to be absent in the mutant cells. Bar, 5 μm.
Mentions: To gain a better understanding of how a lack of PCS1 function perturbs mitosis, we visualized DNA and microtubules using fluorescence microscopy in pcs1td cells that had accumulated in the terminal arrest described by flow cytometry (see above). After 9 h at 37°C, the DNA remained unsegregated and was localized in a region immediately adjacent to the bud neck in ∼80% of these cells (Fig. 6, B–E). Nuclei occupying this position in PCS1 cells grown at 37°C were shown by tubulin antibody staining to contain a short intranuclear spindle (Fig. 6 A), as is characteristic of yeast cells in G2 or mitotic metaphase. However, the arrested pcs1td cells did not contain discrete spindles and generally had fewer detectable intranuclear microtubules. The most common phenotype is shown in Fig. 6 B, in which a single bundle of cytoplasmic microtubules extended away from the nucleus and through the bud neck. Additionally, some cells contained a relatively bright focus of microtubule staining at the nuclear periphery, as well as cytoplasmic microtubules emanating away from this focus (Fig. 6 C). Less frequently, we observed cells in which microtubules appeared to lie along the surface of the nucleus (Fig. 6 D), and other cells in which two bundles of cytoplasmic microtubules extended away from the nucleus in an antennae-like array (Fig. 6 E). (Significant numbers of cells lacking spindle structures were not observed at earlier time points in this experiment, presumably because the first cycle proceeded normally, albeit slowly.) These findings suggest that cells deprived of PCS1 function fail to form a mitotic spindle while retaining the ability to assemble cytoplasmic microtubule arrays.

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