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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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Multiple sequence  alignment of Pcs1p, p42,  Sug1/Cim3 protein, and S4.  (Black boxes) Amino acid  identity; (grey boxes) similarity. This display was created  using the program BOXSHADE.
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Figure 3: Multiple sequence alignment of Pcs1p, p42, Sug1/Cim3 protein, and S4. (Black boxes) Amino acid identity; (grey boxes) similarity. This display was created using the program BOXSHADE.

Mentions: To examine the relationship between Pcs1p and other protein sequences in the databases, we used the BLAST alignment program (2). This analysis confirmed the identity of Pcs1p as a member of the “AAA” (ATPases Associated with diverse cellular Activities [56]) superfamily. Members of this superfamily share a conserved region of ∼200 amino acids that contains motifs A and B characteristic of ATP binding, underlined in Fig. 2 B (99). Pcs1p is most similar to a subclass of this superfamily composed of proteasomal 19S cap subunits. Fig. 3 shows an alignment of Pcs1p with three of the highest scoring proteins in the BLAST search. p42 (25) and S4 (23) are subunits of the human 26S proteasome, and their sequences are respectively 67% and 38% identical to the Pcs1p sequence as determined by the pairwise alignment program BESTFIT (36). SUG1 (92)/CIM3 (31) (and CIM5 [31]) are S. cerevisiae genes; the sequences of their predicted products are 43% and 39% identical to the Pcs1p sequence. Homologs of the SUG1/CIM3 and CIM5 gene products were identified in purified preparations of 26S proteasomes from Drosophila (31), and the Sug1/Cim3 protein has been directly shown to be a subunit of the yeast 26S proteasome (82). Pcs1p is also closely related to the S. pombe mts2+ gene product, a 26S proteasome component necessary for completion of mitosis (35). The very high degree of sequence similarity between these known proteasome cap subunits and Pcs1p provides strong evidence that Pcs1p is itself a 19S cap subunit. This identity has been confirmed by the recent work of Russell et al. (83), who demonstrated that the protein encoded by SUG2 (which is identical to PCS1) is a component of the 26S proteasome, while our work was in progress.


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

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

Multiple sequence  alignment of Pcs1p, p42,  Sug1/Cim3 protein, and S4.  (Black boxes) Amino acid  identity; (grey boxes) similarity. This display was created  using the program BOXSHADE.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Multiple sequence alignment of Pcs1p, p42, Sug1/Cim3 protein, and S4. (Black boxes) Amino acid identity; (grey boxes) similarity. This display was created using the program BOXSHADE.
Mentions: To examine the relationship between Pcs1p and other protein sequences in the databases, we used the BLAST alignment program (2). This analysis confirmed the identity of Pcs1p as a member of the “AAA” (ATPases Associated with diverse cellular Activities [56]) superfamily. Members of this superfamily share a conserved region of ∼200 amino acids that contains motifs A and B characteristic of ATP binding, underlined in Fig. 2 B (99). Pcs1p is most similar to a subclass of this superfamily composed of proteasomal 19S cap subunits. Fig. 3 shows an alignment of Pcs1p with three of the highest scoring proteins in the BLAST search. p42 (25) and S4 (23) are subunits of the human 26S proteasome, and their sequences are respectively 67% and 38% identical to the Pcs1p sequence as determined by the pairwise alignment program BESTFIT (36). SUG1 (92)/CIM3 (31) (and CIM5 [31]) are S. cerevisiae genes; the sequences of their predicted products are 43% and 39% identical to the Pcs1p sequence. Homologs of the SUG1/CIM3 and CIM5 gene products were identified in purified preparations of 26S proteasomes from Drosophila (31), and the Sug1/Cim3 protein has been directly shown to be a subunit of the yeast 26S proteasome (82). Pcs1p is also closely related to the S. pombe mts2+ gene product, a 26S proteasome component necessary for completion of mitosis (35). The very high degree of sequence similarity between these known proteasome cap subunits and Pcs1p provides strong evidence that Pcs1p is itself a 19S cap subunit. This identity has been confirmed by the recent work of Russell et al. (83), who demonstrated that the protein encoded by SUG2 (which is identical to PCS1) is a component of the 26S proteasome, while our work was in progress.

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