Two-hybrid analysis of the Saccharomyces cerevisiae 26S proteasome.
A two-hybrid screen against an activation domain array of Saccharomyces cerevisiae proteins was carried out for 31 yeast proteasome proteins. Fifty-five putative interactions were identified: 21 between components of the proteasome complex and 34 between proteasome proteins and other proteins. Many of these latter interactions involved either proteins of the ubiquitin pathway, cell cycle proteins, protein kinases or a translation initiation factor subunit. The role of eleven proteins associated with proteasome function by these screens was analyzed by examining the corresponding deletion strains for temperature sensitivity and canavanine sensitivity and for the stability of a ubiquitin-beta-galactosidase fusion protein. These assays additionally implicated three proteins, Bim1, Ump1, and YKL171W, in proteasome function. This study demonstrates the utility of genome-wide two-hybrid assays as an entry point for the further analysis of a large protein complex.
Cagney
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Departments of Genetics and Medicine, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195-7360, USA.
Uetz
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P
Fields
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D002384:Catalysis; D017353:Gene Deletion; D015723:Gene Library; D046911:Macromolecular Substances; D010447:Peptide Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D010641:Phenotype; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013696:Temperature; D013816:Thermodynamics; D020798:Two-Hybrid System Techniques; D025801:Ubiquitin / Q000378:metabolism
D046911:Macromolecular Substances; D021122:Protein Subunits; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D025801:Ubiquitin; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease