Inhibition of proteolysis and cell cycle progression in a multiubiquitination-deficient yeast mutant.
The degradation of many proteins requires their prior attachment to ubiquitin. Proteolytic substrates are characteristically multiubiquitinated through the formation of ubiquitin-ubiquitin linkages. Lys-48 of ubiquitin can serve as a linkage site in the formation of such chains and is required for the degradation of some substrates of this pathway in vitro. We have characterized the recessive and dominant effects of a Lys-48-to-Arg mutant of ubiquitin (UbK48R) in Saccharomyces cerevisiae. Although UbK48R is expected to terminate the growth of Lys-48 multiubiquitin chains and thus to exert a dominant negative effect on protein turnover, overproduction of UbK48R in wild-type cells results in only a weak inhibition of protein turnover, apparently because the mutant ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin gene (UB14) deletion mutants. However, UbK48R cannot serve as a sole source of ubiquitin in S. cerevisiae, as evidenced by its inability to rescue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provided solely with UbK48R, cells undergo cell cycle arrest with a terminal phenotype characterized by replicated DNA, mitotic spindles, and two-lobed nuclei. Under these conditions, degradation of amino acid analog-containing proteins is severely inhibited. Thus, multiubiquitin chains containing Lys-48 linkages play a critical role in protein degradation in vivo.
Department of Cell Biology, Harvard Medical School, Boston, Massachusetts 02115.
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Finley
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Sadis
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Monia
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Boucher
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Ecker
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Crooke
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Chau
D002453:Cell Cycle; D010450:Endopeptidases / Q000378:metabolism; D005656:Fungal Proteins / Q000378:metabolism*; D005799:Genes, Dominant; D005816:Genetic Complementation Test; D008239:Lysine / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D012441:Saccharomyces cerevisiae; D014452:Ubiquitins / Q000378:metabolism*
D005656:Fungal Proteins; D014452:Ubiquitins; D010450:Endopeptidases; D008239:Lysine