INHIBITION OF PROTEOLYSIS AND CELL-CYCLE PROGRESSION IN A MULTIUBIQUITINATION-DEFICIENT YEAST MUTANT

The degradation of many proteins requires their prior attachment to ub iquitin. Proteolytic substrates are characteristically multiubiquitina ted through the formation of ubiquitin-ubiquitin linkages. Lys-48 of u biquitin can serve as a linkage site in the formation of such chains a nd 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 Sacckaromyces cerevisi ae. Although UbK48R is expected to terminate the growth of Lys-48 mult iubiquitin chains and thus to exert a dominant negative effect on prot ein turnover, overproduction of UbK48R in wild-type cells results in o nly a weak inhibition of protein turnover, apparently because the muta nt ubiquitin can be removed from multiubiquitin chains. Surprisingly, expression of UbK48R complements several phenotypes of polyubiquitin g ene (UBI4) deletion mutants. However, UbK48R cannot serve as a sole so urce of ubiquitin in S. cerevisiae, as evidenced by its inability to r escue the growth of ubi1 ubi2 ubi3 ubi4 quadruple mutants. When provid ed 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 chain s containing Lys-48 linkages play a critical role in protein degradati on in vivo.