Eliminating All Obstacles: Regulated Proteolysis in the Eukaryotic Cell Cycle

from E2 to a specific substrate usually requires a ubiquiEukaryotic cell cycle research took a quantum leap fortin-ligase activity (E3). E3 activities are provided by diward approximately ten years ago when it was appreciverse and sometimes large and complex protein assemated that a conserved class of protein kinases propels blies. Substrate specificity is believed to be a major the cell through the periodic events of mitotic division. function of E3 activities. Papers in this issue of Cell The actions of specific cyclin-dependent kinases (CDKs), (Feldman et al., 1997; Skowyra et al., 1997) report the so named for their requisite associated cyclin subunits, characterization of a three-subunit E3 complex required promote events such as DNA replication and chromofor degradation of Sic1 and other proteins that are phossome segregation by the mitotic spindle. The view of phorylated near the G1-S boundary. These papers demthe cell cycle as a series of CDK-activated events is onstrate that the F-box domain protein Cdc4 acts as only partially complete, however. We now understand the specificity-determining subunit of this E3 complex that cycle periodicity requires essential degradative proand that specificity can be altered by the replacement of cesses as well (reviewed in King et al., 1996). Highly Cdc4 with other F-box proteins. For essential M-phase specific proteolytic events initiate cell cycle transitions proteolytic events, an E3 activity is provided by the anaas well as eliminate cyclically acting proteins at stages phase-promoting complex (APC; a.k.a. the cyclosome), when they are no longer required and are possibly delea 20Scomplex consisting of approximately eight distinct terious. subunits. APC-mediated proteolysis is required for both A role for proteolysis in cell cycle progression was entry into anaphase as well as a distinct later step, exit suggested by early observations of cycle-specific flucfrom M phase into G1 of the next cycle. The papers by tuations in the levels of cyclin proteins. Indeed, the inSchwab et al. (1997) and Visintin et al. (1997) reveal ability to eliminate M-phase-promoting cyclins was that the APC may distinguish between different mitotic found to block exit from mitosis, demonstrating a resubstrates through the actions of Cdc20 and the related quirement for specific and temporally regulated proteolHct1/Cdh1. ysis. In more recent studies, additional proteolytically Regulated Proteolysis at the G1-S Transition controlled steps in the cell division program have been In budding yeast, CDK complexes that promote S phase revealed. Some cell cycle transitions are negatively regare assembled during G1, but are maintained in an inaculated by specific inhibitors. These inhibitors must be tive state by the association of the CKI Sic1 (Schwob eliminated in a timely fashion. In budding yeast (Sacet al., 1994). In contrast, CDK complexes containing G1 charomyces cerevisiae), the actions of S-phase-promotcyclins are immune to Sic1 inhibition. The only essential ing CDK complexes are inhibited by the cyclin kinase role for the yeast G1 CDKs is to promote the transition inhibitor (CKI) Sic1. Sic1 is degraded at the G1-S boundto S phase by the phosphorylation of Sic1 (Schneider, ary, triggering the initiation of DNA synthesis (Schwob et al., 1996; Skowyra et al., 1997; Tyers, 1996; Verma et et al., 1994). In this same organism, degradation of the al., 1997). Phosphorylation of Sic1 at multiple positions anaphase inhibitor Pds1 is required for chromosome targets it for ubiquitin-mediated proteolysis. Mutant segregation, although the target of Pds1 inhibitory acforms of Sic1 that are missing multiplephosphoacceptor tion is not known (Cohen-Fix et al., 1996). It is also sites are stabilized in vivo and block S-phase entry. apparent that regulated proteolysis eliminates compoTherefore, the first specific step in this proteolytic pathnents of the cyclically utilized DNA replication and segway is the marking of the substrate by phosphate adregation machinery. For example, proteolytic destrucdition. tion of the budding yeast spindle component Ase1 in The experiments described by Feldman et al. and late M phase is required for proper spindle disassembly Skowyra et al. define in detail the mechanism by which (Juang et al., 1997). The identification of regulatory and phospho-Sic1 is recognized and ubiquitinated. Both pastructural proteins that aredegraded within the cell cycle pers report technically sophisticated studies in which raises questions related to proteolytic specificity. How the entire pathway leading to ubiquitinated Sic1 was are proteolytic substrates selected? How is temporal reproduced in vitro using recombinant protein prepararegulation of specific degradative events achieved? Imtions. Phosphorylation of Sic1 was accomplished by portant insights regarding the specificity of two budding purified G1 CDK complexes, rendering Sic1 susceptible yeast cell cycle proteolytic mechanisms are provided to subsequent ubiquitin addition. Previous studies have by five recently published studies (Feldman et al., 1997; implicated four gene products required for the ubiquitinSchwab et al., 1997; Skowyra et al., 1997; Verma et al., mediated proteolysis of Sic1. The loss of function of