Identification ofProteins WhoseSynthesis IsModulated During theCell Cycle ofSaccharomyces cerevisiae

Weexamined thesynthesis andturnover ofindividual proteins intheSaccharomyces cerevisiae cell cycle. Proteins werepulse-labeled withradioactive isotope (32S or14C) incells atdiscrete cycle stages andthenresolved ontwo-dimensional gels andanalyzed byasemiautomatic procedure forquantitating gelelectropherogram-autoradiographs. Thecells were obtained byone ofthree methods: (i) isolation ofsynchronous subpopulations ofgrowing cells byzonal centrifugation; (ii) fractionation ofpulse-labeled steady-state cultures according tocell age;and (iii) synchronization ofcells withthemating pheromone, a-factor. Inconfirmation ofprevious studies, we foundthatthehistones H4,H2A,andH2B were synthesized almost exclusively inthelate Glandearly Sphases. Inaddition, we identified eight proteins whoseratesofsynthesis weremodulated inthecell cycle, andnineproteins (ofwhichfive, which may wellberelated, were unstable, with half-lives of10to15min)thatmightberegulated inthecell cycle byperiodic synthesis, modification, ordegradation. Based on thetimeofmaximal labeling in thecell cycle andonexperiments witha-factor andhydroxyurea, we assigned the cell cycle proteins totwoclasses: proteins inclass Iwere labeled principally in early Glphaseandata late stageofthecycle, whereas those inclass II were primarily synthesized attimes ranging fromlate GltomidSphase. Atleast one major control point forthecell cycle proteins occurred between "start" andearly Sphase. A setofstress-responsive proteins was also identified andanalyzed. The ratesofsynthesis ofthese proteins were affected bycertain perturbations that resulted during selection ofsynchronous cell populations andbyheatshock. Understanding themolecular basis ofthe manycellular events whichoccuratdiscrete stages oftheprogress ofcells fromonedivision tothenextisthecentral focus ofresearch onthe cell cycle. Saccharomyces cerevisiae hasbeen developed asa modelorganism forstudying eucaryotic cellcycle control (15-17, 34,43). Genetic analysis hasgenerated someofthebasic ideas about functional pathways inthecell cycle andalso provided material forfurther biochemicalstudies. Inthis paper, wereport onasearch forcell cycle-regulated proteins inS.cerevisiae. Inan earlier analysis ofthepatterns ofsynthesis ofS. cerevisiae proteins inthecell cycle (8-10, 22), almost 200proteins wereexamined quantitativelyafter being separated ontwo-dimensional (2