Stage-specific proteolysis of mitotic cyclins is fundamental to eukaryotic cell cycle regulation. We found that yeast Hct1, a conserved protein of eukaryotes, is a necessary and rate-limiting component of this proteolysis pathway. In hct1 mutants, the mitotic cyclin Clb2 is highly stabilized and inappropriately induces DNA replication, while G1 cyclins and other proteolytic substrates remain short-lived. Viability of hct1 mutants depends on SIC1. This and further results suggest that inhibition of cyclin-dependent kinases may compensate for defects in cyclin proteolysis. Remarkably, elevated levels of Hct1 ectopically activate destruction box- and Cdc23-dependent degradation of Clb2 and cause phenotypic effects characteristic for a depletion of M-phase cyclins. Hct1 and the related Cdc20 may function as substrate-specific regulators of proteolysis during mitosis.

Proteolysis of mitotic cyclins depends on a multisubunit ubiquitin-protein ligase, the anaphase promoting complex (APC). Proteolysis commences during anaphase, persisting throughout G1 until it is terminated by cyclin-dependent kinases (CDKs) as cells enter S phase. Proteolysis of mitotic cyclins in yeast was shown to require association of the APC with the substrate-specific activator Hct1 (also called Cdh1). Phosphorylation of Hct1 by CDKs blocked the Hct1-APC interaction. The mutual inhibition between APC and CDKs explains how cells suppress mitotic CDK activity during G1 and then establish a period with elevated kinase activity from S phase until anaphase.

Ubiquitin‐conjugating enzymes catalyse the covalent attachment of ubiquitin to target proteins. Members of this enzyme family are involved in strikingly diverse cellular functions: UBC2 (RAD6) is central to DNA repair, UBC3 (CDC34) is involved in cell cycle control. We have cloned the genes for two novel ubiquitin‐conjugating enzymes, UBC4 and UBC5, from the yeast Saccharomyces cerevisiae. These enzymes mediate selective degradation of short‐lived and abnormal proteins. UBC4 and UBC5 are closely related in sequence and complementing in function. Expression of UBC4 and UBC5 genes is heat inducible. UBC4 and UBC5 enzymes generate high mol. wt ubiquitin‐protein conjugates in vivo consistent with previous studies which suggested that attachment of multiple ubiquitin molecules to proteolytic substrates is required for their selective degradation. UBC4 and UBC5 enzymes comprise a major part of total ubiquitin‐conjugation activity in stressed cells. Turnover of short‐lived proteins and canavanyl‐peptides but not of long‐lived proteins is markedly reduced in ubc4ubc5 mutants. Loss of UBC4 and UBC5 activity impairs cell growth, leads to inviability at elevated temperatures or in the presence of an amino acid analog, and induces the stress response.

Cell cycle progression in eukaryotes is controlled by the p34cdc2/CDC28 protein kinase and its short-lived, phase-specific regulatory subunits called cyclins. In Xenopus oocytes, degradation of M-phase (B-type) cyclins is required for exit from mitosis and is mediated by the ubiquitin-dependent proteolytic system. Here we show that B-type-cyclin degradation in yeast involves an essential nuclear ubiquitin-conjugating enzyme, UBC9. Repression of UBC9 synthesis prevents cell cycle progression at the G2 or early M phase, causing the accumulation of large budded cells with a single nucleus, a short spindle and replicated DNA. In ubc9 mutants both CLB5, an S-phase cyclin, and CLB2, an M-phase cyclin, are stabilized. In wild-type cells the CLB5 protein is unstable throughout the cell cycle, whereas CLB2 turnover occurs only at a specific cell-cycle stage. Thus distinct degradation signals or regulated interaction with the ubiquitin-protein ligase system may determine the cell-cycle specificity of cyclin proteolysis.

Epitope tagging is the insertion of a short stretch of amino acids constituting an epitope into another protein. Tagged proteins can be identified by Western, immunoprecipitation and immunofluorescence assays using pre-existing antibodies. We have designed vectors containing the URA3 gene flanked by direct repeats of epitope tags. We use the polymerase chain reaction (PCR) to amplify the tag-URA3-tag cassette such that the ends of the PCR fragments possess homology to the gene of interest. In vivo recombination is then used to direct integration of the fragment to the location of interest, and transformants are selected by their Ura+ phenotype. Finally, selection for Ura- cells on 5-fluoro-orotic acid plates yields cells where recombination between the repeated epitopes has 'popped out' the URA3 gene, leaving a single copy of the epitope at the desired location. PCR epitope tagging (PET) provides a rapid and direct technique for tagging that does not require any cloning steps. We have used PET to tag three Saccharomyces cerevisiae proteins, Cln1, Sic1 and Est1.

For many short‐lived eukaryotic proteins, conjugation to ubiquitin, yielding a multiubiquitin chain, is an obligatory pre‐degradation step. The conjugated ubiquitin moieties function as a ‘secondary’ signal for degradation, in that their posttranslational coupling to a substrate protein is mediated by amino acid sequences of the substrate that act as a primary degradation signal. We report that the fusion protein ubiquitin‐‐proline‐‐beta‐galactosidase (Ub‐P‐beta gal) is short‐lived in the yeast Saccharomyces cerevisiae because its N‐terminal ubiquitin moiety functions as an autonomous, primary degradation signal. This signal mediates the formation of a multiubiquitin chain linked to Lys48 of the N‐terminal ubiquitin in Ub‐P‐beta gal. The degradation of Ub‐P‐beta gal is shown to require Ubc4, one of at least seven ubiquitin‐conjugating enzymes in S.cerevisiae. Our findings provide the first direct evidence that a monoubiquitin moiety can function as an autonomous degradation signal. This generally applicable, cis‐acting signal can be used to manipulate the in vivo half‐lives of specific intracellular proteins.

Ubiquitin‐mediated proteolysis has emerged as a key mechanism of regulation in eukaryotic cells. During cell division, a multi‐subunit ubiquitin ligase termed the anaphase promoting complex (APC) targets critical regulatory proteins such as securin and mitotic cyclins, and thereby triggers chromosome separation and exit from mitosis. Previous studies in the yeast Saccharomyces cerevisiae identified the conserved WD40 proteins Cdc20 and Hct1 (Cdh1) as substrate‐specific activators of the APC, but their precise mechanism of action has remained unclear. This study provides evidence that Hct1 functions as a substrate receptor that recognizes target proteins and recruits them to the APC for ubiquitylation and subsequent proteolysis. By co‐immunoprecipitation, we found that Hct1 interacted with the mitotic cyclins Clb2 and Clb3 and the polo‐related kinase Cdc5, whereas Cdc20 interacted with the securin Pds1. Failure to interact with Hct1 resulted in stabilization of Clb2. Analysis of Hct1 derivatives identified the C‐box, a motif required for APC association of Hct1 and conserved among Cdc20‐related proteins. We propose that proteins of the Cdc20 family are substrate recognition subunits of the ubiquitin ligase APC.

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