Wolfgang Seufert

YEAST HCT1 IS A REGULATOR OF CLB2 CYCLIN PROTEOLYSIS

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.

Ubiquitin-conjugating enzymes UBC4 and UBC5 mediate selective degradation of short-lived and abnormal proteins.

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.

Ubiquitin as a degradation signal.

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.

Yeast Hct1 recognizes the mitotic cyclin Clb2 and other substrates of the ubiquitin ligase APC

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.

Genetic analysis of the ubiquitin system

Here we review the genetic analysis of the ubiquitin system with an emphasis on the study of the functions of the enzymatic components. Additionally, we will compile recent data on ubiquitin-like proteins encoded by cellular and viral genes. Furthermore, we will present and discuss models for the mechanisms which might control the remarkable selectivity of this posttranslational modification system

In vivo function of the proteasome in the ubiquitin pathway.

A major eukaryotic proteolytic system is known to require the covalent attachment of ubiquitin to substrates prior to their degradation, yet the proteinase involved remains poorly defined. The proteasome, a large conserved multi‐subunit protein complex of the cytosol and the nucleus, has been implicated in a variety of cellular functions. It is shown here that a yeast mutant with a defective proteasome fails to degrade proteins which are subject to ubiquitin‐dependent proteolysis in wild‐type cells. Thus, the proteasome is part of the ubiquitin system and mediates the degradation of ubiquitin‐protein conjugates in vivo.

Ubiquitin-conjugating enzymes: novel regulators of eukaryotic cells

Covalent attachment of ubiquitin to cellular proteins is essential for cell viability and is catalysed by a set of distinct ubiquitin-conjugating enzymes. Individual members of this novel enzyme family mediate strikingly diverse functions, including DNA repair, cell cycle control, selective protein degradation and essential functions of the stress response.

Asymmetric spindle pole localization of yeast Cdc15 kinase links mitotic exit and cytokinesis.

The inactivation of mitotic cyclin-dependent kinases (CDKs) during anaphase is a prerequisite for the completion of nuclear division and the onset of cytokinesis [1, 2]. In the budding yeast Saccharomyces cerevisiae, the essential protein kinase Cdc15 [3] together with other proteins of the mitotic exit network (Tem1, Lte1, Cdc5, and Dbf2/Dbf20 [4-7]) activates Cdc14 phosphatase, which triggers cyclin degradation and the accumulation of the CDK inhibitor Sic1 [8]. However, it is still unclear how CDK inactivation promotes cytokinesis. Here, we analyze the properties of Cdc15 kinase during mitotic exit. We found that Cdc15 localized to the spindle pole body (SPB) in a unique pattern. Cdc15 was present at the SPB of the mother cell until late mitosis, when it also associated with the daughter pole. High CDK activity inhibited this association, while dephosphorylation of Cdc15 by Cdc14 phosphatase enabled it. The analysis of Cdc15 derivatives indicated that SPB localization was specifically required for cytokinesis but not for mitotic exit. These results show that Cdc15 has two separate functions during the cell cycle. First, it is required for the activation of Cdc14. CD14, in turn, promotes CDK inactivation and also dephosphorylates of Cdc15. As a consequence, Cdc15 binds to the daughter pole and triggers cytokinesis. Thus, Cdc15 helps to coordinate mitotic exit and cytokinesis.

Establishment and maintenance of alternative chromatin states at a multicopy gene locus.

In eukaryotes, each of the more than 100 copies of ribosomal RNA (rRNA) genes exists in either an RNA polymerase I transcribed open chromatin state or a nucleosomal, closed chromatin state. Open rRNA genes guarantee the cells supply with structural components of the ribosome, whereas closed rRNA genes ensure genomic integrity. We report that the observed balance between open and closed rRNA gene chromatin states in proliferating yeast cells is due to a dynamic equilibrium of transcription-dependent removal and replication-dependent assembly of nucleosomes. Pol I transcription is required for the association of the HMG box protein Hmo1 with open rRNA genes, counteracting replication-independent nucleosome deposition and maintaining the open rRNA gene chromatin state outside of S phase. The findings indicate that the opposing effects of replication and transcription lead to a de novo establishment of chromatin states for rRNA genes during each cell cycle.

DnaA protein binding to the plasmid origin region can substitute for primosome assembly during replication of pBR322 in vitro

We analyzed the significance of DnaA protein binding to the origin region of pBR322. Replication of pBR322 in vitro was stimulated by DnaA protein. Moreover, the primosomal component protein i was no longer essential for replication after addition of DnaA protein, whereas, among others, proteins DnaB and DnaG were still required. Complete replication products were synthesized under these conditions. We constructed pBR322 deletion derivatives missing the primosome assembly sites. Efficient replication of these deletion plasmids was dependent on the presence of DnaA protein and its binding site, but independent of protein i activity. We conclude that DnaA protein binding to the pBR322 origin region substitutes for primosome assembly by directing DnaB, DnaC, and DnaG proteins to the origin. We term this process DnaA-directed pre-replisome formation.