A Role for Saccharomyces cerevisiae Cul8 Ubiquitin Ligase in Proper Anaphase Progression

Abstract

We have undertaken a study of the yeast cullin family members Cul3 and Cul8, as little is known about their biochemical and physiological functions. We demonstrate that these cullins are associated in vivo with ubiquitin ligase activity. We show that Cul3 and Cul8 are functionally distinct from Cdc53 and do not interact with ySkp1, suggesting that they target substrates by Skp1-and possibly F-box protein-independent mechanisms. Whereas null mutants of CUL3 appear normal, yeast cells lacking CUL8 have a slower growth rate and are delayed in their progress through anaphase. The anaphase delay phenotype can be complemented by ectopic expression of Cul8 but not by any other yeast or human cullins, nor by a cul8 mutant deficient in binding to RING finger protein Roc1. Deletion of the RAD9 gene suppressed the anaphase delay phenotype of cul8⌬, suggesting that loss of Cul8 function may compromise genomic integrity. These results indicate that in addition to the anaphase promoting complex, mitotic progression may involve another E3 ubiquitin ligase mediated by Cul8 protein.Ubiquitin-mediated proteolysis is a rapid and irreversible mechanism used by eukaryotic cells to ensure unidirectional cell cycle progression and proper cell division. This process requires a cascade of three enzymatic activities: E1 1 (ubiquitin activating), E2 (ubiquitin conjugating), and E3 (ubiquitin ligase) enzymes (1-3). Unlike E1 and E2, the E3 ubiquitin ligases have been loosely defined to comprise at least two distinct functions: 1) an enzyme activity that catalyzes isopeptide bond formation, and 2) a substrate-targeting function that recruits substrates to this catalytic activity. The mechanisms by which E3 ligases target substrates and the resulting biological functions they mediate are currently under intensive investigation.The transitions from G 1 to S and from G 2 /M to G 1 require two different multisubunit E3 ligases, the Skp1-Cdc53/CUL1-F box (SCF) complex and the anaphase-promoting complex (APC) or cyclosome, respectively (4, 5). The SCF E3 ligase targets the ubiquitination of G 1 cyclin-dependent kinase inhibitors and G 1 cyclins, whereas the APC E3 ligase targets the ubiquitination of anaphase inhibitors and B-type cyclins to allow sister chromatid separation and exit from mitosis, respectively. SCF and APC represent two major E3 ubiquitin ligase activities identified thus far that are required for cell cycle progression.The Cdc53 component of the yeast SCF E3 ligase belongs to an evolutionarily conserved family of proteins known as cullins that contains three related genes in both budding and fission yeast and at least six genes in worm, fruit fly, and human (6, 7). The functional importance and conservation of cullins is underscored by the finding that a subunit of APC E3 ligase, APC2, contains limited sequence homology to cullins (8, 9). Ample genetic evidence establishes that cullin proteins perform various physiological functions critical not only to the cell cycle but also to cell growth and animal development. Mutations in ...

Functional Rescue of Mutant Human Cystathionine β-Synthase by Manipulation of Hsp26 and Hsp70 Levels in Saccharomyces cerevisiae

“…We hypothesize that there may also be a ubiquitin-protein isopeptide ligase that interacts with yeast Hsp26 and directs ubiquitination of the complex. One possible candidate is Cul8, a ubiquitin-protein isopeptide ligase involved in anaphase progression that was found to interact with Hsp26 in a large scale proteomics screen using tandem affinity purification (25,26). One interesting difference between the CHIP system and the one described here is that in CHIP-mediated degradation the levels of the accompanying Hsp protein do not change, whereas in the system described here both the misfolded protein and the accompanying Hsp26s are degraded.…”

Defective Break-Induced Replication Leads to Half-Crossovers inSaccharomyces cerevisiae

“…However, the fraction of chromosome loss events (Ade Àr Leu À ) was not dramatically different from wild type and, therefore, the efficiency of BIR was only mildly affected in this mutant. Deletion of RTT101 leads to several known phenotypes in budding yeast, including an increase in transposition of Ty1 elements (Scholes et al 2001) and delayed anaphase progression (Michel et al 2003). Recently, Rtt101p was shown to be required for progression of replication through damaged DNA and through natural replication-impeding loci (Luke et al 2006;Roberts et al 2008).…”

Nedd8 on cullin: building an expressway to protein destruction

“…h (Kohrman and Imperiale, 1992;Tsai et al, 2000;Dias et al, 2002;Arai et al, 2003;Dias and Pan, unpublished results). i (Michel et al, 2003). j (Nikolaev et al, 2003;Gu, personal communication) Role of Nedd8 in proteolysis Z-Q Pan et al CUL7 assembles an SCF-ROC1-like E3 ligase complex comprising Skp1, CUL7, the F-box protein Fbx29 and ROC1/Rbx1 (Dias et al, 2002;Arai et al, 2003).…”

“…Using site-directed mutagenesis, neddylation sites have been determined for human (h) CUL1 (K720; Read et al, 2000), hCUL2 (K689; Wada et al, 1999), S. pombe (sp) CUL3/Pcu3 (K729; Zhou et al, 2001), spCUL4/Pcu4 (K680; Osaka et al, 2000) and S. cerevisiae (sc) CUL8 (K791; Michel et al, 2003). It was initially noted by Wada et al (1999) that the neddylation site is located within a conserved sequence, IVRIMKMR (Figure 1), although the significance of the amino acids flanking the lysine residue in neddylation has yet to be determined.…”