Two Ubiquitin-Conjugating Enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, Have Distinct Functions for Ubiquitination of Mitotic Cyclin

Abstract

Cell cycle events are regulated by sequential activation and inactivation of Cdk kinases. Mitotic exit is accomplished by the inactivation of mitotic Cdk kinase, which is mainly achieved by degradation of cyclins. The ubiquitin-proteasome system is involved in this process, requiring APC/C (anaphase-promoting complex/ cyclosome) as a ubiquitin ligase. In Xenopus and clam oocytes, the ubiquitin-conjugating enzymes that function with APC/C have been identified as two proteins, UBC4 and UBCx/E2-C. Previously we reported that the fission yeast ubiquitin-conjugating enzyme UbcP4/Ubc11, a homologue of UBCx/E2-C, is required for mitotic transition. Here we show that the other fission yeast ubiquitin-conjugating enzyme, UbcP1/Ubc4, which is homologous to UBC4, is also required for mitotic transition in the same manner as UbcP4/Ubc11. Both ubiquitinconjugating enzymes are essential for cell division and directly required for the degradation of mitotic cyclin Cdc13. They function nonredundantly in the ubiquitination of CDC13 because a defect in ubcP1/ubc4؉ cannot be suppressed by high expression of UbcP4/Ubc11 and a defect in ubcP4/ubc11؉ cannot be suppressed by high expression of UbcP1/Ubc4. In vivo analysis of the ubiquitinated state of Cdc13 shows that the ubiquitin chains on Cdc13 were short in ubcP1/ubc4 mutant cells while ubiquitinated Cdc13 was totally reduced in ubcP4/ubc11 mutant cells. Taken together, these results indicate that the two ubiquitin-conjugating enzymes play distinct and essential roles in the degradation of mitotic cyclin Cdc13, with the UbcP4/Ubc11-pathway initiating ubiquitination of Cdc13 and the UbcP1/Ubc4-pathway elongating the short ubiquitin chains on Cdc13.

An E2 enzyme Ubc11 is required for ubiquitination of Slp1/Cdc20 and spindle checkpoint silencing in fission yeast

“…The cold-sensitive nda3-KM311 mutation defective in the spindle formation 42 was introduced into each of the ubc11-P93L mutant, ubc4-P61S mutant 37 and ubc4-P61S ubc11-P93L double mutant, respectively. In the nda3-KM311 single mutant after incubation at its restrictive temperature (20°C), the three proteins accumulated.…”

“…The level of Cdc13, E2s, fission yeast Ubc11 and Ubc4, each of which has an essential and incompatible role in polyubiquitin-chain formation. 37 The B-type cyclin/Cdc13 and securin/Cut2 cause a mitotic arrest if not properly degraded in the APC/C-dependent manner. [38][39][40][41] We therefore attempted to examine how the ubc11-P93L mutation affects the stability of proteins normally degraded in the APC/C-dependent manner.…”

“…34,42,[49][50][51]63,64 The mutant ubc4-P61S was a generous gift from Dr Hiroaki Seino. 37 Yeast transformations were performed by lithium acetate methods. 65,66 Total RNAs were prepared as previously described.…”

UbcH10 has a rate-limiting role in G1 phase but might not act in the spindle checkpoint or as part of an autonomous oscillator

“…We are forced to conclude that in mammalian cells UbcH10 is either not essential for the APC/C to ubiquitylate its substrates in mitosis -for example, the APC/C may be able to use UbcH5 and E2-25K, as it can in vitro (Rodrigo-Brenni and Morgan, 2007) -or UbcH10 is not rate limiting and the low levels remaining in the cells are sufficient for the normal kinetics of the APC/C. Completely eliminating UbcH10 might inactivate the APC/C because cells of vihar mutants in Drosophila arrest in mitosis with defective spindles (Mathe et al, 2004), and fission yeast cells lacking the UbcH10 orthologue UbcP4/Ubc11 are unable to degrade cyclin B (Cdc13) (Seino et al, 2003).…”

Effete-mediated degradation of Cyclin A is essential for the maintenance of germline stem cells in Drosophila

“…However, an in vivo study showed that these two classes of E2 are not functionally equivalent but exhibit distinct functions in mitotic cyclin degradation, suggesting that different E2 family members probably execute distinct functions (Seino et al, 2003). The Drosophila ortholog of UBCx/E2-C, Vihar E2, has been reported to be involved in Cyclin B degradation during the metaphase-anaphase transition (Mathe et al, 2004).…”