A. DEPARTMENT OF MOLECULAR GENETICS
A-c. Molecular Mechanism Laboratory - Hiroaki Seino Group

RESEARCH ACTIVITIES

(1) An in vitro ubiquitination assay of mitotic cyclin

Hiroaki Seino

--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 by a ubiquitin-proteasome system.
--Previously we reported that two ubiquitin-conjugating enzymes, UbcP1/Ubc4 and UbcP4/Ubc11, were responsible for degradation of mitotic cyclin Cdc13 in fission yeast. Each of these two ubiquitin-conjugating enzymes is essential for cell viability and responsible for degradation of Cdc13. These results suggest that the functions of these two ubiquitin-conjugating enzymes are not redundant and they have distinct functions for ubiquitination of Cdc13. Furthermore, we found that ubiquitin chains of Cdc13 were totally reduced in ubcP4/ubc11 mutant cells, whereas ubiquitin chains were short and not reduced in ubcP1/ubc4 mutant cells. Thus, we proposed a hypothesis that UbcP4/Ubc11 might be involved in initiation of ubiquitination, and UbcP1/Ubc4 might be involved in elongation of ubiquitin chains of Cdc13. However, this hypothesis has not been elucidated yet.
--To clarify the functional differences between UbcP1/Ubc4 and UbcP4/Ubc11 for degradation of Cdc13, development of an in vitro assay system for ubiquitination for Cdc13 by using fission yeast components is required. Currently, I am attempting to develop this assay system for Cdc13. A ubiquitin-activating enzyme, these two ubiquitin-conjugating enzymes and substrate Cdc13 were expressed as recombinant proteins in bacterial cells and purified. One component of Cdc13-specific ubiquitin ligase anaphase promoting complex/cyclosome (APC/C) was tagged and expressed in fission yeast cells, and APC/C was purified from fission yeast cells. Now I am examining the conditions for reconstitution of a ubiquitination reaction of Cdc13.

(2) Ubiquitin-conjugating enzyme(s) responsible for degradation of SCF substrates

Hiroaki Seino

--One of the important ubiquitin pathways responsible for cell cycle regulation is the pathway involving APC ubiquitin ligase. Another important pathway for cell cycle regulation is the pathway involving SCF (Skp1-Cullin-F-box) ubiquitin ligase. The SCF-pathway mainly regulates the progression of G1- and S-phases of the cell cycle. Furthermore, several putative SCF components have been reported to be involved in many cell regulations, e.g., a DNA damage checkpoint mechanism, maintenance of chromosomes and other regulations in fission yeast and other organisms.
--However, ubiquitin-conjugating enzyme(s) responsible for degradation of substrates of SCF ubiquitin ligase in fission yeast have not been elucidated yet. We found that polyubiquitinated proteins were significantly reduced in whole cell extract from ubcP1/ubc4 mutant. It is possible that the substrates of SCF are also ubiquitinated by the UbcP1/Ubc4-pathway. Thus, I am examining the stability of the substrates of SCF ubiquitin ligase Cdc18 in ubcP1/ubc4 mutant cells, and the results are now being obtained.