The CYC3 gene of Trypanosoma brucei encodes a cyclin with a short half-life

Abstract

Recently, we identified two Trypanosoma brucei cyclin genes, CYC2 and CYC3, by rescue of the Saccharomyces cerevisiae mutant DL1, which is deficient in CLN G1 cyclin function. CYC3 has a low level of sequence identity to mitotic B-type cyclins from a variety of organisms. In order to examine whether CYC3 associates in vivo with a trypanosome cdc2-related kinase (CRK), the CYC3 gene was fused with the TY-epitope tag, integrated into the trypanosome genome and expressed under inducible control. CYC3ty was demonstrated to associate with the CRK-binding factor p12 cks1 and histone H1 kinase activity could be detected in CYC3ty immune precipitated fractions, which demonstrates that CYC3ty associates in vivo with an active trypanosome CRK. Both CYC3ty and CYC2ty were shown to have a half-life of less than one cell cycle, which was significantly elongated by specific proteasome inhibitors, strongly suggesting that CYC3ty and CYC2ty are substrates for proteasome degradation. This is consistent with the presence in CYC3 of a putative destruction box motif that defines proteins for degradation via the ubiquitin degradation pathway. These results are consistant with proteolysis by the proteasome being involved in regulation of the cellular cyclin concentration in trypanosomes.