RING box protein-1 gene involved in flagellar disassembly of Dunaliella salina.

Abstract

Ring box protein-1 (RBX1), also called Regulator of Cullins-1 (ROC1), is a key component of SCF (Skp-1, cullins, F-box proteins) E3 ubiquitin ligases, which regulate diverse cellular processes by targeting protein substrates for degradation. Although RBX1 plays an important role in ubiquitination machinery of both prokaryotes and eukaryotes, studies on the RBX1 have not been involved in the unicellular green alga Dunaliella salina. In this study, a full-length RBX1 cDNA fragment of 817bp was cloned using rapid amplification of cDNA end (RACE) technique. The full-length sequence contained an open reading frame of 411bp encoding 136 amino acids. The predicted protein had a molecular molar mass of 14.8kDa and pI of 5.9 with a high degree of homology to RBX1 from Chlamydomonas reinhardtii (92%). Recombinant RBX1 was expressed in Escherichia coli BL21 and was purified and characterized. The apparent molecular mass of the recombinant protein was approximately 17kDa, and the optimal induction time and concentration were 3h and 0.1mmol/L IPTG, respectively. The predicted 3D structures of RBX1 proteins contained RING-H2 finger domain including "Cys59-X2-Cys62-X30-Cys93-X1-His95-X2-His98-X2-Cys101-X10-Cys112-X2-Cys115." The expression of RBX1 protein was increased by 132% during flagellar disassembly and decreased by 76% during flagellar assembly of D. salina. The expression of RBX1 mRNA had a similar tendency with the expression of RBX1 protein. The results indicated that RBX1 responded to flagellar disassembly of D. salina.