The two yeast members of the CLIP-170/Bik1p and Lis1/Pac1p families of microtubule-associated proteins are shown to interact with the sumoylation machinery and the STUbL complex Ris1p–Nis1p. Pac1p can be modified by both SUMO and ubiquitin. The She1 regulator of dynactin is identified as a novel inhibitor of Pac1p modification.

Stu2p is the yeast member of the XMAP215/Dis1/ch-TOG family of microtubule-associated proteins that promote microtubule polymerization. However, the factors that regulate its activity are not clearly understood. Here we report that Stu2p in the budding yeast Saccharomyces cerevisiae interacts with SUMO by covalent and noncovalent mechanisms. Stu2p interacted by two-hybrid analysis with the yeast SUMO Smt3p, its E2 Ubc9p, and the E3 Nfi1p. A region of Stu2p containing the dimerization domain was both necessary and sufficient for interaction with SUMO and Ubc9p. Stu2p was found to be sumoylated both in vitro and in vivo. Stu2p copurified with SUMO in a pull-down assay and vice versa. Stu2p also bound to a nonconjugatable form of SUMO, suggesting that Stu2p can interact noncovalently with SUMO. In addition, Stu2p interacted with the STUbL enzyme Ris1p. Stu2p also copurified with ubiquitin in a pull-down assay, suggesting that it can be modified by both SUMO and ubiquitin. Tubulin, a major binding partner of Stu2p, also interacted noncovalently with SUMO. By two-hybrid analysis, the beta-tubulin Tub2p interacted with SUMO independently of the microtubule stressor, benomyl. Together, these findings raise the possibility that the microtubule polymerization activities mediated by Stu2p are regulated through sumoylation pathways.

A crucial step in the initiation of cap dependent translation of eukaryotic mRNA is the interaction of the mRNA cap-binding protein, eIF4E, with eIF4G, two integral components of the mRNA cap-binding complex. The present study was aimed to express human eIF4E at high level in E. coli. For high-level expression, the E. coli cells were first transformed with an expression vector that contains the cloned gene, human eIF4E. Following transformation, the recombinant plasmid was purified and stepwise digestion of the recombinant plasmid revealed that the plasmid was harboring an insert, equal to the length of the eIF4E gene. As the vector contained a heat-inducible promoter, high-level expression of human eIF4E was carried out by heat induction at 42°C. Expression of recombinant protein was observed by comparing the cell extracts collected before and after heat induction and their subsequent analysis on a denatured polyacrylamide gel. The appearance of a 24-kDa protein following induction of transformed cells confirmed the expression of the cloned gene, human eIF4E. Key words: Translation initiation, cap-binding protein, eIF4E Dhaka Univ. J. Pharm. Sci. Vol.5(1-2) 2006 The full text is of this article is available at the Dhaka Univ. J. Pharm. Sci. website