Covalent attachment of the small, highly-conserved protein ubiquitin (Ub) to other cellular proteins appears to mediate the role of Ub in diverse biological processes, including progression of the cell cycle, DNA repair, and intracellular protein degradation. The applicant's long-term research goal is to understand how specific Ub-conjugating enzymes mediate these diverse physiological functions of Ub. This RCDA application is motivated by applicant's need to develop new expertise, in the area of molecular biology in order to realize this research goal. In the event of an award, the Department of Biochemistry will provide the applicant with release time from teaching and service, and support for a postdoctoral fellow. This will allow the applicant to develop new approaches in her research as a result of expanded interactions with other investigators in the Department and in the University, and through the opportunity to devote maximum effort to research during the period of the award. The proposed studies address the role for Ub in erythroid differentiation which is mediated by a novel, membrane-associated Ub-conjugating enzyme (E2-14Kp). It is proposed that this E2 specifically ubiquitinates mitochondrial proteins in erythroid cells, and thereby targets them for degradation during erythroid differentiation and maturation. Murine erythroleukemic (MEL) cells will be used to elucidate aspects of the molecular physiology of this E2 which are directly relevant to its proposed function; this will include in imunocytochemically localizing the E2 at the subcellular level, and determining the pattern of its expression and localization during differentiation in vitro. The question of whether E2- 14Kp mediates conjugation and degradation of mitochondrial proteins in differentiating MEL cells will analyzed, in part, by comparing normal and mutant MEL cells, the latter bearing null alleles at the E2 coding loci. The generation of the mutant cells will be carried out during a sabbatical away from Buffalo. This leave is a critical element in the area of molecular genetics. An RCDA award will make this research year possible.
| Wilkinson, K D; Tashayev, V L; O'Connor, L B et al. (1995) Metabolism of the polyubiquitin degradation signal: structure, mechanism, and role of isopeptidase T. Biochemistry 34:14535-46 |
