NSF Award Abstract - #0215924

AWSFL008-DS3

C-RUI Collaborative Project: Intracellular and Structural Aalysis of Roles of a
Cytokine Precursor in Aminoacyl-tRNA Synthetase Complexes

Abstract

Aminoacyl-tRNA synthetase complexes play a key role in protein biosynthesis. An understanding of the structure and distribution of these complexes and their variants is fundamental to understanding the mechanisms and regulation of this basic biological process, which critically impacts cell growth, maintenance and death. The core multisynthetase complex contains nine aminoacyl-tRNA synthetase activities and three auxiliary proteins, p43, p38, and p18. A tRNA binding protein, p43 enhances aminoacylation activity of specific synthetases within the complex. Metabolic stress induces cleavage of p43 at a caspase-like site as well as release and secretion of the C-terminal half of the protein (EMAPII), which functions as an inflammatory cytokine. Due to its central location in the core multienzyme aminoacyl-tRNA synthetase complex, a likely function of p43 is to maintain structural integrity of this particle. This project will determine the effect of metabolic stress on p43 cleavage and stability of the complex. Specifically, mammalian and non-mammalian cells will be grown under various adverse conditions. Multisynthetase complexes will be purified from these cultures and their compositions and structures characterized. The cellular localization and distribution of complexes, of tagged p43 and of its processed components will be quantitated by immunoblot analysis of subcellular fractions. This collaborative research project will be performed primarily by undergraduate students who will receive training in molecular and structural biology.