Renal proximal tubule epithelial cell is actively engaged in reabsorption of a large number of compounds and fluid. This transport occurs across a specialized structure of the cell, the brush border, which consists of numerous, packed microvilli facing the renal tubular lumen. The biochemical processes by which the epithelial cell generates and maintains the microvillus membranes distinct from the basal-lateral membranes is unknown. The overal objectives of this research proposal are to study mechanisms whereby the cell achieves and maintains this polarity. Three microvillus enzymes involved in the cell surface degradation of glutathione, gamma-glutamyl transpeptidase, aminopeptidase M, and a dipeptidase, will be used as model systems to study the biogenesis, processing, and intracellular topogenesis of microvillus proteins.
The specific aims i nclude: 1. Structural and Topological Studies isolation, characterization, and processing of the single-chain precursor of the dimeric enzyme, gamma-glutamyl transpeptidase; the structural basis for the association of these enzymes with the membranes and their topology within the membranes. 2. Topogenesis of the Microvillus Enzymes - intracellular transport and sorting mechanisms for achieving their final destination; the initial events in the biosynthesis and processing of the three enzymes. The methods of procedure include classical methods of protein and enzyme chemistry, immunochemistry, immunoelectron microscopy, pulse-chase studies with kidney slices, and studies on the in vitro translation products of the kidney mRNA.
