BIOSYNTHESIS OF RENAL GAMMA-GLUTAMYLTRANSPEPTIDASE

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The proximal tubule cell of the kidney, which is responsible for the vectorial transport of solutes and fluid, is characterized by an asymmetric plasma membrane which gives the cell distinct polarity. The mechanism by which the cell achieves and maintains a basal lateral plasma membrane distinct from an apical lumenal brush border membrane is unknown. Recent experiments in an analogous cell type (intestinal villus cells) suggest that glycoproteins are inserted directly into both membranes, but some in the basal lateral membrane migrate by a microtubule-dependent process into the brush border membrane. The objective of this proposal is to study renal brush border membrane biogenesis utilizing Gamma-glutamyltranspeptidase as a specific probe. This brush border membrane hydrolase is synthesized as a single glycoprotein (propeptide) and the majority of it is cleaved either co- or posttranslationally to yield a heterodimer structure. The larger subunit contains the membrane anchor while the smaller subunit contains a portion of the active site. The residual uncleaved propeptide is apparently processed and inserted into the brush border membrane with kinetics similar to the heterodimer structure before it is cleaved by a secondary proteolytic reaction. The specific aims of this proposal include: 1) determination of the subcellular route of insertion of transpeptidase into the brush border membrane of cultured renal cells; 2) partial characterization of the oligosaccharide moieties on purified transpeptidase and determination of the kinetics of their processing in vivo; 3) identification of the primary translation product of the transpeptidase and the basis for incomplete heterodimer production in the endoplasmic reticulum; and 4) determination of the fate of transpeptidase propeptide in the brush border membrane.