C-TERMINAL PROCESSING OF NASCENT PROPROTEINS

Case Western Reserve University, Cleveland OH

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Abstract

Membrane anchoring via glycosylphosphatidyl inositol (GPI) structures is a ubiquitous mechanism whereby many functionally diverse proteins are linked to cell surfaces, and by means of which any protein of interest can be experimentally attached to cells. From previous work by several labs including our own, the biosynthetic pathway that provides for GPI assembly has been characterized and the genes that encode several of the enzymes that mediate these reactions have been cloned. In recent studies, our laboratory has generated a novel GPI-anchoring-defective mutant line (designated K) which accumulates complete GPI precursors but does not transfer them to acceptor proproteins. Our previous studies have established that this mutant is defective in a transamidation reaction which mediates this transfer. We have cloned the affected gene and shown that it corresponds to hGPI8, a homologue of a yeast gene, yGPI8, which appears to be the transamidase. In other collaborative work we have cloned a second human gene termed hGAA1 which encodes another factor required for the transamidation. In further work we have shown that a deacylation reaction which removes a fatty acid from the transfered GPI is closely coupled to the transamidation reaction. The purpose of the current proposal is to build upon this work so as to further clarify the post-assembly steps in GPI anchoring. Specifically, our aims are 1) To map the active sites of hGpi8p/hGaa1p and investigate their substrate specificity, 2) To utilize complementary yeast and mammalian systems to search for associated components, 3) To develop a solubilized/reconstituted system to study the transamidation reaction, and 4) To characterize the deacylase activity that removes inositol-linked acyl chains from the GPI moiety after its transfer. The data obtained should contribute to basic understanding of GPI-anchored protein metabolism. It could also have therapeutic relevance for fungal and parasitic infectious diseases as well as relevance for engineering cell surface expression of GPI-anchored proteins.