Processing of Nascent Proteins to Glycosylphosphatidylinositol-Anchored forms in Cell-Free Systems

Abstract

This chapter discusses the processing of nascent proteins to glycosylphosphatidylinositol (GPI)-anchored forms in cell-free systems. Many cell types express GPI proteins on the cell surface or when transfected with an appropriate cDNA. Thus, the enzymes, the GPI precursor, and any coenzymes or accessory factors required for processing a nascent protein to the GPI form are present in most cells. Additional requirements for carrying out cell-free studies are a nascent protein or its equivalent and methods for specifically identifying the products. In the case of NH2-terminal signal processing by rough microsomal membranes (RM) or purified signal peptidase, it is possible to use small synthetic peptides containing a typical NH2-terminal signal peptide and the appropriate –1, –3 substituents as substrates. The corresponding type of experiments is feasible with small synthetic peptides containing a typical COOH-terminal signal peptide and appropriate ω and ω + 2 substituents. The 40- to 60-mers representing the COOH terminus of placental alkaline phosphatase (PLAP) are synthesized and exposed to rough microsomal membranes (RM). In both cases, cleavage products are formed; they migrate with Mr values consistent with appropriate cleavage. © 1995 Elsevier Inc.