Abstract
Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.
Highlights
Proteins that are anchored to membranes via a glycosylphosphatidylinositol (GPl)l linkage, discovered relatively recently [1, 2], are known to be ubiquitous among eukaryotes where they serve a variety of important functions [3,4,5,6,7,8,9]
As the nascent chain is translocated into the endoplasmic reticulum the NH2-terminal signal peptide is removed giving rise to the proprotein
Tide from the proprotein occurs concomitantly with the condensation of the ethanolamine group of GPI with the COOH group of the newly formed COOH-terminal amino acid (w site). Both processes occur in a single step catalyzed by what has been assumed to be a transamidase. We have studied this process using rough microsomal membranes (RM) from various cells coupled to a translational system with mRNA coding for an engineered form of human placental alkaline phosphatase (PLAP) as a template
Summary
Vol 270, No 33, Issue of August 18, pp. 19576-19582, 1995 Printed in U.S.A. An Active Carbonyl Formed during Glycosylphosphatidylinositol Addition to a Protein Is Evidence of Catalysis by a Transamidase*. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. We have studied this process using rough microsomal membranes (RM) from various cells coupled to a translational system with mRNA coding for an engineered form of human placental alkaline phosphatase (PLAP) as a template In the latter, the catalytic domain and all the glycosylation sites were removed to form a much smaller product (miniPLAP). To obtain additional evidence that processing to GPI proteins involves a transamidase, we investigated hydrazine and hydroxylamine as possible substrates in our cell-free system
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