Early Events in Glycosylphosphatidylinositol Anchor Addition: SUBSTRATE PROTEINS ASSOCIATE WITH THE TRANSAMIDASE SUBUNIT Gpi8p

Tracey D Spurway,Jane A Dalley,Stephen High,Neil J Bulleid

doi:10.1074/jbc.m010128200

Tracey D Spurway, Jane A Dalley + Show 2 more

Open Access

https://doi.org/10.1074/jbc.m010128200

Copy DOI

Abstract

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p. By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and conversely that GPI anchor addition does not require protein folding. These results demonstrate that folding of the ectodomain and GPI addition are two distinct processes and can be mutually exclusive. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The ability of the transamidase to recognize and associate with GPI anchor signal sequences provides an explanation for the retention of GPI-anchored protein within the ER in the absence of GPI anchor addition.

Highlights

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation
We show that placental alkaline phosphatase becomes efficiently GPI-anchored when translated in the presence of semipermeabilized K562 cells but is not GPI-anchored in cell lines defective in the transamidase subunit hGpi8p
By studying the synthesis of placental alkaline phosphatase, we demonstrate that folding of the protein is not influenced by the addition of a GPI anchor and that GPI anchor addition does not require protein folding

Summary

Introduction

The addition of glycosylphosphatidylinositol (GPI) anchors to proteins occurs by a transamidase-catalyzed reaction mechanism soon after completion of polypeptide synthesis and translocation. When GPI addition is prevented, either by synthesis of the protein in the presence of cell lines defective in GPI addition or by mutation of the GPI carboxyl-terminal signal sequence cleavage site, the substrate forms a prolonged association with the transamidase subunit hGpi8p. The amino group on the terminal ethanolamine of GPI attacks this intermediate to produce GPI-anchored protein [12, 13] This proposed reaction mechanism for GPI addition is widely accepted, isolation of an active enzyme capable of such anchor attachment has not been achieved to date. Stages of GPI Anchor Addition to Proteins with each other and with the precursor protein and GPI substrates remains to be elucidated

Objectives

Methods

Results