The Endoplasmic Reticulum (ER) Translocon Can Differentiate between Hydrophobic Sequences Allowing Signals for Glycosylphosphatidylinositol Anchor Addition to Be Fully Translocated into the ER Lumen

Jane A Dalley,Neil J Bulleid

doi:10.1074/jbc.m303978200

Abstract

The signal sequence within polypeptide chains that designates whether a protein is to be anchored to the membrane by a glycosylphosphatidylinositol (GPI) anchor is characterized by a carboxyl-terminal hydrophobic domain preceded by a short hydrophilic spacer linked to the GPI anchor attachment (omega) site. The hydrophobic domain within the GPI anchor signal sequence is very similar to a transmembrane domain within a stop transfer sequence. To investigate whether the GPI anchor signal sequence is translocated across or integrated into the endoplasmic reticulum membrane we studied the translocation, GPI anchor addition, and glycosylation of different variants of a model GPI-anchored protein. Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence. However, a single amino acid change within the hydrophobic domain of the GPI-signal converts it into a transmembrane domain that is fully integrated into the endoplasmic reticulum membrane. These results demonstrated that the translocation machinery can recognize and differentiate subtle changes in hydrophobic sequence allowing either full translocation or membrane integration.

Highlights

Introduction of glycosylation sequons within miniPLAP and minimembrane was achieved by site-directed mutagenesis using the following sense primers: G1 glycosylation mutant A175N, 5Ј-CTGGCGCCCCCCAACGGCACCACCGAC-3Ј; G2 glycosylation mutant T204N, 5Ј-CTGCTGGAGAACGCCACTGCTCCC-3Ј
Our results unequivocally demonstrated that the hydrophobic domain within a GPI signal cannot act as a transmembrane domain and is fully translocated even when followed by an authentic charged cytosolic tail sequence
We have previously shown that the initial stages of GPI anchor addition can be faithfully reconstituted by translating a model GPI-anchored protein in the presence of SP cells derived from cells grown in culture [16]

Summary

Introduction

PLAP and minimembrane was achieved by site-directed mutagenesis using the following sense primers: G1 glycosylation mutant A175N, 5Ј-CTGGCGCCCCCCAACGGCACCACCGAC-3Ј; G2 glycosylation mutant T204N, 5Ј-CTGCTGGAGAACGCCACTGCTCCC-3Ј. Introduction of the glycosylation sequon within TM-VSVG (Ile 3 Asn) was achieved by site-directed mutagenesis using the following sense primer: 5Ј-CCCGGGGCGGTCTAACGCCTCTTTT-3Ј. Mutagenesis of Glu-203 to Leu in signal VSVG was achieved by site-directed mutagenesis using sense primer 5Ј-CTGCTGCTGCTGCTGACGGCCACTGCTCCC-3Ј, and in signal VSVG-G was achieved using 5Ј-CTGCTGCTGCTGCTGAACGCCACTGCTCCC-3Ј. Transcription in Vitro—Transcription reactions were carried out as described previously [23]. Preparation of Semipermeabilized Cells—The human lymphoblastoid cell line K562 was obtained from the European collection of animal cell cultures. The cell line was cultured in RPMI 1640 medium supplemented with 10% fetal calf serum. Semipermeabilized (SP) cells were prepared by treatment with digitonin as described previously [24]

Methods

Results

Conclusion