A cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of glycosylphosphatidylinositol-anchored proteins.

C Lanctôt,S Howell,P Crine,G Boileau

doi:10.1016/s0021-9258(17)32508-5

C Lanctôt, S Howell + Show 2 more

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https://doi.org/10.1016/s0021-9258(17)32508-5

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Journal: Journal of Biological Chemistry	Publication Date: Jun 1, 1994
Citations: 21	License type: cc-by

Affiliation: Université de Montréal

Abstract

During the biosynthesis of glycosylphosphatidylinositol (GPI)-anchored proteins, an N-terminal signal peptide is used to direct biosynthesis to the endoplasmic reticulum. It was previously unknown whether or not this signal must be removed during the biosynthesis of GPI-anchored proteins. Using neutral endopeptidase (EC 3.4.24.11), a well characterized type II membrane protein that is attached to the membrane via an uncleaved N-terminal signal peptide, we extended its C terminus with 33 of the 37 amino acids of the GPI anchor signal sequence of decay-accelerating factor. When expressed in COS-1 and Chinese hamster fibroblast (CHW) cells, the protein was shown to possess both transmembrane and GPI anchors, indicating that a cleavable N-terminal signal peptide is not a prerequisite for the biosynthesis of GPI-anchored proteins.

Highlights

Neutral endopeptidase (NEP) ias type I1 membrane protein anchored to the cell surfaceby an uncleaved N-terminal signal peptide [8].By extending the C terminus of NEP with p a r t of the GPI anchor attachment sequencoef decay-accelerating factor, we showthat a cleavable N-terminal signal peptide is naot prerequisite for the biosynthesis of a GPI-anchored protein
PSVDANEP should result in theproduction of terminal signal peptide, we extenidtseCd terminus with NEP attached to the cell surface via an uncleaved N-terminal signal
The presence of the N-terminal cytoplasmic tail was only detected upon permeabilization of transfected cells prior to im

Summary

Introduction

Neutral endopeptidase (NEP) ias type I1 membrane protein anchored to the cell surfaceby an uncleaved N-terminal signal peptide [8].By extending the C terminus of NEP with p a r t of the GPI anchor attachment sequencoef decay-accelerating factor, we showthat a cleavable N-terminal signal peptide is naot prerequisite for the biosynthesis of a GPI-anchored protein. Ethanolamine labeling was performed as described previously [11]on COS-1 cells transfected with pSVEnk, pSVGPINEP, and pSVDANEP, on wild-typeCHW cells, and on CHW cells expressing DA-NEP. Immunoprecipitated incubation of microsomal membranes from the CHW cell line express- proteins were subjected to 7.5% SDS-PAGE and fluorography.

Results

Conclusion