GSBS Student Publications

Title

Integration of membrane proteins into the endoplasmic reticulum requires GTP

UMMS Affiliation

Graduate School of Biomedical Sciences; Department of Biochemistry and Molecular Biology; Department of Molecular Genetics and Microbiology

Date

7-1-1988

Document Type

Article

Medical Subject Headings

Cell Membrane; Centrifugation, Density Gradient; Chromatography, Gel; Electrophoresis, Polyacrylamide Gel; Endoplasmic Reticulum; Guanosine Triphosphate; Guanylyl Imidodiphosphate; HN Protein; Hemagglutinins, Viral; Membrane Glycoproteins; Microsomes; Newcastle disease virus; Protein Biosynthesis; Protein Processing, Post-Translational; Protein Sorting Signals; RNA, Messenger; Ribosomes; Transcription, Genetic; Vesicular stomatitis Indiana virus; Viral Envelope Proteins; Viral Matrix Proteins

Disciplines

Life Sciences | Medicine and Health Sciences

Abstract

We have examined the requirement for ribonucleotides and ribonucleotide triphosphate hydrolysis during early events in the membrane integration of two membrane proteins: the G protein of vesicular stomatitis virus and the hemagglutinin-neuraminidase (HN) glycoprotein of Newcastle disease virus. Both proteins contain a single transmembrane-spanning segment but are integrated in the membrane with opposite orientations. The G protein has an amino-terminal signal sequence and a stop-transfer sequence located near the carboxy terminus. The HN glycoprotein has a single sequence near the amino terminus that functions as both a signal-sequence and a transmembrane-spanning segment. Membrane insertion was explored using a cell-free system directed by transcribed mRNAs encoding amino-terminal segments of the two proteins. Ribosome-bound nascent polypeptides were assembled, ribonucleotides were removed by gel filtration chromatography, and the ribosomes were incubated with microsomal membranes under conditions of defined ribonucleotide content. Nascent chain insertion into the membrane required the presence of both the signal recognition particle and a functional signal recognition particle receptor. In the absence of ribonucleotides, insertion of nascent membrane proteins was not detected. GTP or nonhydrolyzable GTP analogues promoted efficient insertion, while ATP was comparatively ineffective. Surprisingly, the majority of the HN nascent chain remained ribosome associated after puromycin treatment. Ribosome-associated HN nascent chains remained competent for membrane insertion, while free HN chains were not competent. We conclude that a GTP binding protein performs an essential function during ribosome-dependent insertion of membrane proteins into the endoplasmic reticulum that is unrelated to protein synthesis.

Rights and Permissions

Citation: J Cell Biol. 1988 Jul;107(1):69-77.

Related Resources

Link to Article in PubMed

Journal Title

The Journal of cell biology

PubMed ID

2839521