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© The Rockefeller University Press, 0021-9525/1999//257 $5.00
The Journal of Cell Biology, Volume 147, Number 2, , 1999 257-266

Original Article

Glycosylation Can Influence Topogenesis of Membrane Proteins and Reveals Dynamic Reorientation of Nascent Polypeptides within the Translocon



Veit Godera, Christoph Bieria, and Martin Spiessa

a Biozentrum, University of Basel, CH-4056 Basel, Switzerland
Department of Biochemistry, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland.41-61-267214941-61-2672164

spiess{at}ubaclu.unibas.ch

The topology of multispanning membrane proteins in the mammalian endoplasmic reticulum is thought to be dictated primarily by the first hydrophobic sequence. We analyzed the in vivo insertion of a series of chimeric model proteins containing two conflicting signal sequences, i.e., an NH2-terminal and an internal signal, each of which normally directs translocation of its COOH-terminal end. When the signals were separated by more than 60 residues, linear insertion with the second signal acting as a stop-transfer sequence was observed. With shorter spacers, an increasing fraction of proteins inserted with a translocated COOH terminus as dictated by the second signal. Whether this resulted from membrane targeting via the second signal was tested by measuring the targeting efficiency of NH2-terminal signals followed by polypeptides of different lengths. The results show that targeting is mediated predominantly by the first signal in a protein. Most importantly, we discovered that glycosylation within the spacer sequence affects protein orientation. This indicates that the nascent polypeptide can reorient within the translocation machinery, a process that is blocked by glycosylation. Thus, topogenesis of membrane proteins is a dynamic process in which topogenic information of closely spaced signal and transmembrane sequences is integrated.

Key Words: endoplasmic reticulum • glycosylation • integral membrane protein • signal recognition particle • signal sequence

© 1999 The Rockefeller University Press

1.used in this paper: ASGP, asialoglycoprotein; endo H, endo-β-d-N-acetyl glucosaminidase H; SRP, signal recognition particle


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