Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition

Silvia Brambillasca, Monica Yabal, Paolo Soffientini, Sandra Stefanovic, Marja Makarow, Ramanujan S. Hegde, Nica Borgese

Research output: Contribution to journalArticlepeer-review

Abstract

A large class of proteins with cytosolic functional domains is anchored to selected intracellular membranes by a single hydrophobic segment close to the C-terminus. Although such tail-anchored (TA) proteins are numerous, diverse, and functionally important, the mechanism of their transmembrane insertion and the basis of their membrane selectivity remain unclear. To address this problem, we have developed a highly specific, sensitive, and quantitative in vitro assay for the proper membrane-spanning topology of a model TA protein, cytochrome b5 (b5). Selective depletion from membranes of components involved in cotranslational protein translocation had no effect on either the efficiency or topology of b5 insertion. Indeed, the kinetics of transmembrane insertion into protein-free phospholipid vesicles was the same as for native ER microsomes. Remarkably, loading of either liposomes or microsomes with cholesterol to levels found in other membranes of the secretory pathway sharply and reversibly inhibited b5 transmembrane insertion. These results identify the minimal requirements for transmembrane topogenesis of a TA protein and suggest that selectivity among various intracellular compartments can be imparted by differences in their lipid composition.

Original languageEnglish
Pages (from-to)2533-2542
Number of pages10
JournalEMBO Journal
Volume24
Issue number14
DOIs
Publication statusPublished - Jul 20 2005

Keywords

  • Endoplasmic reticulum
  • Membrane proteins
  • Protein targeting
  • Protein translocation
  • Sec61 translocon

ASJC Scopus subject areas

  • Genetics
  • Cell Biology

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