Article
- The EMBO Journal (2005) 24, 2533 - 2542
- doi:10.1038/sj.emboj.7600730
Published online: 23 June 2005
Subject Categories:
Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition
Silvia Brambillasca1, Monica Yabal2, Paolo Soffientini1,a, Sandra Stefanovic3, Marja Makarow2, Ramanujan S Hegde3 and Nica Borgese1,4
- CNR Institute of Neuroscience – Cell Mol Pharmacology – and Department of Medical Pharmacology, University of Milan, Milan, Italy
- Program of Cellular Biotechnology, Institute of Biotechnology and Department of Applied Chemistry and Microbiology, University of Helsinki, Helsinki, Finland
- Cell Biology and Metabolism Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, USA
- Faculty of Pharmacy, University of Catanzaro Magna Graecia, Roccelletta di Borgia (CZ), Italy
Correspondence to:
Ramanujan S Hegde, Cell Biology and Metabolism Branch, NICHD, National Institutes of Health, 18 Library Drive, Bldg. 18, Room 101, Bethesda, MD 20892, USA. Tel.: +1 301 496 4855; Fax: +1 301 402 0078; E-mail: hegder@mail.nih.gov
Nica Borgese, CNR Institute of Neuroscience/Cell Mol Pharmacology, via Vanvitelli 32, 20129 Milano, Italy. Tel.: +39 02 503 16971; Fax: +39 02 749 0574; E-mail: n.borgese@in.cnr.it
aPresent address: Istituto Nazionale per lo Studio e la Cura dei Tumori – Unità Trapianto Midollo 'Cristina Ghandini', 20133 Milano, Italy
Received 1 February 2005; Accepted 6 June 2005
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.
Keywords:
- endoplasmic reticulum,
- membrane proteins,
- protein targeting,
- protein translocation,
- Sec61 translocon
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