Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition.
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.
CNR Institute of Neuroscience - Cell Mol Pharmacology - and Department of Medical Pharmacology, University of Milan, Milan, Italy.
Silvia
S
Brambillasca
Monica
M
Yabal
Paolo
P
Soffientini
Sandra
S
Stefanovic
Marja
M
Makarow
Ramanujan S
RS
Hegde
Nica
N
Borgese
D000818:Animals; D015786:Cytochromes b5 / Q000378:metabolism*; D004285:Dogs; D006801:Humans; D007425:Intracellular Membranes / Q000201:enzymology / Q000378:metabolism*; D008563:Membrane Lipids / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D018832:Molecular Chaperones; D017434:Protein Structure, Tertiary; D021381:Protein Transport / Q000502:physiology; D011510:Proteolipids / Q000378:metabolism; D016601:RNA-Binding Proteins; D013552:Swine
D008563:Membrane Lipids; D008565:Membrane Proteins; D018832:Molecular Chaperones; D011510:Proteolipids; D016601:RNA-Binding Proteins; C487464:SEC63 protein, human; C020485:proteoliposomes; D015786:Cytochromes b5