TY - JOUR
T1 - Transmembrane topogenesis of a tail-anchored protein is modulated by membrane lipid composition
AU - Brambillasca, Silvia
AU - Yabal, Monica
AU - Soffientini, Paolo
AU - Stefanovic, Sandra
AU - Makarow, Marja
AU - Hegde, Ramanujan S.
AU - Borgese, Nica
PY - 2005/7/20
Y1 - 2005/7/20
N2 - 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.
AB - 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.
KW - Endoplasmic reticulum
KW - Membrane proteins
KW - Protein targeting
KW - Protein translocation
KW - Sec61 translocon
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UR - http://www.scopus.com/inward/citedby.url?scp=23044492259&partnerID=8YFLogxK
U2 - 10.1038/sj.emboj.7600730
DO - 10.1038/sj.emboj.7600730
M3 - Article
C2 - 15973434
AN - SCOPUS:23044492259
VL - 24
SP - 2533
EP - 2542
JO - EMBO Journal
JF - EMBO Journal
SN - 0261-4189
IS - 14
ER -