Unassisted translocation of large polypeptide domains across phospholipid bilayers.
Although transmembrane proteins generally require membrane-embedded machinery for integration, a few can insert spontaneously into liposomes. Previously, we established that the tail-anchored (TA) protein cytochrome b(5) (b5) can posttranslationally translocate 28 residues downstream to its transmembrane domain (TMD) across protein-free bilayers (Brambillasca, S., M. Yabal, P. Soffientini, S. Stefanovic, M. Makarow, R.S. Hegde, and N. Borgese. 2005. EMBO J. 24:2533-2542). In the present study, we investigated the limits of this unassisted translocation and report that surprisingly long (85 residues) domains of different sequence and charge placed downstream of b5's TMD can posttranslationally translocate into mammalian microsomes and liposomes at nanomolar nucleotide concentrations. Furthermore, integration of these constructs occurred in vivo in translocon-defective yeast strains. Unassisted translocation was not unique to b5 but was also observed for another TA protein (protein tyrosine phosphatase 1B) whose TMD, like the one of b5, is only moderately hydrophobic. In contrast, more hydrophobic TMDs, like synaptobrevin's, were incapable of supporting unassisted integration, possibly because of their tendency to aggregate in aqueous solution. Our data resolve long-standing discrepancies on TA protein insertion and are relevant to membrane evolution, biogenesis, and physiology.
Brambillasca
Silvia
S
Cellular and Molecular Pharmacology Section, Consiglio Nazionale delle Ricerche Institute of Neuroscience, University of Milan, 20129 Milan, Italy.
Yabal
Monica
M
Makarow
Marja
M
Borgese
Nica
N
D002462:Cell Membrane / Q000378:metabolism*; D008051:Lipid Bilayers / Q000737:chemistry*; D008081:Liposomes / Q000378:metabolism; D008957:Models, Genetic; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D010743:Phospholipids / Q000737:chemistry*; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D017027:Protein Tyrosine Phosphatases / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
D008051:Lipid Bilayers; D008081:Liposomes; D010455:Peptides; D010743:Phospholipids; D021382:Protein Sorting Signals; D029701:Saccharomyces cerevisiae Proteins; D017027:Protein Tyrosine Phosphatases