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Published online 27 November 2006. doi:10.1083/jcb.200608101
The Rockefeller University Press, 0021-9525 $8.00
JCB, Volume 175, Number 5, 767-777
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Article

 Unassisted translocation of large polypeptide domains across phospholipid bilayers

Silvia Brambillasca1,2, Monica Yabal3,4, Marja Makarow3,4, and Nica Borgese1,2,5

1 Cellular and Molecular Pharmacology Section, Consiglio Nazionale delle Ricerche Institute of Neuroscience, and 2 Department of Medical Pharmacology, University of Milan, 20129 Milan, Italy
3 Program of Cellular Biotechnology, Institute of Biotechnology, and 4 Department of Applied Chemistry and Microbiology, 00014 University of Helsinki, Helsinki, Finland
5 Faculty of Pharmacy, University of Catanzaro Magna Graecia, Roccelletta di Borgia, 88021 Catanzaro, Italy

Correspondence to Nica Borgese: n.borgese{at}in.cnr.it

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.

Abbreviations used in this paper: b5, cytochrome b(5); CPY, carboxypeptidase Y; PC, phosphatidylcholine; PF, protected fragment; PK, protease K; PTP1B, protein tyrosine phosphatase 1B; RM, rough microsome; SRP, signal recognition particle; Syb, synaptobrevin; TA, tail anchored; TB, translocation buffer; TMD, transmembrane domain; VSVG, vesicular stomatitis virus glycoprotein.


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