Transposition of domains between the M2 and HN viral membrane proteins results in polypeptides which can adopt more than one membrane orientation.

Abstract

Abstract. The influenza A virus M2 polypeptide is a small integral membrane protein that does not contain a cleaved signal sequence, but is unusual in that it assumes the membrane orientation of a class I integral membrane protein with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail. To determine the domains of M2 involved in specifying membrane orientation, hybrid genes were constructed and expressed in which regions of the ME protein were linked to portions of the paramyxovirus HN and SH proteins, two class II integral membrane proteins that adopt the opposite orientation in membranes from M2. A hybrid protein (MgMH) consisting of the M2 NH2-terminal and membrane-spanning domains linked precisely to the HN COOH-terminal ectodomain was found in cells in two forms: integrated into membranes in the ME topology or completely translocated across the endoplasmic reticulum membrane and ultimately secreted from the cell. The finding of a soluble form suggested that in this hybrid protein the anchor function of the M2 signal/anchor domain can be overridden. A second hybrid which contained the M2 NH2 terminus linked to the HN signal anchor and ectodomain (MgHH) was found in both the M2 and the HN orientation, suggesting that the M2 NH2 terminus was capable of reversing the topology of a class II membrane protein. The exchange of the M2 signal/anchor domain with that of SH resulted in a hybrid protein which assumed only the M2 topology. Thus, all these data suggest that the NH2-terminal 24 residues of M2 are important for directing the unusual membrane topology of the M2 protein. These data are discussed in relationship to the loop model for insertion of proteins into membranes and the role of charged residues as a factor in determining orientation. INTF.GRAL membrane proteins that span the lipid bilayer once can be divided into two major groups based on their topology with respect to the membrane (3, 37). The most common are the class I proteins, which are orientated with an NH2-terminal ectodomain and a COOH-terminal cytoplasmic tail, e.g., the low density lipoprotein receptor (26), vesicular stomatitis virus (VSV) G surface glycoprotein (25), and the influenza virus hemagglutinin (37). Class I proteins contain an NH2-terminal signal sequence which initiates the interaction of the growing polypeptide with the ER through binding with signal recognition particle (reviewed in reference 36), and this signal is ultimately cleaved in the lumen of the ER by signal peptidase. Translocation of class I proteins across the ER membrane is halted by a COOHterminal stop-transfer sequence which anchors the protein in the membrane (27,37).Class II membrane proteins are orientated with the NH2-terminal domain in the cytoplasm and a COOH-terminal ectodomain (37). By definition, class II membrane proteins do not contain a cleavable NH2-terminal signal peptide, but instead contain a stretch of hydrophobic residues near the NH2 terminus which acts as both an internal signal sequence directing integration into the membrane a...

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