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

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, hybdrid genes were constructed and expressed in which regions of the M₂ 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 M₂ 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.