Integration of a small integral membrane protein, M₂, of influenza virus into the endoplasmic reticulum: Analysis of the internal signal-anchor domain of a protein with an ectoplasmic NH₂ terminus

The M₂ protein of influenza A virus is a small integral membrane protein of 97 residues that is expressed on the surface of virus-infected cells. M₂ has an unusual structure as it lacks a cleavable signal sequence yet contains an ectoplasmic amino-terminal domain of 23 residues, a 19 residue hydrophobic transmembrane spanning segment, and a cytoplasmic carboxyl-terminal domain of 55 residues. Oligonucleotide-mediated deletion mutagenesis was used to construct a series of M₂ mutants lacking portions of the hydrophobic segment. Membrane integration of the M₂ protein was examined by in vitro translation of synthetic mRNA transcripts prepared using bacteriophage T₇ RNA polymerase. After membrane integration, M₂ was resistant to alkaline extraction and was converted to an M(r) ≃ 7,000 membrane-protected fragment after digestion with trypsin. In vitro integration of M₂ requires the cotranslational presence of the signal recognition particle. Deletion of as few as two residues from the hydrophobic segment of M₂ markedly decreases the efficiency of membrane integration, whereas deletion of six residues completely eliminates integration. M₂ proteins containing deletions that eliminate stable membrane anchoring are apparently not recognized by signal recognition particles, as these polypeptides remain sensitive to protease digestion, indicating that in addition they do not have a functional signal sequence. These data thus indicate that the signal sequence that initiates membrane integration of M₂ resides within the transmembrane spanning segment of the polypeptide.