Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.
Bottom Line: This places stringent hydrophobicity requirements on transmembrane domains (TMDs) from single-spanning membrane proteins.On examining the single-spanning influenza A membrane proteins, we found that the strict hydrophobicity requirement applies to the N(out)-C(in) HA and M2 TMDs but not the N(in)-C(out) TMDs from the type II membrane protein neuraminidase (NA).To investigate this discrepancy, we analyzed NA TMDs of varying hydrophobicity, followed by increasing polypeptide lengths, in mammalian cells and ER microsomes.
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Mentions: To examine whether the cotranslational integration process contributes to TMD inversion more directly, we tested whether elongating the C-tail following the Nout-Cin M2 TMD could facilitate its inversion. As shown by the oxidation of M2 into homodimers and tetramers via its N-terminal Cys residues, full-length M2 with a 70-aa C-tail (including the epitope tag) possessed an Nout-Cin orientation at steady-state (Figure 8A). Similarly, when the 76-aa NA C-tail was fused to the M2 N-terminus and TMD, the Nout-Cin orientation remained unchanged, as no glycosylation of the NA C-tail was observed (Figure 8B, lanes 3 and 4). However, when the full-length 440-aa NA C-tail was fused to the M2 N-terminus and TMD, a reasonable amount of inversion to an Nin-Cout orientation occurred, based on glycosylation (Figure 8B, lanes 7 and 8) and the production of enzymatically active NA (Figure 8C). The ability to invert the hydrophobic M2 TMD in the absence of its single C-terminal positive charge upon substantially lengthening the 76-aa NA C-tail to 440 aa indicates that cotranslational synthesis and/or integration contributes to the inversion process. However, the production of mixed orientated species also implies that TMDs likely possess their own topology determinants in addition to their cytoplasmic-localized flanking residues.