Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.
Bottom Line: Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity.Overall the cotranslational inversion of marginally hydrophobic NA TMDs initiates once ~70 amino acids past the TMD are synthesized, and the efficiency reaches 50% by ~100 amino acids, consistent with the positioning of this TMD class in type II human membrane proteins.Inversion of the M2 TMD, achieved by elongating its C-terminus, underscores the contribution of cotranslational synthesis to TMD inversion.
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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.