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: This approach was first applied to evaluate the localization of full-length NA with natural hydrophobic (ΔGapp = −0.7 kcal/mol) and marginally hydrophobic (ΔGapp = +1.3) Nin-Cout TMDs. Including the epitope tag, both constructs possess a long, 440-aa C-tail that follows the TMD, and from here on are referred to by the nomenclature TM∆G XNAZaa, where X is the predicted TMD hydrophobicity (ΔGapp value in kcal/mol) and Z is the C-tail length in amino acids (aa). As expected, both full-length NA constructs (TM∆G −0.7NA440aa and TM∆G +1.3NA440aa) localized to the PM with an average PM/IC ratio >2 (Figure 2A). Similarly, full-length M2 with a hydrophobic TMD (ΔGapp = −1.1 kcal/mol) and a C-tail of 70 aa (including the epitope tag) also localized to the PM.