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: 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.