title: Type II transmembrane domain hydrophobicity dictates the cotranslational dependence for inversion.
abstract: Membrane insertion by the Sec61 translocon in the endoplasmic reticulum (ER) is highly dependent on hydrophobicity. 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. Our results show that the marginally hydrophobic NA TMDs (ΔG(app) > 0 kcal/mol) require the cotranslational insertion process for facilitating their inversion during translocation and a positively charged N-terminal flanking residue and that NA inversion enhances its plasma membrane localization. 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.
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,  SE-106 91 Stockholm, Sweden.
authors.forename: Dan
authors.initials: D
authors.lastname: Dou
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,  SE-106 91 Stockholm, Sweden.
authors.forename: Diogo V
authors.initials: DV
authors.lastname: da Silva
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,  SE-106 91 Stockholm, Sweden.
authors.forename: Johan
authors.initials: J
authors.lastname: Nordholm
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,  SE-106 91 Stockholm, Sweden.
authors.forename: Hao
authors.initials: H
authors.lastname: Wang
authors.affiliation: Center for Biomembrane Research, Department of Biochemistry and Biophysics, Stockholm University,  SE-106 91 Stockholm, Sweden robertd@dbb.su.se.
authors.forename: Robert
authors.initials: R
authors.lastname: Daniels
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002462:Cell Membrane / Q000378:metabolism; D002522:Chlorocebus aethiops; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D053118:Influenza A Virus, H1N1 Subtype / Q000737:chemistry; D008861:Microsomes / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D012270:Ribosomes / Q000378:metabolism; D059010:Single-Cell Analysis; D014709:Vero Cells; D014763:Viral Matrix Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism; D000086082:Viroporin Proteins
chemical_list: D009439:Neuraminidase; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C488937:M2 protein, Influenza A virus; D000086082:Viroporin Proteins