title: Transmembrane protein insertion orientation in yeast depends on the charge difference across transmembrane segments, their total hydrophobicity, and its distribution.
abstract: The determinants of transmembrane protein insertion orientation at the endoplasmic reticulum have been investigated in Saccharomyces cerevisiae using variants of a Type III (naturally exofacial N terminus (Nexo)) transmembrane fusion protein derived from the N terminus of Ste2p, the alpha-factor receptor. Small positive and negative charges adjacent to the transmembrane segment had equal and opposite effects on orientation, and this effect was independent of N- or C-terminal location, consistent with a purely electrostatic interaction with response mechanisms. A 3:1 bias toward Nexo insertion, observed in the absence of a charge difference, was shown to reflect the Nexo bias conferred by longer transmembrane segments. Orientation correlated best with total hydrophobicity rather than length, but it was also strongly affected by the distribution of hydrophobicity within the transmembrane segment. The most hydrophobic terminus was preferentially translocated. Insertion orientation thus depends on integration of responses to at least three parameters: charge difference across a transmembrane segment, its total hydrophobicity, and its hydrophobicity gradient. Relative signal strengths were estimated, and consequences for topology prediction are discussed. Responses to transmembrane sequence may depend on protein-translocon interactions, but responses to charge difference may be mediated by the electrostatic field provided by anionic phospholipids.
authors.affiliation: Department of Molecular Genetics and Microbiology, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
authors.forename: C A
authors.initials: CA
authors.lastname: Harley
authors.forename: J A
authors.initials: JA
authors.lastname: Holt
authors.forename: R
authors.initials: R
authors.lastname: Turner
authors.forename: D J
authors.initials: DJ
authors.lastname: Tipper
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004721:Endoplasmic Reticulum / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D014644:Genetic Variation; D007425:Intracellular Membranes / Q000502:physiology* / Q000648:ultrastructure; D008561:Membrane Fusion; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology* / Q000648:ultrastructure; D055672:Static Electricity; D014157:Transcription Factors
chemical_list: D017931:DNA Primers; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors