title: The role of charged residues in determining transmembrane protein insertion orientation in yeast.
abstract: The first 79 residues of the yeast Ste2p G protein-coupled pheromone receptor, including the negatively charged N-terminal domain, the first transmembrane segment, and the following positively charged cytoplasmic loop, has been fused to a Kex2p-cleavable beta-lactamase reporter. Insertion orientation was determined by analysis of cell-associated and secreted beta-lactamase activities and independently corroborated by analysis of membrane association and glycosylation patterns. This fusion inserts with exclusively N terminus exofacial (Nexo) topology, serving as a model type III membrane protein. Orientation is unaffected by removal of all three positively charged residues in the cytoplasmic loop or by deletion of all but 12 residues from the N-terminal domain. The residual -2 N-terminal charge apparently provides a signal sufficient to determine Nexo topology. This is entirely consistent with the statistically derived rule in which the charge difference, Delta(C-N), counted for the 15 immediately flanking residues, is the primary topology determinant. Mutations altering Delta(C-N) to zero favors Nexo insertion by 3 to 1, whereas increasingly negative values cause increasing inversion of orientation. All results are consistent with the charge difference rule and indicate that whereas positive charges promote cytoplasmic retention, negative charges promote translocation.
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: D J
authors.initials: DJ
authors.lastname: Tipper
mesh_terms: D000595:Amino Acid Sequence; D003593:Cytoplasm / Q000201:enzymology; D004563:Electrochemistry; D004795:Enzyme Stability; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide / Q000737:chemistry / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D014157:Transcription Factors; D001618:beta-Lactamases / Q000378:metabolism
chemical_list: D008565:Membrane Proteins; D051964:Receptors, Mating Factor; D018000:Receptors, Peptide; D014157:Transcription Factors; D001618:beta-Lactamases