title: Mitochondrial Mas70p signal anchor sequence. Mutations in the transmembrane domain that disrupt dimerization but not targeting or membrane insertion.
abstract: Mas70p is an integral membrane protein in Saccharomyces cerevisiae that is targeted and inserted into the mitochondrial outer membrane in an N(in)-Ccyto orientation by its NH2-terminal 29-amino acid signal anchor sequence. Recently, we demonstrated that the signal anchor was capable of mediating homo-oligomerization of a fusion protein, pOMD29, in the outer membrane in vitro (Millar, D. G., and Shore, G. C. (1992) J. Biol. Chem. 268, 18403-18406). Consistent with this finding, we show here that a synthetic peptide corresponding to the Mas70p signal anchor is capable of independent membrane insertion and dimerization with pOMD29. To further map the oligomerization domain in the signal anchor sequence, a deletion mutant of pOMD29 that lacks amino acids 2-10 was constructed. This protein, pOMD29 delta 2-10, efficiently participated in dimer formation following import, indicating that dimerization was mediated by the putative membrane spanning segment (amino acids 11-29). This segment is predicted to form an alpha-helix that has an alanine-rich face and contains multiple copies of a pentapeptide dimerization motif that is widespread among members of the receptor tyrosine kinase family. Substitution of the alanine residues in one of these copies with isoleucine, producing a potentially bulkier contact surface, resulted in a protein which was targeted and inserted into the outer membrane but failed to assemble into dimers. Taken together, these results identify a structural feature of the signal anchor transmembrane domain that is important for oligomerization but is not required for targeting and membrane insertion.
authors.affiliation: Department of Biochemistry, McGill University, Montreal, Canada.
authors.forename: D G
authors.initials: DG
authors.lastname: Millar
authors.forename: G C
authors.initials: GC
authors.lastname: Shore
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D046911:Macromolecular Substances; D008565:Membrane Proteins / Q000096:biosynthesis*; D008928:Mitochondria / Q000378:metabolism*; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D010455:Peptides / Q000138:chemical synthesis; D014176:Protein Biosynthesis; D011499:Protein Processing, Post-Translational; D021382:Protein Sorting Signals / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*; D017433:Protein Structure, Secondary; D011956:Receptors, Cell Surface / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification; D012156:Reticulocytes / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins; D017384:Sequence Deletion; D013462:Sulfur Radioisotopes
chemical_list: D005656:Fungal Proteins; D046911:Macromolecular Substances; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D010455:Peptides; D021382:Protein Sorting Signals; D011956:Receptors, Cell Surface; D029701:Saccharomyces cerevisiae Proteins; D013462:Sulfur Radioisotopes; C489039:TOM70 protein, S cerevisiae