title: A Targeted Mutation Identified through pKa Measurements Indicates a Postrecruitment Role for Fis1 in Yeast Mitochondrial Fission.
abstract: The tail-anchored protein Fis1 is implicated as a passive tether in yeast mitochondrial fission. We probed the functional role of Fis1 Glu-78, whose elevated side chain pKa suggests participation in protein interactions. Fis1 binds partners Mdv1 or Dnm1 tightly, but mutation E78A weakens Fis1 interaction with Mdv1, alters mitochondrial morphology, and abolishes fission in a growth assay. In fis1Δ rescue experiments, Fis1-E78A causes a novel localization pattern in which Dnm1 uniformly coats the mitochondria. By contrast, Fis1-E78A at lower expression levels recruits Dnm1 into mitochondrial punctate structures but fails to support normal fission. Thus, Fis1 makes multiple interactions that support Dnm1 puncta formation and may be essential after this step, supporting a revised model for assembly of the mitochondrial fission machinery. The insights gained by mutating a residue with a perturbed pKa suggest that side chain pKa values inferred from routine NMR sample pH optimization could provide useful leads for functional investigations.
authors: From the Department of Biology and.
authors: Marijke
authors: M
authors: Koppenol-Raab
authors: the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and.
authors: Megan Cleland
authors: MC
authors: Harwig
authors: From the Department of Biology and the Program in Molecular Biophysics, Johns Hopkins University, Baltimore, Maryland 21218.
authors: Ammon E
authors: AE
authors: Posey
authors: the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and.
authors: John M
authors: JM
authors: Egner
authors: the Department of Pathology, Baylor College of Medicine, Houston, Texas 77030.
authors: Kevin R
authors: KR
authors: MacKenzie
authors: the Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, and rbhill@mcw.edu.
authors: R Blake
authors: RB
authors: Hill
mesh_terms: D019943:Amino Acid Substitution; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D063154:Mitochondrial Dynamics / Q000502:physiology*; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D020125:Mutation, Missense; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: C464859:FIS1 protein, S cerevisiae; D024101:Mitochondrial Proteins; D029701:Saccharomyces cerevisiae Proteins; D020558:GTP Phosphohydrolases; C094774:DNM1 protein, S cerevisiae
keywords: confocal microscopy; macromolecular machine; mitochondria; mitochondrial dynamics; nuclear magnetic resonance (NMR); pKa measurements; protein assembly; protein-protein interaction; structural model; tetratricopeptide repeat (TPR) domains