Ugo1p is a multipass transmembrane protein with a single carrier domain required for mitochondrial fusion.
The outer mitochondrial membrane protein Ugo1 forms a complex with the Fzo1p and Mgm1p GTPases that regulates mitochondrial fusion in yeast. Ugo1p contains two putative carrier domains (PCDs) found in mitochondrial carrier proteins (MCPs). Mitochondrial carrier proteins are multipass transmembrane proteins that actively transport molecules across the inner mitochondrial membrane. Mitochondrial carrier protein transport requires functional carrier domains with the consensus sequence PX(D/E)XX(K/R). Mutation of charged residues in this consensus sequence disrupts transport function. In this study, we used targeted mutagenesis to show that charge reversal mutations in Ugo1p PCD2, but not PCD1, disrupt mitochondrial fusion. Ugo1p is reported to be a single-pass transmembrane protein despite the fact that it contains several additional predicted transmembrane segments. Using a combination of protein targeting and membrane extraction experiments, we provide evidence that Ugo1p contains additional transmembrane domains and is likely a multipass transmembrane protein. These studies identify PCD2 as a functional domain of Ugo1p and provide the first experimental evidence for a multipass topology of this essential fusion component.
Coonrod
Emily M
EM
Institute of Molecular Biology, University of Oregon, Eugene, OR 97401, USA.
Karren
Mary Anne
MA
Shaw
Janet M
JM
D000595:Amino Acid Sequence; D019286:Endopeptidase K / Q000378:metabolism; D008561:Membrane Fusion / Q000502:physiology*; D050576:Membrane Fusion Proteins / Q000235:genetics / Q000502:physiology*; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D008928:Mitochondria / Q000737:chemistry / Q000502:physiology*; D000226:Mitochondrial ADP, ATP Translocases / Q000737:chemistry / Q000235:genetics / Q000502:physiology; D033681:Mitochondrial Membrane Transport Proteins / Q000737:chemistry / Q000187:drug effects / Q000502:physiology; D024101:Mitochondrial Proteins; D008958:Models, Molecular; D009154:Mutation; D017434:Protein Structure, Tertiary / Q000235:genetics / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000502:physiology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid
D050576:Membrane Fusion Proteins; D008565:Membrane Proteins; D033681:Mitochondrial Membrane Transport Proteins; D024101:Mitochondrial Proteins; C112225:PET9 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C423986:UGO1 protein, S cerevisiae; D000226:Mitochondrial ADP, ATP Translocases; D019286:Endopeptidase K