Ugo1p Is a Multipass Transmembrane Protein with a Single Carrier Domain Required for Mitochondrial Fusion

Abstract

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.

Molecular Machinery of Mitochondrial Fusion and Fission

“…Metazoan mitofusins share the same topology and domain organization with yeast Fzo1, with the exception that they lack the most N-terminal coiled-coil region (24). Ugo1 is a yeast mitochondrial outer membrane protein that contains up to five transmembrane regions (20,25). Homologs of Ugo1 in higher organisms are unknown.…”

Ugo1 and Mdm30 act sequentially during Fzo1-mediated mitochondrial outer membrane fusion

“…Ugo1 is a modified carrier protein in the OM that appears to operate after OM tethering (Coonrod et al, 2007;Hoppins et al, 2009). Ugo1 possesses three transmembrane domains and exposes its N-terminus to the cytosol (Coonrod et al, 2007;Hoppins et al, 2009).…”

“…Ugo1 is a modified carrier protein in the OM that appears to operate after OM tethering (Coonrod et al, 2007;Hoppins et al, 2009). Ugo1 possesses three transmembrane domains and exposes its N-terminus to the cytosol (Coonrod et al, 2007;Hoppins et al, 2009). In vitro binding assays with recombinant proteins revealed that the N-terminus of Ugo1 directly interacts with the cytosolic regions adjacent to the transmembrane domains in Fzo1 (Sesaki and Jensen, 2004).…”

In VivoAnalysis of Membrane Fusion

“…Direct interactions between Mgm1p and Fzo1p have not yet been demonstrated. In yeast, it is possible that the coordination is accomplished in part by Ugo1p which binds both Mgm1p and Fzo1p (Coonrod et al, 2007;Sesaki and Jensen, 2004). Peroxisomes participate in the metabolism of fatty acids and other metabolites.…”