title: Localization of the Carnation Italian ringspot virus replication protein p36 to the mitochondrial outer membrane is mediated by an internal targeting signal and the TOM complex.
abstract: BACKGROUND
  Carnation Italian ringspot virus (CIRV) is a positive-strand RNA virus that causes massive structural alterations of mitochondria in infected host cells, the most conspicuous being the formation of numerous internal vesicles/spherules that are derived from the mitochondrial outer membrane and serve as the sites for viral RNA replication. While the membrane-bound components of the CIRV replication complex, including a 36-kD RNA-binding protein (p36), are known to be essential for these changes in mitochondrial morphology and are relatively well characterized in terms of their roles in nascent viral RNA synthesis, how these proteins are specifically targeted and inserted into mitochondria is poorly defined.
  
  
  RESULTS
  Here we report on the molecular signal responsible for sorting p36 to the mitochondrial outer membrane. Using a combination of gain-of-function assays with portions of p36 fused to reporter proteins and domain-swapping assays with p36 and another closely-related viral RNA-binding protein, p33, that sorts specifically to the peroxisomal boundary membrane, we show that the mitochondrial targeting information in p36 resides within its two transmembrane domains (TMDs) and intervening hydrophilic loop sequence. Comprehensive mutational analysis of these regions in p36 revealed that the primary targeting determinants are the moderate hydrophobicity of both TMDs and the positively-charged face of an amphipathic helix within the intervening loop sequence. We show also using bimolecular fluorescence complementation (BiFC) that p36 interacts with certain components of the translocase complex in the mitochondrial outer membrane (TOM), but not with the sorting and assembly machinery (SAM).
  
  
  CONCLUSIONS
  Our results provide insight to how viruses, such as CIRV, exploit specific host-cell protein sorting pathways to facilitate their replication. The characterization of the targeting and insertion of p36 into the mitochondrial outer membrane also sheds light on the mechanisms involved in sorting of host-cell membrane proteins to mitochondria, a process that has been largely unexplored in plants.
authors.affiliation: Department of Molecular and Cellular Biology, University of Guelph, Guelph, Ontario, N1G 2W1, Canada. hwangy@uoguelph.ca
authors.forename: Yeen Ting
authors.initials: YT
authors.lastname: Hwang
authors.forename: Andrew W
authors.initials: AW
authors.lastname: McCartney
authors.forename: Satinder K
authors.initials: SK
authors.lastname: Gidda
authors.forename: Robert T
authors.initials: RT
authors.lastname: Mullen
mesh_terms: D000595:Amino Acid Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D008565:Membrane Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D051336:Mitochondrial Membranes / Q000378:metabolism*; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D008969:Molecular Sequence Data; D021382:Protein Sorting Signals; D017434:Protein Structure, Tertiary; D012367:RNA, Viral; D017864:Tombusvirus / Q000502:physiology*; D014764:Viral Proteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D014779:Virus Replication / Q000502:physiology*
chemical_list: D002352:Carrier Proteins; D008565:Membrane Proteins; D000090622:Mitochondrial Precursor Protein Import Complex Proteins; D021382:Protein Sorting Signals; D012367:RNA, Viral; D014764:Viral Proteins; D049452:Green Fluorescent Proteins