Subcellular localization of host and viral proteins associated with tobamovirus RNA replication

• Published in: The EMBO journal Vol. 22; no. 2; pp. 344 - 353
• Main Authors: Hagiwara, Yuka, Komoda, Keisuke, Yamanaka, Takuya, Tamai, Atsushi, Meshi, Tetsuo, Funada, Ryo, Tsuchiya, Tomohiro, Naito, Satoshi, Ishikawa, Masayuki
• Format: Journal Article
• Language: English
• Published: Chichester, UK John Wiley & Sons, Ltd 15.01.2003; Blackwell Publishing Ltd; Oxford University Press
• Subjects: Arabidopsis - genetics; Arabidopsis - metabolism; Arabidopsis - ultrastructure; Arabidopsis - virology; Arabidopsis Proteins - genetics; Arabidopsis Proteins - metabolism; Carrier Proteins - metabolism; Cell Fractionation; Cell Line; Cell Membrane - metabolism; Fractionation; Genes, Plant; Green Fluorescent Proteins; host factor; Intracellular Membranes - metabolism; Luminescent Proteins - genetics; Luminescent Proteins - metabolism; Membrane Proteins - genetics; Membrane Proteins - metabolism; Membranes; Nicotiana - cytology; Nicotiana - genetics; Nicotiana - metabolism; Organelles - metabolism; Plant Proteins - genetics; Plant Proteins - metabolism; Plants, Genetically Modified; Proteins; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; replication complex; RNA, Viral - metabolism; RNA-Dependent RNA Polymerase - metabolism; tobamovirus; Tobamovirus - genetics; Tobamovirus - metabolism; vacuolar membrane; Viral Proteins - genetics; Viral Proteins - metabolism; Virus Replication
• ISSN: 0261-4189; 1460-2075; 1460-2075
• DOI: 10.1093/emboj/cdg033

Arabidopsis TOM1 (AtTOM1) and TOM2A (AtTOM2A) are integral membrane proteins genetically identified to be necessary for efficient intracellular multiplication of tobamoviruses. AtTOM1 interacts with the helicase domain polypeptide of tobamovirus‐encoded replication proteins and with AtTOM2A, suggesting that both AtTOM1 and AtTOM2A are integral components of the tobamovirus replication complex. We show here that AtTOM1 and AtTOM2A proteins tagged with green fluorescent protein (GFP) are targeted to the vacuolar membrane (tonoplast)‐like structures in plant cells. In subcellular fractionation analyses, GFP–AtTOM2A, AtTOM2A and its tobacco homolog NtTOM2A were predominantly fractionated to low‐density tonoplast‐rich fractions, whereas AtTOM1–GFP, AtTOM1 and its tobacco homolog NtTOM1 were distributed mainly into the tonoplast‐rich fractions and partially into higher‐buoyant‐density fractions containing membranes from several other organelles. The tobamovirus‐encoded replication proteins were co‐fractionated with both NtTOM1 and viral RNA‐dependent RNA polymerase activity. The replication proteins were also found in the fractions containing non‐membrane‐bound proteins, but neither NtTOM1 nor the polymerase activity was detected there. These observations suggest that the formation of tobamoviral RNA replication complex occurs on TOM1‐containing membranes and is facilitated by TOM2A.