Subcellular localization of host and viral proteins associated with tobamovirus RNA replication.
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.
Hagiwara
Yuka
Y
Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
Komoda
Keisuke
K
Yamanaka
Takuya
T
Tamai
Atsushi
A
Meshi
Tetsuo
T
Funada
Ryo
R
Tsuchiya
Tomohiro
T
Naito
Satoshi
S
Ishikawa
Masayuki
M
D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D002352:Carrier Proteins / Q000378:metabolism*; D002458:Cell Fractionation; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D017343:Genes, Plant; D049452:Green Fluorescent Proteins; D007425:Intracellular Membranes / Q000378:metabolism; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D015388:Organelles / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D012367:RNA, Viral / Q000378:metabolism*; D012324:RNA-Dependent RNA Polymerase / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D017898:Tobamovirus / Q000235:genetics* / Q000378:metabolism*; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*; D014779:Virus Replication
D029681:Arabidopsis Proteins; D002352:Carrier Proteins; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010940:Plant Proteins; D012367:RNA, Viral; D011993:Recombinant Fusion Proteins; C413802:TOM1 protein, Arabidopsis; C471269:TOM2 protein, Arabidopsis; D014764:Viral Proteins; D049452:Green Fluorescent Proteins; D012324:RNA-Dependent RNA Polymerase