2FNC1†Present address: Fraunhofer USA Center for Molecular Biotechnology, 9 Innovation Way, Newark, DE 19711, USA.
2FNC‡Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, -1 Yamadaoka, Suita 565-0871, Osaka, Japan.
2FNC3§Present address: W. M. Keck Center for Comparative and Functional Genomics, 39 Edward R. Madigan Laboratory (MC 051), 101 West Gregory Drive, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
A series of deletion mutants of tobacco mosaic virus movement protein (TMV-MP) was used to identify domains of the protein necessary for membrane association. A membrane fraction was isolated from tobacco BY-2 protoplasts infected with wild-type and mutant TMV that produce MP carrying a 3 aa deletion. Deletions that affected membrane association were clustered around the two major hydrophobic regions of MP that are predicted to be transmembrane. Deletions in other hydrophobic regions also reduced membrane association. In addition, a non-functional mutant of MP, in which one of the known phosphorylation sites was eliminated, was not associated with cellular membranes, while a functional second site revertant restored membrane association. This indicates that MP function requires interaction with membrane; however, membrane association was not sufficient for function. Results are consistent with the hypothesis that TMV-MP is an integral or tightly associated membrane protein that includes two hydrophobic transmembrane domains.
AsurmendiS.,
BergR. H.,
KooJ. C.,
BeachyR. N.2004; Coat protein regulates formation of replication complexes during tobacco mosaic virus infection. Proc Natl Acad Sci U S A 101:1415–1420[CrossRef]
BernaA.1995; Involvement of residues within putative α -helix motifs in the behavior of the alfalfa and tobacco mosaic virus movement proteins. Phytopathology 85:1441–1448[CrossRef]
BernaA.,
GafnyR.,
WolfS.,
LusasW. J.,
HoltC. A.,
BeachyR. N.1991; The TMV movement protein: role of the C-terminal 73 amino acids in subcellular localization and function. Virology 182:682–689[CrossRef]
Bleve-ZacheoT.,
RubinoL.,
MelilloM. T.,
RussoM.1997; The 33K protein encoded by cymbidium ringspot virus localizes to modified peroxisomes of infected cells and of uninfected transgenic plants. J Plant Pathol 79:197–202
BoykoV.,
van der LaakJ.,
FerralliJ.,
SuslovaE.,
KwonM.-O.,
HeinleinM.2000; Cellular targets of functional and dysfunctional mutants of tobacco mosaic virus movement protein fused to green fluorescent protein. J Virol 74:11339–11346[CrossRef]
BrillL. M.,
NunnR. S.,
KahnT. W.,
YeagerM.,
BeachyR. N.2000; Recombinant tobacco mosaic virus movement protein is an RNA-binding, α -helical membrane protein. Proc Natl Acad Sci U S A 97:7112–7117[CrossRef]
CitovskyV.,
McLeanB. G.,
ZupanJ. R.,
ZambryskiP.1993; Phosphorylation of tobacco mosaic virus cell-to-cell movement protein by a developmentally regulated plant cell wall-associated protein kinase. Genes Dev 7:904–910[CrossRef]
CserzoM.,
WallinE.,
SimonI.,
von HeijneG.,
ElofssonA.1997; Prediction of transmembrane α -helices in prokaryotic membrane proteins: the dense alignment surface method. Protein Eng 10:673–676[CrossRef]
DeomC. M.,
WolfS.,
HoltC. A.,
LucasW. J.,
BeachyR. N.1991; Altered function of the tobacco mosaic virus movement protein in a hypersensitive host. Virology 180:251–256[CrossRef]
FroshauerS.,
KartenbeckJ.,
HeleniusA.1988; Alphavirus RNA replicase is located on the cytoplasmic surface of endosomes and lysosomes. J Cell Biol 107:2075–2086[CrossRef]
GafnyR.,
LapidotM.,
BernaA.,
HoltC. A.,
DeomC. M.,
BeachyR. N.1992; Effects of terminal deletion mutations on function of the movement protein of tobacco mosaic virus. Virology 187:499–507[CrossRef]
HeinleinM.,
PadgettH. S.,
GensJ. S.,
PickardB. G.,
CasperS. J.,
EpelB. L.,
BeachyR. N.1998; Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection. Plant Cell 10:1107–1120[CrossRef]
HoltC. A.,
BeachyR. N.1991; In vivo complementation of infectious transcripts from mutant tobacco mosaic virus cDNAs in transgenic plants. Virology 181:109–117[CrossRef]
KahnT. W.,
LapidotM.,
HeinleinM.,
ReichelC.,
CooperB.,
GafnyR.,
BeachyR. N.1998; Domain of the TMV movement protein involved in subcellular localization. Plant J 15:15–25[CrossRef]
KawakamiS.,
PadgettH. S.,
HososkawaD.,
OkadaY.,
BeachyR. N.,
WatanabeY.1999; Phosphorylation and/or presence of serine 37 in the movement protein of tomato mosaic tobamovirus is essential for intracellular localization and stability in vivo
. J Virol 73:6831–6840
KawakamiS.,
HoriK.,
HosokawaD.,
OkadaY.,
WatanabeY.2003; Defective tobamovirus movement protein lacking wild-type phosphorylation sites can be complemented by substitutions found in revertants. J Virol 77:1452–1461[CrossRef]
KawakamiS.,
WatanabeY.,
BeachyR. N.2004; Tobacco mosaic virus infection spreads cell to cell as intact replication complexes. Proc Natl Acad Sci U S A 101:6291–6296[CrossRef]
KroghA.,
LarssonB.,
von HeijneG.,
SonnhammerE. L. L.2001; Predicting transmembrane protein topology with a hidden Markov model: application to complete genomes. J Mol Biol 305:567–580[CrossRef]
LaporteC.,
VetterG.,
LoudesA.-M.,
RobinsonD. G.,
HillmerS.,
Stussi-GaraudC.,
RitzenthalerC.2003; Involvement of secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells. Plant Cell 15:2058–2075[CrossRef]
LazarowitzS. G.,
BeachyR. N.1999; Viral movement proteins as probes for intracellular and intercellular trafficking in plants. Plant Cell 11:535–548[CrossRef]
MasP.,
BeachyR. N.1999; Replication of tobacco mosaic virus on endoplasmic reticulum and role of the cytoskeleton and virus movement protein in intracellular distribution of viral RNA. J Cell Biol 147:945–958[CrossRef]
MillerD. J.,
SchwartsM. D.,
AhlquistP.2001; Flock house virus RNA replicates on outer mitochondrial membranes in Drosophila cells. J Virol 75:11664–11676[CrossRef]
OparkaK. J.,
PriorD. A.,
Santa CruzS.,
PadgettH. S.,
BeachyR. N.1997; Gating of epidermal plasmodesmata is restricted to the leading edge of expanding infection sites of tobacco mosaic virus (TMV). Plant J 12:781–789[CrossRef]
OparkaK. J.,
RobertsA. G.,
BoevinkP.& 7 other authors1999; Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves. Cell 97:743–754[CrossRef]
Prod'hommeD.,
JakubiecA.,
TournierV.,
DrugeonG.,
JupinI.2003; Targeting of the turnip yellow mosaic virus 66K replication protein to the chloroplast envelope is mediated by the 140K protein. J Virol 77:9124–9135[CrossRef]
ReichelC.,
BeachyR. N.1998; Tobacco mosaic virus infection induces severe morphological changes of the endoplasmic reticulum. Proc Natl Acad Sci U S A 95:11169–11174[CrossRef]
SaurìA.,
SaksenaS.,
SalgadoJ.,
JohnsonA. E.,
MingarroI.2005; Double-spanning plant viral movement protein integration into the endoplasmic reticulum membrane is signal recognition particle-dependent, translocon-mediated, and concerted. J Biol Chem 280:25907–25912[CrossRef]
SonnhammerE. L. L.,
von HeijneG.,
KroghA.1998; A hidden Markov model for predicting transmembrane helices in protein sequences. Proc Int Conf Intell Syst Mol Biol 6:175–182
Van der HeijdenM. W.,
CaretteJ. E.,
ReinhoudP. J.,
HaegiA.,
BolJ. F.2001; Alfalfa mosaic virus replicase proteins P1 and P2 interact and colocalize at the vacuolar membrane. J Virol 75:1879–1887[CrossRef]
VilarM.,
SauríA.,
MonnéM.,
MarcosJ. F.,
von HeijneG.,
Pérez-PayáE.,
MingarroI.2002; Insertion and topology of a plant viral movement protein in the endoplasmic reticulum membrane. J Biol Chem 277:23447–23452[CrossRef]
WaigmannE.,
ChenM. H.,
BachmaierR.,
GhoshroyS.,
CitovskyV.2000).Regulation; of plasmodesmal transport by phosphorylation of tobacco mosaic virus cell-to-cell movement protein. EMBO J 19:4875–4885[CrossRef]
WatanabeY.,
MeshiT.,
OkadaY.1987; Infection of tobacco protoplasts with in vitro transcribed tobacco mosaic virus RNA using an improved electroporation method. FEBS Lett 219:65–69[CrossRef]