Evidence that insertion of Tomato ringspot nepovirus NTB-VPg protein in endoplasmic reticulum membranes is directed by two domains: a C-terminal transmembrane helix and an N-terminal amphipathic helix.
The NTB-VPg protein of Tomato ringspot nepovirus is an integral membrane protein found in association with endoplasmic reticulum (ER)-derived membranes active in virus replication. A transmembrane helix present in a hydrophobic region at the C terminus of the NTB domain was previously shown to traverse the membranes, resulting in the translocation of the VPg domain in the lumen. We have now conducted an in planta analysis of membrane-targeting domains within NTB-VPg using in-frame fusions to the green fluorescent protein (GFP). As expected, the entire NTB-VPg protein directed the GFP fluorescence to ER membranes. GFP fusion proteins containing the C-terminal 86 amino acids of NTB-VPg also associated with ER membranes, resulting in ER-specific glycosylation at a naturally occurring glycosylation site in the VPg domain. Deletion of the hydrophobic region prevented the membrane association. The N-terminal 80 amino acids of NTB were also sufficient to direct the GFP fluorescence to intracellular membranes. A putative amphipathic helix in this region was necessary and sufficient to promote membrane association of the fusion proteins. Using in vitro membrane association assays and glycosylation site mapping, we show that the N terminus of NTB can be translocated in the lumen at least in vitro. This translocation was dependent on the presence of the putative amphipathic helix, suggesting that oligomeric forms of this helix traverse the membrane. Taken together, our results suggest that at least two distinct elements play a key role in the insertion of NTB-VPg in the membranes: a C-terminal transmembrane helix and an N-terminal amphipathic helix. An updated model of the topology of the protein in the membrane is presented.
Zhang
Shuo Cheng
SC
Pacific Agri-Food Research Centre, 4200 Highway 97, Summerland, BC, Canada V0H 1Z0.
Zhang
Guangzhi
G
Yang
Lanying
L
Chisholm
Joan
J
Sanfaçon
Hélène
H
D000595:Amino Acid Sequence; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics; D004721:Endoplasmic Reticulum / Q000821:virology; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018613:Microscopy, Confocal; D008954:Models, Biological; D008969:Molecular Sequence Data; D017860:Nepovirus / Q000235:genetics / Q000472:pathogenicity / Q000502:physiology*; D018515:Plant Leaves / Q000821:virology; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000821:virology; D014162:Transfection; D014764:Viral Proteins / Q000737:chemistry / Q000235:genetics / Q000502:physiology*
D004279:DNA, Viral; D011993:Recombinant Fusion Proteins; D014764:Viral Proteins; D049452:Green Fluorescent Proteins