Abstract:
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[EN] Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via
routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus
p7B, a ...[+]
[EN] Viral movement proteins exploit host endomembranes and the cytoskeleton to move within the cell via
routes that, in some cases, are dependent on the secretory pathway. For example, melon necrotic spot virus
p7B, a type II transmembrane protein, leaves the endoplasmic reticulum (ER) through the COPII-dependent
Golgi pathway to reach the plasmodesmata. Here we investigated the sequence requirements and putative
mechanisms governing p7B transport through the early secretory pathway. Deletion of either the cytoplasmic
N–terminal region (CR) or the luminal C–terminal region (LR) led to ER retention, suggesting that they
are both essential for ER export. Through alanine-scanning mutagenesis, we identified residues in the CR
and LR that are critical for both ER export and for viral cell-to-cell movement. Within the CR, alanine substitution
of aspartic and proline residues in the DSSP b–turn motif (D7AP10A) led to movement of discrete
structures along the cortical ER in an actin-dependent manner. In contrast, alanine substitution of a lysine
residue in the LR (K49A) resulted in a homogenous ER distribution of the movement protein and inhibition
of ER–Golgi traffic. Moreover, the ability of p7B to recruit Sar1 to the ER membrane is lost in the D7AP10A
mutant, but enhanced in the K49A mutant. In addition, fluorescence recovery after photobleaching revealed
that K49A but not D7AP10A dramatically diminished protein lateral mobility. From these data, we propose a
model whereby the LR directs actin-dependent mobility toward the cortical ER, where the cytoplasmic
DSSP b–turn favors assembly of COPII vesicles for export of p7B from the ER.
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Thanks:
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We are grateful to C. Hawes (Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington, Oxford, UK) and V. V. Dolja (Department of Botany and Plant Pathology ...[+]
We are grateful to C. Hawes (Department of Biological and Medical Sciences, Faculty of Health and Life Sciences, Oxford Brookes University, Headington, Oxford, UK) and V. V. Dolja (Department of Botany and Plant Pathology and Center for Genome Research and Biocomputing, Oregon State University, Corvallis, OR, USA) for providing the Sar1-YFP construct and some fluorescent-tagged organelle markers, respectively. We would like to thank F. Aniento (Departmento de Bioquimica y Biologia Molecular, Facultad de Farmacia, Universidad de Valencia, Burjassot, Valencia, Spain) and E. Rojo (Departament of Plant Molecular Genetics, Centro Nacional de Biotecnologia, Consejo Superior de Investigaciones Cientificas, 28049 Madrid, Spain) for critical reading and valuable comments on this manuscript. This work was funded by grant BIO2011-25018 from the Spanish Departmento Governmental de Investigaciones Cientificas y Tecnologicas and Prometeo Program GV2011/003 from the Generalitat Valenciana. J.A.N. and M. S. are the recipients of a postdoctoral contract and a PhD fellowship from the Ministerio de Educacion y Ciencia of Spain. We thank L. Corachan for her technical assistance.
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