A model for transport of a viral membrane protein through the early secretory pathway: minimal sequence and endoplasmic reticulum lateral mobility requirements.
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 β-turn motif (D7 AP10 A) 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 (K49 A) 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 D7 AP10 A mutant, but enhanced in the K49 A mutant. In addition, fluorescence recovery after photobleaching revealed that K49 A but not D7 AP10 A 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 β-turn favors assembly of COPII vesicles for export of p7B from the ER.
Instituto de Biología Molecular y Celular de Plantas, Universitat Politècnica de València/Consejo Superior de Investigaciones Científicas, Avenida Ingeniero Fausto Elio s/n, 46022, Valencia, Spain.
Marta
M
Serra-Soriano
Vicente
V
Pallás
José A
JA
Navarro
D000595:Amino Acid Sequence; D022181:COP-Coated Vesicles / Q000378:metabolism; D028463:Cucumis melo / Q000166:cytology / Q000235:genetics / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D017930:Genes, Reporter; D006056:Golgi Apparatus / Q000378:metabolism; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism*; D008954:Models, Biological; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D053830:Plant Viral Movement Proteins / Q000235:genetics; D010942:Plant Viruses / Q000502:physiology*; D031425:Plasmodesmata / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D055571:Secretory Pathway; D016415:Sequence Alignment; D017384:Sequence Deletion; D014764:Viral Proteins / Q000737:chemistry / Q000378:metabolism
D008565:Membrane Proteins; D053830:Plant Viral Movement Proteins; D014764:Viral Proteins
Cucumis melo; cargo concentration; endoplasmic reticulum sorting signal; lateral diffusion; movement proteins; secretory pathway; transmembrane protein; virus movement