title: An intact dilysine-like motif in the carboxyl terminus of MAL is required for normal apical transport of the influenza virus hemagglutinin cargo protein in epithelial Madin-Darby canine kidney cells.
abstract: The MAL proteolipid, a component of the integral protein sorting machinery, has been demonstrated as being necessary for normal apical transport of the influenza virus hemagglutinin (HA) and the overall apical membrane proteins in Madin-Darby canine kidney (MDCK) cells. The MAL carboxy terminus ends with the sequence Arg-Trp-Lys-Ser-Ser (RWKSS), which resembles dilysine-based motifs involved in protein sorting. To investigate whether the RWKSS pentapeptide plays a role in modulating the distribution of MAL and/or its function in apical transport, we have expressed MAL proteins with distinct carboxy terminus in MDCK cells whose apical transport was impaired by depletion of endogenous MAL. Apical transport of HA was restored to normal levels by expression of MAL with an intact but not with modified carboxyl terminal sequences bearing mutations that impair the functioning of dilysine-based sorting signals, although all the MAL proteins analyzed incorporated efficiently into lipid rafts. Ultrastructural analysis indicated that compared with MAL bearing an intact RWKSS sequence, a mutant with lysine -3 substituted by serine showed a twofold increased presence in clathrin-coated cytoplasmic structures and a reduced expression on the plasma membrane. These results indicate that the carboxyl-terminal RWKSS sequence modulates the distribution of MAL in clathrin-coated elements and is necessary for HA transport to the apical surface.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Puertollano
authors.forename: J A
authors.initials: JA
authors.lastname: Martínez-Menárguez
authors.forename: A
authors.initials: A
authors.lastname: Batista
authors.forename: J
authors.initials: J
authors.lastname: Ballesta
authors.forename: M A
authors.initials: MA
authors.lastname: Alonso
mesh_terms: D033982:Adaptor Protein Complex gamma Subunits; D020816:Amino Acid Motifs; D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism; D001692:Biological Transport; D019921:Biotinylation; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D003902:Detergents / Q000494:pharmacology; D004151:Dipeptides / Q000737:chemistry*; D004285:Dogs; D004591:Electrophoresis, Polyacrylamide Gel; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism*; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000378:metabolism*; D015151:Immunoblotting; D007668:Kidney / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D026901:Membrane Transport Proteins; D018613:Microscopy, Confocal; D016253:Microscopy, Immunoelectron; D009154:Mutation; D009185:Myelin Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D017434:Protein Structure, Tertiary; D011510:Proteolipids / Q000737:chemistry*; D014162:Transfection; D014168:Transferrin / Q000378:metabolism
chemical_list: D033982:Adaptor Protein Complex gamma Subunits; D000911:Antibodies, Monoclonal; D051242:Caveolin 1; D022461:Caveolins; D003902:Detergents; D004151:Dipeptides; D019267:Hemagglutinin Glycoproteins, Influenza Virus; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009185:Myelin Proteins; D062806:Myelin and Lymphocyte-Associated Proteolipid Proteins; D011510:Proteolipids; D014168:Transferrin; D004247:DNA; C024575:lysyllysine