title: Recombinant expression of the MAL proteolipid, a component of glycolipid-enriched membrane microdomains, induces the formation of vesicular structures in insect cells.
abstract: The MAL proteolipid has been identified as a component of glycolipid-enriched membrane microdomains resistant to detergent solubilization in epithelial Madin-Darby canine cells, as well as in T lymphocytes and in myelin-forming cells. To study the function of the MAL proteolipid we have ectopically expressed a tagged form of MAL in both mammalian and insect cellular backgrounds. Immunofluorescence analysis in transiently transfected COS-7 cells showed the presence of MAL in large vesicular structures, and biochemical analysis identified MAL in the fraction of membranes resistant to Triton X-100 solubilization. Electron microscopic analysis showed that the expression of MAL in Sf21 cells morphologically resulted in the intracellular accumulation of large vesicles with a diameter from 200 to greater than 700 nm that were absent in uninfected or control infected cultures. Thus, ectopic expression of MAL in this heterologous expression system was sufficient to drive the formation of vesicles with a size similar to that of the vesicles detected in mammalian cells. These vesicles were clearly different from the caveolae-like vesicles induced by caveolin expression, as evidenced by co-infection experiments using a recombinant caveolin baculovirus. Taken together, these results suggest that the MAL proteolipid might play a role as a component of the machinery of vesiculation of glycolipid-enriched membranes.
authors.affiliation: Centro de Biología Molecular "Severo Ochoa," Universidad Autónoma de Madrid, Consejo Superior de Investigaciones Científicas, Cantoblanco, 28049 Madrid, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Puertollano
authors.forename: S
authors.initials: S
authors.lastname: Li
authors.forename: M P
authors.initials: MP
authors.lastname: Lisanti
authors.forename: M A
authors.initials: MA
authors.lastname: Alonso
mesh_terms: D000818:Animals; D019556:COS Cells; D051242:Caveolin 1; D022461:Caveolins; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D003001:Cloning, Molecular; D004285:Dogs; D004848:Epithelium; D006017:Glycolipids / Q000032:analysis / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000096:biosynthesis; D008854:Microscopy, Electron; D016133:Polymerase Chain Reaction; D017433:Protein Structure, Secondary; D011510:Proteolipids / Q000096:biosynthesis* / Q000737:chemistry / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D018411:Spodoptera; D013601:T-Lymphocytes / Q000378:metabolism; D014162:Transfection
chemical_list: C495540:CAV1 protein, human; D051242:Caveolin 1; D022461:Caveolins; D006017:Glycolipids; D008565:Membrane Proteins; D011510:Proteolipids; D011994:Recombinant Proteins