title: A cell-free biochemical complementation assay reveals complex and redundant cytosolic requirements for LRP endocytosis.
abstract: The low density lipoprotein receptor-related protein (LRP) binds multiple, distinct ligands and participates in constitutive endocytosis and signal transduction. Using an in vitro reconstitution system and a new biochemical complementation assay, we have explored the limiting cytosolic requirements for endocytosis of LRP from isolated plasma membranes. We find that clathrin, AP2 and dynamin do not support efficient LRP uptake and that additional factors present in a 30% ammonium sulfate supernatant fraction of bovine brain cytosol (AS supt) are required. Fractionation of the AS supt revealed that multiple and redundant factors are required to support LRP endocytosis. Among these, we identified Hsc70, synaptojanin1 and CRMP-2 by mass spectrometry. Our data suggest that LRP, which bears several distinct endocytic motifs in its cytoplasmic domain, may use multiple pathways for endocytosis in vitro.
authors.affiliation: Department of Cell Biology, The Scripps Research Institute, 10550 N. Torrey Pines Rd., La Jolla, CA 92037, USA.
authors.forename: Ishido
authors.initials: I
authors.lastname: Miwako
authors.forename: Sandra L
authors.initials: SL
authors.lastname: Schmid
mesh_terms: D020816:Amino Acid Motifs / Q000502:physiology; D000818:Animals; D001681:Biological Assay / Q000379:methods; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism* / Q000648:ultrastructure; D002474:Cell-Free System / Q000737:chemistry / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism* / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology*; D050883:HSC70 Heat-Shock Proteins / Q000378:metabolism; D036341:Intercellular Signaling Peptides and Proteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1 / Q000737:chemistry / Q000378:metabolism*; D009419:Nerve Tissue Proteins / Q000378:metabolism; D010744:Phosphoric Monoester Hydrolases / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D051381:Rats; D015398:Signal Transduction / Q000502:physiology; D013347:Subcellular Fractions; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D050883:HSC70 Heat-Shock Proteins; D036341:Intercellular Signaling Peptides and Proteins; D026503:Low Density Lipoprotein Receptor-Related Protein-1; D009419:Nerve Tissue Proteins; C095114:collapsin response mediator protein-2; C097702:synaptojanin; D010744:Phosphoric Monoester Hydrolases