title: Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery.
abstract: An increased understanding of cellular uptake mechanisms of macromolecules remains an important challenge in cell biology with implications for viral infection and macromolecular drug delivery. Here, we report a strategy based on antibody-conjugated magnetic nanoparticles for the isolation of endocytic vesicles induced by heparan sulfate proteoglycans (HSPGs), key cell-surface receptors of macromolecular delivery. We provide evidence for a role of the glucose-regulated protein (GRP)75/PBP74/mtHSP70/mortalin (hereafter termed "GRP75") in HSPG-mediated endocytosis of macromolecules. GRP75 was found to be a functional constituent of intracellular vesicles of a nonclathrin-, noncaveolin-dependent pathway that was sensitive to membrane cholesterol depletion and that showed colocalization with the membrane raft marker cholera toxin subunit B. We further demonstrate a functional role of the RhoA GTPase family member CDC42 in this transport pathway; however, the small GTPase dynamin appeared not to be involved. Interestingly, we provide evidence of a functional role of GRP75 using RNAi-mediated down-regulation of GRP75 and GRP75-blocking antibodies, both of which inhibited macromolecular endocytosis. We conclude that GRP75, a chaperone protein classically found in the endoplasmic reticulum and mitochondria, is a functional constituent of noncaveolar, membrane raft-associated endocytic vesicles. Our data provide proof of principle of a strategy that should be generally applicable in the molecular characterization of selected endocytic pathways involved in macromolecular uptake by mammalian cells.
authors.affiliation: Section of Oncology, Department of Clinical Sciences, Lund University, SE-221 85, Lund, Sweden.
authors.forename: Anders
authors.initials: A
authors.lastname: Wittrup
authors.forename: Si-He
authors.initials: SH
authors.lastname: Zhang
authors.forename: Katrin J
authors.initials: KJ
authors.lastname: Svensson
authors.forename: Paulina
authors.initials: P
authors.lastname: Kucharzewska
authors.forename: Maria C
authors.initials: MC
authors.lastname: Johansson
authors.forename: Matthias
authors.initials: M
authors.lastname: Mörgelin
authors.forename: Mattias
authors.initials: M
authors.lastname: Belting
mesh_terms: D000818:Animals; D019138:Antibodies, Blocking / Q000276:immunology / Q000494:pharmacology; D002460:Cell Line; D004705:Endocytosis / Q000187:drug effects / Q000502:physiology; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D006367:HeLa Cells; D019812:Heparan Sulfate Proteoglycans / Q000378:metabolism; D006801:Humans; D015151:Immunoblotting; D046911:Macromolecular Substances / Q000378:metabolism*; D008280:Magnetics; D008565:Membrane Proteins / Q000235:genetics / Q000276:immunology / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008854:Microscopy, Electron; D053758:Nanoparticles / Q000737:chemistry*; D034622:RNA Interference; D011956:Receptors, Cell Surface / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D022161:Transport Vesicles / Q000378:metabolism* / Q000648:ultrastructure; D020764:cdc42 GTP-Binding Protein / Q000235:genetics / Q000378:metabolism
chemical_list: D019138:Antibodies, Blocking; D018840:HSP70 Heat-Shock Proteins; D019812:Heparan Sulfate Proteoglycans; D046911:Macromolecular Substances; D008565:Membrane Proteins; D011956:Receptors, Cell Surface; C018551:glucose-regulated proteins; D049452:Green Fluorescent Proteins; D020764:cdc42 GTP-Binding Protein