Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery.
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.
Wittrup
Anders
A
Section of Oncology, Department of Clinical Sciences, Lund University, SE-221 85, Lund, Sweden.
Zhang
Si-He
SH
Svensson
Katrin J
KJ
Kucharzewska
Paulina
P
Johansson
Maria C
MC
Mörgelin
Matthias
M
Belting
Mattias
M
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
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