Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery

• Published in: Proceedings of the National Academy of Sciences - PNAS Vol. 107; no. 30; pp. 13342 - 13347
• Main Authors: Wittrup, Anders, Zhang, Si-He, Svensson, Katrin J., Kucharzewska, Paulina, Johansson, Maria C., Mörgelin, Matthias, Belting, Mattias, Ruoslahti, Erkki
• Format: Journal Article
• Language: English
• Published: United States National Academy of Sciences 27-07-2010; National Acad Sciences
• Subjects: Animals; Antibodies; Antibodies, Blocking - immunology; Antibodies, Blocking - pharmacology; Antigen presenting cells; Biological Sciences; cdc42 GTP-Binding Protein - genetics; cdc42 GTP-Binding Protein - metabolism; Cell Line; Cell membranes; Cells; Cytometry; Endocytosis; Endocytosis - drug effects; Endocytosis - physiology; Green Fluorescent Proteins - genetics; Green Fluorescent Proteins - metabolism; HeLa Cells; Heparan Sulfate Proteoglycans - metabolism; HSP70 Heat-Shock Proteins - genetics; HSP70 Heat-Shock Proteins - immunology; HSP70 Heat-Shock Proteins - physiology; Humans; Immunoblotting; Macromolecular Substances - metabolism; Magnetics; Medical and Health Sciences; Medicin och hälsovetenskap; Membrane Proteins - genetics; Membrane Proteins - immunology; Membrane Proteins - physiology; Membranes; Mice; Mice, Knockout; Microscopy, Electron; Molecules; Nanoparticles; Nanoparticles - chemistry; Proteins; Rafts; Receptors, Cell Surface - metabolism; RNA Interference; Signal Transduction - drug effects; Signal Transduction - physiology; Toxins; Transport vesicles; Transport Vesicles - metabolism; Transport Vesicles - ultrastructure
• ISSN: 0027-8424; 1091-6490; 1091-6490
• DOI: 10.1073/pnas.1002622107

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.