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

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Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery. / Wittrup, Anders; Zhang, Sihe; Svensson, Katrin; Kucharzewska, Paulina; Johansson, Maria C; Mörgelin, Matthias; Belting, Mattias.

I: Proceedings of the National Academy of Sciences, Vol. 107, Nr. 30, 2010, s. 13342-13347.

Forskningsoutput: TidskriftsbidragArtikel i vetenskaplig tidskrift

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T1 - Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery.

AU - Wittrup, Anders

AU - Zhang, Sihe

AU - Svensson, Katrin

AU - Kucharzewska, Paulina

AU - Johansson, Maria C

AU - Mörgelin, Matthias

AU - Belting, Mattias

PY - 2010

Y1 - 2010

N2 - 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.

AB - 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.

U2 - 10.1073/pnas.1002622107

DO - 10.1073/pnas.1002622107

M3 - Article

VL - 107

SP - 13342

EP - 13347

JO - Proceedings of the National Academy of Sciences

JF - Proceedings of the National Academy of Sciences

SN - 1091-6490

IS - 30

ER -