Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery.
(2010) In Proceedings of the National Academy of Sciences 107(30). p.13342-13347- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/1645029
- author
- Wittrup, Anders LU ; Zhang, Sihe LU ; Svensson, Katrin LU ; Kucharzewska, Paulina LU ; Johansson, Maria C LU ; Mörgelin, Matthias LU and Belting, Mattias LU
- organization
- publishing date
- 2010
- type
- Contribution to journal
- publication status
- published
- subject
- in
- Proceedings of the National Academy of Sciences
- volume
- 107
- issue
- 30
- pages
- 13342 - 13347
- publisher
- National Academy of Sciences
- external identifiers
-
- wos:000280602800030
- pmid:20624969
- scopus:77955788277
- ISSN
- 1091-6490
- DOI
- 10.1073/pnas.1002622107
- language
- English
- LU publication?
- yes
- id
- e19fe073-069f-4ce9-b246-73f7a308283b (old id 1645029)
- alternative location
- http://www.ncbi.nlm.nih.gov/pubmed/20624969?dopt=Abstract
- date added to LUP
- 2016-04-01 10:49:45
- date last changed
- 2022-04-04 21:45:57
@article{e19fe073-069f-4ce9-b246-73f7a308283b,
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.}},
author = {{Wittrup, Anders and Zhang, Sihe and Svensson, Katrin and Kucharzewska, Paulina and Johansson, Maria C and Mörgelin, Matthias and Belting, Mattias}},
issn = {{1091-6490}},
language = {{eng}},
number = {{30}},
pages = {{13342--13347}},
publisher = {{National Academy of Sciences}},
series = {{Proceedings of the National Academy of Sciences}},
title = {{Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery.}},
url = {{http://dx.doi.org/10.1073/pnas.1002622107}},
doi = {{10.1073/pnas.1002622107}},
volume = {{107}},
year = {{2010}},
}