Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery
- 12 July 2010
- journal article
- research article
- Published by Proceedings of the National Academy of Sciences in Proceedings of the National Academy of Sciences of the United States of America
- Vol. 107 (30), 13342-13347
- https://doi.org/10.1073/pnas.1002622107
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.Keywords
This publication has 38 references indexed in Scilit:
- ScFv Antibody-induced Translocation of Cell-surface Heparan Sulfate Proteoglycan to Endocytic VesiclesJournal of Biological Chemistry, 2009
- Syndecan-1 and FGF-2, but Not FGF Receptor-1, Share a Common Transport Route and Co-Localize with Heparanase in the Nuclei of Mesenchymal Tumor CellsPLOS ONE, 2009
- Clathrin-independent endocytosis: from nonexisting to an extreme degree of complexityHistochemistry and Cell Biology, 2008
- Signal sequence- and translation-independent mRNA localization to the endoplasmic reticulumRNA, 2008
- PTRF-Cavin, a Conserved Cytoplasmic Protein Required for Caveola Formation and FunctionCell, 2008
- Pathways of clathrin-independent endocytosisNature Reviews Molecular Cell Biology, 2007
- The multiple faces of caveolaeNature Reviews Molecular Cell Biology, 2007
- Alternative splicing generates a novel non-secretable cytosolic isoform of NELL2Biochemical and Biophysical Research Communications, 2007
- Ligands for Clathrin-Mediated Endocytosis Are Differentially Sorted into Distinct Populations of Early EndosomesCell, 2006
- Extracellular HSP70 binding to surface receptors present on antigen presenting cells and endothelial/epithelial cellsFEBS Letters, 2005