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We provide evidence for a role of the glucose-regulated protein (GRP)75\/PBP74\/mtHSP70\/mortalin (hereafter termed \u201cGRP75\u201d) 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.<\/jats:p>","DOI":"10.1073\/pnas.1002622107","type":"journal-article","created":{"date-parts":[[2010,7,12]],"date-time":"2010-07-12T23:40:19Z","timestamp":1278978019000},"page":"13342-13347","update-policy":"https:\/\/doi.org\/10.1073\/pnas.cm10313","source":"Crossref","is-referenced-by-count":62,"title":["Magnetic nanoparticle-based isolation of endocytic vesicles reveals a role of the heat shock protein GRP75 in macromolecular delivery"],"prefix":"10.1073","volume":"107","author":[{"given":"Anders","family":"Wittrup","sequence":"first","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Si-He","family":"Zhang","sequence":"additional","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Katrin J.","family":"Svensson","sequence":"additional","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paulina","family":"Kucharzewska","sequence":"additional","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Maria C.","family":"Johansson","sequence":"additional","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthias","family":"M\u00f6rgelin","sequence":"additional","affiliation":[{"name":"Clinical and Experimental Infectious Medicine, Department of Clinical Sciences, Lund University, SE-221 85, Lund, Sweden"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mattias","family":"Belting","sequence":"additional","affiliation":[{"name":"Sections of aOncology and"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"341","published-online":{"date-parts":[[2010,7,12]]},"reference":[{"key":"e_1_3_3_1_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2005.12.038"},{"key":"e_1_3_3_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.addr.2004.10.004"},{"key":"e_1_3_3_3_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2216"},{"key":"e_1_3_3_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/nature01451"},{"key":"e_1_3_3_5_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0968-0004(03)00031-8"},{"key":"e_1_3_3_6_2","doi-asserted-by":"publisher","DOI":"10.1007\/s00418-007-0376-5"},{"key":"e_1_3_3_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2007.11.042"},{"key":"e_1_3_3_8_2","doi-asserted-by":"publisher","DOI":"10.1038\/nrm2122"},{"key":"e_1_3_3_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2006.10.030"},{"key":"e_1_3_3_10_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.0308186101"},{"key":"e_1_3_3_11_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.012346499"},{"key":"e_1_3_3_12_2","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.93.22.12349"},{"key":"e_1_3_3_13_2","doi-asserted-by":"publisher","DOI":"10.1074\/jbc.M109.036129"},{"key":"e_1_3_3_14_2","doi-asserted-by":"crossref","first-page":"500","DOI":"10.4049\/jimmunol.146.2.500","article-title":"Cellular and subcellular distribution of PBP72\/74, a peptide-binding protein that plays a role in antigen processing","volume":"146","author":"VanBuskirk AM","year":"1991","unstructured":"AM VanBuskirk, DC DeNagel, LE Guagliardi, FM Brodsky, SK Pierce, Cellular and subcellular distribution of PBP72\/74, a peptide-binding protein that plays a role in antigen processing. 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