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PMID: 15292453 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Elevated endosomal cholesterol levels in Niemann-Pick cells inhibit rab4 and perturb membrane recycling.

Molecular biology of the cell ·Vol. 15 ·No. 10 ·2004-10-00 ·Pages 4500-11

Choudhury A, Sharma DK, Marks DL, Pagano RE

Abstract

In normal human skin fibroblasts (HSFs), fluorescent glycosphingolipid analogues are endocytosed and sorted into two pools, one that is recycled to the plasma membrane and one that is transported to the Golgi complex. Here, we investigated glycosphingolipid recycling in Niemann-Pick type A and C lipid storage disease fibroblasts (NPFs). Cells were incubated with a fluorescent analogue of lactosylceramide (LacCer) at 16 degrees C to label early endosomes (EEs), shifted to 37 degrees C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 approximately 8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 approximately 30-40 min), and rab4-dependent recycling was absent, whereas rab11-dependent recycling predominated. Transferrin recycling via the rab4 pathway was similarly perturbed in NPFs. Compared with normal HSFs, EEs in NPFs showed high cholesterol levels and an altered organization of rab4. In vitro extraction of rab4 (but not rab11) with GDP dissociation inhibitor was severely attenuated in NPF endosomal fractions. This impairment was reversed with cholesterol depletion of isolated endosomes or with high-salt treatment of endosomes. These data suggest that abnormal membrane recycling in NPFs results from specific inhibition of rab4 function by excess cholesterol in EEs.

MeSH Terms
Antigens, CD/chemistry,metabolism Boron Compounds/chemistry,metabolism Cell Membrane/metabolism Cells, Cultured Cholesterol/metabolism Endocytosis/physiology Endosomes/chemistry,metabolism Fibroblasts/cytology,physiology Fluorescent Dyes/chemistry,metabolism Humans Lactosylceramides/chemistry,metabolism Niemann-Pick Diseases/metabolism rab4 GTP-Binding Proteins/genetics,metabolism
Chemicals
4,4-difluoro-4-bora-3a,4a-diaza-s-indacene Antigens, CD Boron Compounds Fluorescent Dyes Lactosylceramides CDw17 antigen Cholesterol rab4 GTP-Binding Proteins
Authors & Affiliations
4 authors, click to expand affiliations / ORCID
Choudhury Amit
Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
Sharma Deepak K
Marks David L
Pagano Richard E
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Article Info
Journal
Molecular biology of the cell
Abbr.
Mol Biol Cell
ISSN
1059-1524
DOI
10.1091/mbc.e04-05-0432
Published
2004-10-00
Epub
2004-00-03
Pages
4500-11
Language
English
Region
United States
NLM ID
9201390
PMCID
PMC519144
Subset
IM
Grants
NIGMS NIH HHS · R01 GM022942 · United States
NIGMS NIH HHS · R01 GM060934 · United States
NIGMS NIH HHS · GM-60934 · United States
NIGMS NIH HHS · GM22942 · United States
External Links
PubMed source DOI: 10.1091/mbc.e04-05-0432 PMC Full Text View journal details
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