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Vol. 15, Issue 10, 4500-4511, October 2004
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Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905
Submitted May 25, 2004;
Revised July 22, 2004;
Accepted July 23, 2004
Monitoring Editor: Suzanne Pfeffer
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°C to label early endosomes (EEs), shifted to 37°C, and lipid recycling was quantified. Using dominant negative rabs, we showed that, in normal HSFs, LacCer recycling was rapid (t1/2 
8 min) and mainly rab4-dependent. In NPFs, LacCer recycling was delayed (t1/2 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.
Abbreviations used: BODIPY-LacCer, N-[5-(5,7-dimethylborondipyrromethenedifluoride)-1-penanoyl]-lactosylsphingosine; DF-BSA, defatted bovine serum albumin; DN, dominant negative; EE, early endosome; FITC, fluorescein isothiocyanate; GDI, GDP dissociation inhibitor; GFP, gene fluorescent protein; GSL, glycosphingolipid; GST, glutathione S-transferase; HSF, human skin fibroblast; LacCer, lactosylceramide; m
-CD, methyl-B-cyclodextrin; NP-A, Niemann-Pick disease, type A; NP-C, Niemann-Pick disease, type C; NPF, Niemann-Pick fibroblast; P13K, phosphoinositide 3-kinase; PM, plasma membrane; RE, recycling endosome; SL, sphingolipid; SLSD, sphingolipid storage disease; Tfn, transferrin; WM, wortmannin; WT, wild-type.
The online version of this article contains supplemental material accessible through http://www.molbiolcell.org.
* These authors contributed equally to this work.
Corresponding author. E-mail address: pagano.richard{at}mayo.edu.
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