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MBC in Press, published online ahead of print August 3, 2004
Mol. Biol. Cell 10.1091/mbc.E04-05-0432

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Submitted on May 25, 2004
Revised on July 22, 2004
Accepted on July 23, 2004

Elevated Endosomal Cholesterol in Niemann-Pick Cells Inhibits Rab4 and Perturbs Membrane Recycling

Amit Choudhury,* Deepak K. Sharma,* David L. Marks, and Richard E. Pagano{dagger}

Department of Biochemistry and Molecular Biology, Mayo Clinic and Foundation, Rochester, MN 55905

Monitoring Editor: Suzanne Pfeffer

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

*These authors contributed equally to this work.

{dagger}Corresponding author. E-mail: pagano.Richard{at}mayo.edu






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