Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells
Amit Choudhury, … , David L. Marks, Richard E. Pagano
Amit Choudhury, … , David L. Marks, Richard E. Pagano
Published June 15, 2002
Citation Information: J Clin Invest. 2002;109(12):1541-1550. https://doi.org/10.1172/JCI15420.
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Article Genetics

Rab proteins mediate Golgi transport of caveola-internalized glycosphingolipids and correct lipid trafficking in Niemann-Pick C cells

Abstract

We recently showed that human skin fibroblasts internalize fluorescent analogues of the glycosphingolipids lactosylceramide and globoside almost exclusively by a clathrin-independent mechanism involving caveolae. In contrast, a sphingomyelin analogue is internalized approximately equally via clathrin-dependent and caveolar routes. Here, we further characterized the caveolar pathway for glycosphingolipids, showing that Golgi targeting of sphingolipids internalized via caveolae required microtubules and phosphoinositol 3-kinases and was inhibited in cells expressing dominant-negative Rab7 and Rab9 constructs. In addition, overexpression of wild-type Rab7 or Rab9 (but not Rab11) in Niemann-Pick type C (NP-C) lipid storage disease fibroblasts resulted in correction of lipid trafficking defects, including restoration of Golgi targeting of fluorescent lactosylceramide and endogenous GM1 ganglioside, and a dramatic reduction in intracellular cholesterol stores. Our results demonstrate a role for Rab7 and Rab9 in the Golgi targeting of glycosphingolipids and suggest a new therapeutic approach for restoring normal lipid trafficking in NP-C cells.

Authors

Amit Choudhury, Michel Dominguez, Vishwajeet Puri, Deepak K. Sharma, Keishi Narita, Christine L. Wheatley, David L. Marks, Richard E. Pagano

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Fluorescent LacCer is transported from the PM to the Golgi apparatus in ...
Fluorescent LacCer is transported from the PM to the Golgi apparatus in normal HSFs and is dependent on microtubules and activity of PI3Ks. (a) Cells grown on etched grid coverslips were incubated with 2.5 μM BODIPY-LacCer for 30 minutes at 4°C in HMEM, washed, further incubated for 45 minutes at 37°C, and then back-exchanged at 10°C to remove any fluorescent lipid present at the PM. Live cell imaging (left panel) revealed a perinuclear distribution of the LacCer analogue. Cells were then fixed and immunolabeled with an antibody against the Golgi marker mannosidase II, and the same field was rephotographed for mannosidase II staining (MannII; right panel). (b) Cells were preincubated with wortmannin or nocodazole and subsequently pulse-labeled with BODIPY-LacCer as above. Golgi targeting of LacCer was inhibited in 80% (nocodazole), 50% (wortmannin), 70% (LY294002, not shown), or 5% (untreated) of the cells (n = 20 for each). No disruption of Golgi morphology was detected for the indicated inhibitors using BODIPY-Cer as a vital stain for this organelle. Bar, 10 μM.