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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Figure 4

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Overexpression of WT Rab7 or Rab9 (but not Rab11) restores BODIPY-LacCer...
Overexpression of WT Rab7 or Rab9 (but not Rab11) restores BODIPY-LacCer and fluorescent CtxB targeting to the Golgi apparatus of NP-C fibroblasts. Cells were transfected with plasmids encoding (a) DsRed fusion proteins or (b) EGFP fusion proteins of WT Rab7, Rab9, or Rab11. Forty-eight hours later, the samples were pulse-labeled with (a) BODIPY-LacCer or (b) Rh-CtxB. Golgi targeting of LacCer and CtxB was assessed by fluorescence microscopy. (a and b) Top rows: Transfected cells overexpressing WT Rab7 showed intense Golgi staining of LacCer (green) and CtxB (red), with little punctate cytoplasmic staining. In contrast, untransfected cells showed punctate cytoplasmic staining with either LacCer or CtxB (see insets). Middle rows: Similar results to those obtained with WT Rab7 were seen in cells overexpressing WT Rab9. Bottom rows: Little or no Golgi targeting of fluorescent LacCer or CtxB was observed in transfected cells overexpressing WT Rab11. Bar, 10 μm. (c) Golgi-labeling by BODIPY-LacCer or CtxB in NP-C cells overexpressing WT Rab’s was quantified as described in Figure 3b legend.