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Published online 30 July 2001. doi:10.1083/jcb.200102084
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© The Rockefeller University Press, 0021-9525/2001/8/535 $5.00
The Journal of Cell Biology, Volume 154, Number 3, August 6, 2001 535-548

Article

Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways



Vishwajeet Puri, Rikio Watanabe, Raman Deep Singh, Michel Dominguez, Jennifer C. Brown, Christine L. Wheatley, David L. Marks and Richard E. Pagano

Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905

Address correspondence to Dr. Richard E. Pagano, Mayo Clinic and Foundation, Guggenheim 621-C, 200 First Street, S.W., Rochester, MN 55905-0001. Tel.: (507) 284-8754. Fax: (507) 266-4413. E-mail: pagano.richard{at}mayo.edu

Sphingolipids (SLs) are plasma membrane constituents in eukaryotic cells which play important roles in a wide variety of cellular functions. However, little is known about the mechanisms of their internalization from the plasma membrane or subsequent intracellular targeting. We have begun to study these issues in human skin fibroblasts using fluorescent SL analogues. Using selective endocytic inhibitors and dominant negative constructs of dynamin and epidermal growth factor receptor pathway substrate clone 15, we found that analogues of lactosylceramide and globoside were internalized almost exclusively by a clathrin-independent ("caveolar-like") mechanism, whereas an analogue of sphingomyelin was taken up approximately equally by clathrin-dependent and -independent pathways. We also showed that the Golgi targeting of SL analogues internalized via the caveolar-like pathway was selectively perturbed by elevated intracellular cholesterol, demonstrating the existence of two discrete Golgi targeting pathways. Studies using SL-binding toxins internalized via clathrin-dependent or -independent mechanisms confirmed that endogenous SLs follow the same two pathways. These findings (a) provide a direct demonstration of differential SLs sorting into early endosomes in living cells, (b) provide a "vital marker" for endosomes derived from caveolar-like endocytosis, and (c) identify two independent pathways for lipid transport from the plasma membrane to the Golgi apparatus in human skin fibroblasts.

Key Words: endocytosis; caveolae; cholesterol; Eps15; lipid storage diseases


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