title: Clathrin-dependent and -independent internalization of plasma membrane sphingolipids initiates two Golgi targeting pathways.
abstract: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, Thoracic Diseases Research Unit, Mayo Clinic and Foundation, Rochester, MN 55905, USA.
authors.forename: V
authors.initials: V
authors.lastname: Puri
authors.forename: R
authors.initials: R
authors.lastname: Watanabe
authors.forename: R D
authors.initials: RD
authors.lastname: Singh
authors.forename: M
authors.initials: M
authors.lastname: Dominguez
authors.forename: J C
authors.initials: JC
authors.lastname: Brown
authors.forename: C L
authors.initials: CL
authors.lastname: Wheatley
authors.forename: D L
authors.initials: DL
authors.lastname: Marks
authors.forename: R E
authors.initials: RE
authors.lastname: Pagano
mesh_terms: D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D001896:Boron Compounds / Q000493:pharmacokinetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D051242:Caveolin 1; D022461:Caveolins / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002966:Clathrin / Q000378:metabolism*; D034281:Dynamins; D004705:Endocytosis / Q000502:physiology; D011992:Endosomes / Q000378:metabolism; D005347:Fibroblasts / Q000166:cytology; D005456:Fluorescent Dyes / Q000493:pharmacokinetics; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism; D005733:Gangliosidoses / Q000378:metabolism; D005915:Globosides / Q000493:pharmacokinetics*; D006056:Golgi Apparatus / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D006801:Humans; D007202:Indicators and Reagents / Q000493:pharmacokinetics; D047908:Intracellular Signaling Peptides and Proteins; D007790:Lactosylceramides / Q000493:pharmacokinetics*; D008164:Luminescent Proteins / Q000493:pharmacokinetics; D016296:Mutagenesis / Q000502:physiology; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D021381:Protein Transport / Q000502:physiology; D012867:Skin / Q000166:cytology
chemical_list: C095489:4,4-difluoro-4-bora-3a,4a-diaza-s-indacene; D048868:Adaptor Proteins, Signal Transducing; D015703:Antigens, CD; D001896:Boron Compounds; D002135:Calcium-Binding Proteins; D051242:Caveolin 1; D022461:Caveolins; D002966:Clathrin; C486206:EPS15 protein, human; D005456:Fluorescent Dyes; D005915:Globosides; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; D007790:Lactosylceramides; D008164:Luminescent Proteins; D010750:Phosphoproteins; D049452:Green Fluorescent Proteins; C009744:CDw17 antigen; D020558:GTP Phosphohydrolases; D034281:Dynamins