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Originally published as MBC in Press, 10.1091/mbc.E03-03-0130 on May 3, 2003

Vol. 14, Issue 8, 3482-3493, August 2003

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Association of the Golgi UDP-Galactose Transporter with UDP-Galactose:Ceramide Galactosyltransferase Allows UDP-Galactose Import in the Endoplasmic Reticulum

Hein Sprong * {dagger} {ddagger}, Sophie Degroote * §, Tommy Nilsson ||, Masao Kawakita ¶, Nobuhiro Ishida ¶, Peter van der Sluijs {dagger}, and Gerrit van Meer * § #

* Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, 1100 DE Amsterdam, The Netherlands; {dagger} Department of Cell Biology, University Medical Center G02.525, Institute of Biomembranes, 3584 CX Utrecht, The Netherlands; {ddagger} Max-Planck-Institute of Molecular Cell Biology and Genetics, 01307 Dresden, Germany; § Department of Membrane Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, 3584 CH Utrecht, The Netherlands; || Cell Biology and Biophysics Program, European Molecular Biology Laboratory, 69012 Heidelberg, Germany; and Department of Physiological Chemistry, Tokyo Metropolitan Institute of Medical Science (Rinshoken), Tokyo 113-8613, Japan

Submitted March 5, 2003; Accepted April 11, 2003
Monitoring Editor: Vivek Malhotra

UDP-galactose reaches the Golgi lumen through the UDP-galactose transporter (UGT) and is used for the galactosylation of proteins and lipids. Ceramides and diglycerides are galactosylated within the endoplasmic reticulum by the UDP-galactose:ceramide galactosyltransferase. It is not known how UDP-galactose is transported from the cytosol into the endoplasmic reticulum. We transfected ceramide galactosyltransferase cDNA into CHOlec8 cells, which have a defective UGT and no endogenous ceramide galactosyltransferase. Cotransfection with the human UGT1 greatly stimulated synthesis of lactosylceramide in the Golgi and of galactosylceramide in the endoplasmic reticulum. UDP-galactose was directly imported into the endoplasmic reticulum because transfection with UGT significantly enhanced synthesis of galactosylceramide in endoplasmic reticulum membranes. Subcellular fractionation and double label immunofluorescence microscopy showed that a sizeable fraction of ectopically expressed UGT and ceramide galactosyltransferase resided in the endoplasmic reticulum of CHOlec8 cells. The same was observed when UGT was expressed in human intestinal cells that have an endogenous ceramide galactosyltransferase. In contrast, in CHOlec8 singly transfected with UGT 1, the transporter localized exclusively to the Golgi complex. UGT and ceramide galactosyltransferase were entirely detergent soluble and form a complex because they could be coimmunoprecipitated. We conclude that the ceramide galactosyltransferase ensures a supply of UDP-galactose in the endoplasmic reticulum lumen by retaining UGT in a molecular complex.

Abbreviations used: C6-NBD-, N-6(7-nitro-2,1,3-benzoxadiazol-4-yl)-aminohexanoyl-; CHO, Chinese hamster ovary; GalCer, galactosylceramide; GalT-1, UDP-galactose:ceramide galactosyltransferase; GalT-2, UDP-galactose:glucosylceramide({beta}1–4)galactosyltransferase; GlcCer, glucosylceramide; GM3, sialyllactosylceramide; HA, hemagglutinin epitope; LacCer, lactosylceramide; PDI, protein-disulfide isomerase; p33-PGalT, UDP-galactose:protein({beta}1–4)galactosyltransferase with the cytoplasmic domain of invariant chain p33; PNS, postnuclear supernatant; UGT, UDP-galactose transporter.

Article published online ahead of print. Mol. Biol. Cell 10.1091/mbc.E03-03-0130. Article and publication date are available at www.molbiolcell.org/cgi/doi/10.1091/mbc.E03-03-0130.

# Corresponding author. E-mail address: g.vanmeer{at}chem.uu.nl.




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