DECREASED SYNTHESIS OF GLYCOSPHINGOLIPIDS IN CELLS LACKING VIMENTIN INTERMEDIATE FILAMENTS

We are studying defects in glycosphingolipid synthesis in cells lackin g vimentin intermediate filaments (vimentin-). Sugars can be incorpora ted into glycolipids whose ceramide is synthesized either de novo (pat hway 1) or from sphingoid bases salvaged from hydrolysis of sphingolip ids (pathway 2) and into glycolipids recycling from the endosomal path way through the Golgi (pathway 3). Vimentin- embryonic fibroblasts, ob tained from vimentin-knockout mice, incorporate less sugar into glycol ipids than vimentin+ fibroblasts. Using two inhibitors of ceramide syn thesis, beta-chloroalanine and fumonisin B1, we found the major defect in synthesis to be in pathway 2 and not in de novo synthesis. Pie use d two additional approaches to analyze the functions of pathways 2 and 3, First, we used exogenous glucosylthioceramide ([C-14]C-8-Glc-S-Cer ), a synthetic, nonhydrolyzable glycosphingolipid, as a precursor for synthesis of larger glycolipids. Vimentin- SW13 cells and embryonic fi broblasts glycosylated [C-14]C-8-Glc-S-Cer less extensively than their vimentin+ counterparts. Second, we used chloroquine to inhibit the hy drolysis of sphingolipids in endosomes and lysosomes. Chloroquine mark edly decreased the incorporation of sugars into glycolipids larger tha n glucosylceramide. The defect in glycolipid synthesis in vimentin- ce lls probably results from impaired intracellular transport of glycolip ids and sphingoid bases between the endosomal/lysosomal pathway and th e Golgi apparatus and endoplasmic reticulum, Intermediate filaments ma y accomplish this function by contributing to the organization of subc ellular organelles and/or by binding proteins that participate in tran sport processes. (C) 1998 Academic Press.