PATHWAYS OF GLYCOSPHINGOLIPID BIOSYNTHESIS IN SW13 CELLS IN THE PRESENCE AND ABSENCE OF VIMENTIN INTERMEDIATE FILAMENTS

We reported previously that the incorporation of sugars into glycosphi ngolipids (GSL) is diminished in SW13 cells that lack a vimentin inter mediate filament (IF) network (vim-) compared to vim+ cells. To furthe r analyze the nature of this abnormality, we double-labeled cells with H-3-serine and C-14-sugars, There was no difference between vim+ and vim- cells in the incorporation of serine into GSL, although the usual difference in sugar incorporation was observed. This indicated that t he defect in vim- cells was not in the incorporation of sugars into ce ramide synthesized de novo by acylation of sphinganine (pathway 1), Su gars can also be incorporated into ceramide synthesized from sphingosi ne that is derived from catabolism of sphingolipids (pathway 2), and i nto GSL that recycle through the Golgi apparatus from endosomes (pathw ay 3), The amount of galactose and glucosamine incorporated into GSL i n these three pathways was analyzed by the use of two inhibitors of sp hingolipid biosynthesis. beta-Chloroalanine inhibits the de novo synth esis of sphinganine (pathway 1), and fumonisin B1 inhibits the acylati on of sphinganine and sphingosine (pathways 1 and 2), We were surprise d to observe that in both vim+ and vim- cells only 20-40% of sugar inc orporation into GSL took place in pathway 1, and 60-80% of sugar incor poration took place in the recycling pathways, Moreover, in contrast t o larger GSL, GlcCer was not synthesized in pathway 3, Our observation s indicate that vimentin IF facilitate the recycling of GSL and sphing osine, and that the differences between vim+ and vim- cells are predom inantly in pathways 2 and 3, Furthermore, although it is generally bel ieved that virtually all GSL are synthesized in the de novo pathway, t hese data indicate that the recyling pathways predominate in the incor poration of sugars into GSL in SW13 cells.