Abstract
Ceramide (Cer) transfer from the endoplasmic reticulum (ER) to the Golgi apparatus was measured under conditions that block vesicle-mediated protein transfer. This was done either in intact cells by reducing the incubation temperature to 15 degreesC, or in streptolysin O-permeabilized cells by manipulating the intracellular environment. In both cases, Cer transfer was not inhibited, as demonstrated by the biosynthesis of ceramide monohexosides and sphingomyelin (SM) de novo from metabolically (with [14C]serine) labelled Cer. This assay is based on the knowledge that Cer is synthesized, starting from serine and palmitoyl-CoA, at the ER, whereas glycosphingolipids and SM are synthesized in the (early) Golgi apparatus. Formation of [14C]glycosphingolipids and [14C]SM was observed under conditions that block vesicle-mediated vesicular stomatitis virus glycoprotein transport. These results indicate that [14C]Cer is transferred from ER to Golgi by a non-vesicular mechanism.