Both the Golgi and the endosomes have recently been proposed as the main site of SM-synthase, the enzyme responsible for sphingomyelin (SM) biosynthesis. To settle this confusion, we studied the subcellular distribution of SM-synthase in human liver-derived HepG2 and baby hamster kidney BHK-21 cells. To discriminate between Golgi and endosomes we made use of 3,3-diaminobenzidine (DAB) cytochemistry. Cells were incubated with a conjugate of transferrin (Tf) and horseradish peroxidase (HRP), or with unconjugated HRP, to label the recycling pathway and the complete endocytic pathway (including lysosomes) with peroxidase activity, respectively. After cell homogenization, the peroxidase activity was used to induce a local deposition of DAB-polymer. The total SM-synthase activity was not affected by this procedure, and, in contrast to endosomes labeled with (125)I-Tf, organelles containing SM-synthase did not increase in buoyant density as determined by Percoll density gradient fractionation. Thus, little, if any, SM-synthase localizes to the endocytic pathway of HepG2 and BHK-21 cells. In experiments performed at low temperature to inhibit vesicular transport, we found less than 10% of newly synthesized short-chain SM at the cell surface. We conclude that most SM-synthase activity is present in the Golgi, and to a small extent at the cell surface.

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