Abstract
Sphingomyelin synthases (SMS1 and 2) represent a class of enzymes that transfer a phosphocholine moiety from phosphatidylcholine onto ceramide thus producing sphingomyelin and diacylglycerol (DAG). SMS1 localizes at the Golgi while SMS2 localizes both at the Golgi and the plasma membrane. Previous studies from our laboratory showed that modulation of SMS1 and, to a lesser extent, of SMS2 affected the formation of DAG at the Golgi apparatus. As a consequence, down-regulation of SMS1 and SMS2 reduced the localization of the DAG-binding protein, protein kinase D (PKD), to the Golgi. Since PKD recruitment to the Golgi has been implicated in cellular secretion through the trans golgi network (TGN), the effect of down-regulation of SMSs on TGN-to-plasma membrane trafficking was studied. Down regulation of either SMS1 or SMS2 significantly retarded trafficking of the reporter protein vesicular stomatitis virus G protein tagged with GFP (VSVG-GFP) from the TGN to the cell surface. Inhibition of SMSs also induced tubular protrusions from the trans Golgi network reminiscent of inhibited TGN membrane fission. Since a recent study demonstrated the requirement of PKD activity for insulin secretion in beta cells, we tested the function of SMS in this model. Inhibition of SMS significantly reduced insulin secretion in rat INS-1 cells. Taken together these results provide the first direct evidence that both enzymes (SMS1 and 2) are capable of regulating TGN-mediated protein trafficking and secretion, functions that are compatible with PKD being a down-stream target for SMSs in the Golgi.
Highlights
Sphingomyelin synthase (SMS), known as phosphatidylcholine:ceramide cholinephosphotransferase, is the last enzyme in the sphingomyelin (SM) synthetic pathway
In order to investigate whether SMS1 and SMS2 regulate trans golgi network (TGN) functions in a fashion that is compatible with the potential role of protein kinase D (PKD) as down-stream target of SMSs, we studied the role of SMSs in the vesicular trafficking from the Golgi to the plasma membrane
Since we previously provided direct evidence that both SMS1 and SMS2 possessed the ability of regulating production of DAG at the Golgi and that this pool of DAG is biologically active for recruitment of over-expressed PKD from the cytosol to the Golgi, we hypothesized that PKD might be a downstream target for SMS1 and SMS2 at the Golgi
Summary
Sphingomyelin synthase (SMS), known as phosphatidylcholine:ceramide cholinephosphotransferase, is the last enzyme in the sphingomyelin (SM) synthetic pathway. Medium was replaced with fresh DMEM containing 10% FBS and 0.1 M HEPES (pH 7.4) and the plates were shifted to 40uC (temperature at which VSVG-GFP reversibly misfolds and stays in the ER) for five and half hours (Figure S1).
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