## 3 Role of Lipids for Membrane Function and Intracellular Transport
### Modifications of the Lipidome: Changes as Expected?  
Although interfering with lipid metabolism using inhibitors, siRNA, or by supplying cells with some lipids may provide important information about the role of lipids for endocytosis and intracellular transport, we would also like to warn that the effects observed following such treatments might be due to other changes in the lipidome than those expected. Thus, careful analyses should be carried out to check if the changes in the lipidome are as expected. An excellent example of this is a study by the group of [PERSON] [193]. They used statins, known inhibitors of the rate determining enzyme in cholesterol synthesis, and observed that the retrograde transport of Shiga toxin (and cholera toxin) to Golgi and ER was blocked. By looking carefully into what happens with these cells, they observed that the statin treatment resulted in a partial relocation of the GlcCer synthase, elevated levels of this enzyme, and a several-fold increased level of GlcCer in all nine cell lines studied. There were also some changes in the total level of several other GSL classes but no changes in the relative amounts of the species of each class. The total cellular cholesterol level was unaffected by the statin treatment, which is in agreement with earlier reports that inhibition of cholesterol synthesis triggers an increased expression of the LDL receptor to maintain the cellular cholesterol level [194], while the cholesterol pool in the trans-Golgi network was dissipated. They showed that the changes in GSL and GlcCer synthase were independent of cholesterol synthesis inhibition, but that these effects surprisingly resulted from an aberrant Rab GTPase prenylation. Thus, they revealed an unexpected link between Rab prenylation (geranylgeranyl is an intermediate in the cholesterol biosynthesis) and regulation of GSLs and retrograde trafficking.  
We recently published that inhibitors of diacylglycerol kinase (DAG kinase) and phospholipase D (PLD) strongly increased the retrograde transport of ricin by affecting the endosomal sorting toward the Golgi apparatus [183]. Treatment with these inhibitors strongly affected the endosome morphology by increasing endosomal tubulation and size. Quantitative lipidomic studies of the inhibitor treated cells showed a lipidome as expected when using the DAG kinase inhibitor (increased DAG), whereas the PLD inhibitor resulted in a lipidome very different from that expected. There was a small temporary increase in PA, but it then decreased to control levels after 3 h. Unexpectedly, there was an increase in DAG and phosphatidylinositol(PG), and the species which were increased, clearly indicated that this was due to the metabolism of PC species. We speculate that the use of the PLD inhibitor resulted in compensatory mechanisms where the cells aimed at keeping the cellular PA levels relatively constant. Clearly, these lipid data demonstrate the need to perform quantitative lipidomic analyses before conclusions are drawn from such studies. Furthermore, siRNA was used to knock down different isoenzymes of DAG kinase and PLD. In addition to demonstrating that several of these isoenzymes were involved in regulating ricin transport to the Golgi, the experiments revealed one case where knock-down of one isoenzyme of DAG kinase resulted in increased levels of four other isoenzymes [183], thus demonstrating compensatory mechanisms in such studies.