Diacylglycerol kinase and phospholipase D inhibitors alter the cellular lipidome and endosomal sorting towards the Golgi apparatus

Abstract

The membrane lipids diacylglycerol (DAG) and phosphatidic acid (PA) are important second messengers that can regulate membrane transport by recruiting proteins to the membrane and by altering biophysical membrane properties. DAG and PA are involved in the transport from the Golgi apparatus to endosomes, and we have here investigated whether changes in these lipids might be important for regulation of transport to the Golgi using the protein toxin ricin. Modulation of DAG and PA levels using DAG kinase (DGK) and phospholipase D (PLD) inhibitors gave a strong increase in retrograde ricin transport, but had little impact on ricin recycling or degradation. Inhibitor treatment strongly affected the endosome morphology, increasing endosomal tubulation and size. Furthermore, ricin was present in these tubular structures together with proteins known to regulate retrograde transport. Using siRNA to knock down different isoforms of PLD and DGK, we found that several isoforms of PLD and DGK are involved in regulating ricin transport to the Golgi. Finally, by performing lipidomic analysis we found that the DGK inhibitor gave a weak, but expected, increase in DAG levels, while the PLD inhibitor gave a strong and unexpected increase in DAG levels, showing that it is important to perform lipidomic analysis when using inhibitors of lipid metabolism.

Lyso-Lipid-Induced Oligodendrocyte Maturation Underlies Restoration of Optic Nerve Function

“…We have also investigated the underlying mechanism of LPC 18:1 action. We identified the interaction of SNX4, a protein critical for endocytosis ( Lingelem et al, 2020 ), with LPC 18:1 ( Fig. 4 I ).…”

“…SNX4-mediated endocytosis requires the recruitment of several proteins. The modulation of these interactions depends on membrane lipid composition ( Lingelem et al, 2020 ) and involves a glycosylphosphatidylinositol-anchored proteins-enriched early endosomal compartment ( Kumari et al, 2010 ; Lingelem et al, 2020 ). The presence of SNX4-mediated alkyl-lyso-phospholipid endocytosis, reported earlier, supports our findings ( Cuesta-Marbán et al, 2013 ).…”

The Protein Toxins Ricin and Shiga Toxin as Tools to Explore Cellular Mechanisms of Internalization and Intracellular Transport

“…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.…”

“…Effect increased by combining the inhibitors. No changes in recycling or degradation of ricin [183]. See the main text for further details.…”

“…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.…”