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.

中文翻译：

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二酰基甘油激酶和磷脂酶D抑制剂会改变细胞脂质组和内体向高尔基体的分选。

膜脂质二酰基甘油（DAG）和磷脂酸（PA）是重要的第二信使，可以通过将蛋白质募集到膜上并改变生物物理膜特性来调节膜的运输。DAG和PA参与了从高尔基体到内体的运输，我们在这里研究了这些脂质的变化对于使用蛋白毒素蓖麻毒蛋白调节向高尔基体的运输是否重要。使用DAG激酶（DGK）和磷脂酶D（PLD）抑制剂对DAG和PA水平的调节使逆向蓖麻毒蛋白的转运大大增加，但对蓖麻毒蛋白的回收或降解影响很小。抑制剂治疗强烈影响内体的形态，增加了内体的微管和大小。此外，蓖麻毒蛋白与已知调节逆行转运的蛋白质一起存在于这些管状结构中。使用siRNA敲低PLD和DGK的不同同工型，我们发现PLD和DGK的几种同工型参与调节蓖麻毒蛋白向高尔基体的转运。最后，通过进行脂质组分析，我们发现DGK抑制剂可导致DAG含量微弱但可以预期的增加，而PLD抑制剂可显着提高DAG含量，且出乎意料，这表明使用抑制剂进行脂质组分析非常重要脂质代谢。