National Key Laboratory of Crop Genetic Improvement, Huazhong Agricultural University, Wuhan, 430070, China.
Department of Biology, University of Missouri, St. Louis, MO, 63121, USA.
Plant J. 2021 Jun;106(6):1647-1659. doi: 10.1111/tpj.15260. Epub 2021 May 2.
Non-specific phospholipase C (NPC) is involved in plant growth, development and stress responses. To elucidate the mechanism by which NPCs mediate cellular functions, here we show that NPC4 is S-acylated at the C terminus and that acylation determines its plasma membrane (PM) association and function. The acylation of NPC4 was detected using NPC4 isolated from Arabidopsis and reconstituted in vitro. The C-terminal Cys-533 was identified as the S-acylation residue, and the mutation of Cys-533 to Ala-533 in NPC4 (NPC4 ) led to the loss of S-acylation and membrane association of NPC4. The knockout of NPC4 impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4 to npc4-1 failed to complement this defect, thereby supporting the hypothesis that the non-acylated NPC4 fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4 failed to complement the primary root growth in npc4-1 under stress. In addition, NPC4 in Brassica napus was S-acylated and mutation of the S-acylating cysteine residue of BnaC01.NPC4 led to the loss of S-acylation and its membrane association. Together, our results reveal that S-acylation of NPC4 in the C terminus is conserved and required for its membrane association, phosphosphingolipid hydrolysis and function in plant stress responses.
非特异性磷酯酶 C（NPC）参与植物的生长、发育和应激反应。为了阐明 NPC 介导细胞功能的机制，我们发现 NPC4 在 C 末端发生 S-酰化，并且酰化决定了其质膜（PM）的结合和功能。通过从小麦中分离 NPC4 并在体外重建，检测 NPC4 的酰化。鉴定出 C 末端半胱氨酸 533 是 S-酰化残基，NPC4 中 Cys-533 突变为 Ala-533（NPC4 ）导致 NPC4 的 S-酰化和膜结合丧失。NPC4 的敲除阻碍了磷酸盐缺乏诱导的磷酸鞘氨醇糖基肌醇磷酸神经酰胺（GIPC）的减少，但将 NPC4 引入 npc4-1 未能弥补这一缺陷，从而支持了非酰化 NPC4 在磷酸盐缺乏时不能水解 GIPC 的假设。此外，在胁迫下，npc4-1 中的 NPC4 无法补充主根的生长。此外，油菜中的 NPC4 发生 S-酰化，BnaC01.NPC4 的 S-酰化半胱氨酸残基突变导致 S-酰化及其膜结合丧失。总之，我们的研究结果表明 NPC4 在 C 末端的 S-酰化是保守的，是其质膜结合、磷酸鞘氨醇水解和植物应激反应功能所必需的。
