Recently, the Huazhong Agricultural University canola team and the University of Missouri St. Louis/Donald Danfoss Plant Science Center published the latest results of a collaborative research, revealing the localization mechanism of plant non-specific phospholipase C4.
non-specific phospholipase C (non-specific phospholipase C, NPC) is a family of phospholipases peculiar to plants, so named because it has no specificity to the substrate. There are six NPC genes in Arabidopsis. Studies have shown that different NPCs play completely different roles in the growth and development of plants and in response to stress. The differences in NPC functions may be caused by their different subcellular locations. The amino acid sequence similarity between NPC4 and NPC5 reaches 88%. There is no transmembrane domain in NPC4 and NPC5. NPC5 is located in the cytoplasm, while NPC4 is located in the plasma membrane. The cytoplasmic membrane location mechanism of NPC4 is still unknown.
In this study, the researchers found that the C-terminus of NPC4 was 17 more amino acids than NPC5. The C-terminus of NPC4 was truncated and found that NPC4Δ17 is located in the cytoplasm, indicating that the C-terminus of NPC4 protein has 17 amino acids for its cytoplasm. Membrane positioning is necessary. Further analysis revealed that there is a conservative cysteine (cys) site in the C-terminal sequence of NPC4 of different species. A point mutation was performed on the cysteine (Cys-533) at position 533 of NPC4, and it was found that NPC4C533A was also located in In the cytoplasm, it indicates that Cys at this site determines its cytoplasmic membrane location. Cys site acylation is an important way to determine protein subcellular localization. Through acylation detection, in vitro enzyme activity, mass spectrometry analysis, etc., Cys-533 of Arabidopsis NPC4 was identified as palmitic acid acylation. Further analysis revealed that the cysteine at position 531 of rape BnaC01.NPC4, which is located in the plasma membrane of the cytoplasm, was also modified by palmitic acid acylation.
In order to study whether the acylation of NPC4 is the key to the hydrolysis of sphingolipids in cytoplasmic membrane lipid rafts, the researchers found that the mutation of NPC4C533A does not affect its enzyme activity. Through complementation experiments, it was found that NPC4C533A could not replenish the phosphorus deficiency of npc4 mutants. The defect of sphingolipid metabolism under conditions is due to the fact that NPC4C533A is located in the cytoplasm and cannot hydrolyze the sphingolipids in the lipid rafts of the plasma membrane. The above results indicate that the palmitic acid acylation modification of the protein C-terminal cysteine leads to the localization of NPC4 in the plasma membrane, which in turn determines its function of participating in membrane lipid remodeling under conditions of phosphorus deficiency.
Nonspecific 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 the 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 mutation of Cys-533 to Ala-533 of NPC4 (NPC4C533A) led to the loss of S-acylation and membrane association of NPC4. Knockout of NPC4 impeded the phosphate deficiency-induced decrease of the phosphosphingolipid glycosyl inositol phosphoryl ceramide (GIPC), but introducing NPC4C533A to npc4‐1 failed to complement this defect, supporting that the non‐acylated NPC4C533A fails to hydrolyze GIPC during phosphate deprivation. Moreover, NPC4C533A failed to complement the primary root growth in npc4‐1 under stress. I n 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 reveal association. Together, our results 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.
Link to the paper:https://doi.org/10.1111/tpj.15260