Oilseed rape team reveals the localization mechanism of plant non-specific phospholipase C4

Recently, Plant Journal published online the results of a collaboration between the oilseed rape team and the University of Missouri-Louis/Donald Danforth Plant Science Center entitled "Acylation of nonspecific phospholipase C4 determines its function in plant response to phosphate deficiency. deficiency" was published online by the Plant Journal as a result of a collaboration between the rape team and the University of Missouri-St. Louis/Donald Danforth Plant Science Center.

Non-specific phospholipase C (NPC) is a family of plant-specific phospholipases, named for their lack of substrate specificity. Arabidopsis has six NPC genes, and studies have shown that different NPCs play completely different roles in plant growth and development and in response to adversity, and that differences in NPC function may be due to their different subcellular localizations. 88% amino acid sequence similarity between NPC4 and NPC5, neither NPC4 nor NPC5 has a transmembrane structural domain, and NPC5 is localized in the cytoplasm, whereas NPC5 is localized in the cytoplasm, while NPC4 is localized in the cytoplasmic membrane. The mechanism of cytoplasmic membrane localization of NPC4 remains unknown.

In this study, the researchers analyzed and found that the C-terminus of NPC4 has 17 more amino acids than NPC5, and truncated the C-terminus of NPC4 and found that NPC4Δ17 is localized in the cytoplasm, indicating that the C-terminal 17 amino acids of NPC4 protein are necessary for its cytoplasmic membrane localization. Further analysis revealed a conserved cysteine (cys) site in the C-terminal sequence of NPC4 from different species, and point mutation of the cysteine (Cys-533) at position 533 of NPC4 revealed that NPC4C533A is also localized in the cytoplasm, indicating that this site Cys determines its cytoplasmic membrane localization. acylation modification of the Cys site is Cys-533 of Arabidopsis NPC4 was identified as palmitoylated by acylation assay, in vitro enzymatic activity and mass spectrometry analysis. Further analysis revealed that the cysteine at position 531 of oilseed rape BnaC01.NPC4, which is localized to the cytoplasmic membrane, was also subjected to palmitoylation modification. To investigate whether the acylation modification of NPC4 is essential for its hydrolysis of sphingolipids in cytoplasmic membrane lipid rafts, the investigators found that mutation of NPC4C533A did not affect its enzymatic activity, and complementation experiments revealed that NPC4C533A was unable to backfill the defective sphingolipid metabolism of the npc4 mutant under phosphorus-deficient conditions due to the localization of NPC4C533A in the cytoplasm and its inability to hydrolyze sphingolipids in cytoplasmic membrane lipid rafts. in the sphingolipids of the cytoplasmic membrane. These results suggest that palmitoylation modification of the C-terminal cysteine of the protein leads to the localization of NPC4 to the cytoplasmic membrane, which in turn determines its function to participate in membrane lipid remodeling under phosphorus-deficient conditions.