Recently, the oilseed rape research team of Huazhong Agricultural University (HZAU) and Donald Danfoss Plant Science Center, University of Missouri St. Louis, published their latest cooperative research, revealing the localization mechanism of plant nonspecific phospholipase C4.
Nonspecific phospholipase C (NPC) is a family of plant-specific phospholipases, which is named for its nonspecific substrate. Arabidopsis thaliana has six NPC genes. Studies have shown that different NPCs play completely different roles in plant growth, development and stress response. The difference in the function of NPC may be due to their different subcellular localization. The similarity of amino acid sequence between NPC4 and NPC5 is nearly 88%. There is no transmembrane domain in NPC4 and NPC5. NPC5 is located in the cytoplasm, while NPC4 is in the plasma membrane. The localization mechanism of NPC4 in the plasma membrane is still unknown.
In this study, the researchers found that the C-terminal of NPC4 was 17 amino acids more than that of NPC5. They truncated the C-terminal of NPC4 and found that NPC4 Δ 17 was located in the cytoplasm, which indicated that the C-terminal 17 amino acids of NPC4 protein were necessary for its plasma membrane localization. Further analysis showed that there was a conserved cysteine (Cys) site in the C-terminal sequence of NPC4 in different species. The point mutation of cys-533 in NPC4 showed that NPC4C533A was also located in the cytoplasm, indicating that the Cys site determined the location of its plasma membrane. Acylation of Cys site was an important way to determine the subcellular localization of proteins. The acylation of cys-533 in Arabidopsis NPC4 was identified as palmitic acid acylation by acylation detection, in vitro enzyme activity and mass spectrometry. Further study revealed that cysteine at position 531 of BnaC01.NPC4 located in the plasma membrane of Brassica napus was also modified by palmitic acid acylation.
In order to study whether the acylation of NPC4 was the key to the hydrolysis of sphingolipids in the membrane lipid rafts, researchers found that the mutation of NPC4C533A did not affect its enzyme activity. It was found that NPC4C533A could not make up for the deficiency of sphingolipid metabolism of NPC4 mutant under phosphorus deficiency, because NPC4C533A was located in the cytoplasm and could not hydrolyze the sphingolipid in the membrane lipid rafts. The research suggests that the palmitate acylation of C-terminal cysteine leads to the localization of NPC4 in the plasma membrane, which determines its role in membrane lipid remodeling under phosphorus deficiency.
Paper link：https://doi.org/10.1111/tpj.15260
Source: http://news.hzau.edu.cn/2021/0404/59776.shtml
Translated by: Li Juan
Supervised by: Pan Buhan
