近日，華中農(nóng)業(yè)大學(xué)油菜團隊與美國密蘇里大學(xué)圣路易斯分校/唐納德丹佛斯植物科學(xué)中心的最新合作研究成果發(fā)表，研究揭示了植物非特異性磷脂酶C4的定位機制。
非特異性磷脂酶C（non-specific phospholipase C，NPC）是植物特有的一類磷脂酶家族，因其對底物沒有特異性而得名。擬南芥有6個NPC基因，研究表明，不同NPC在植物的生長發(fā)育和逆境響應(yīng)過程中發(fā)揮著完全不同的作用，NPC功能上的差異可能由于它們的亞細胞定位不同造成的。NPC4與NPC5氨基酸序列相似性達88%，NPC4和NPC5都不存在跨膜結(jié)構(gòu)域，NPC5定位于胞質(zhì)中，而NPC4則定位于細胞質(zhì)膜，NPC4的細胞質(zhì)膜定位機制仍然未知。
在該研究中，研究者分析發(fā)現(xiàn)NPC4的C末端比NPC5多了17個氨基酸，對NPC4的C末端進行截短，發(fā)現(xiàn)NPC4Δ17定位于細胞質(zhì)中，表明NPC4蛋白質(zhì)C末端17氨基酸對于其細胞質(zhì)膜定位是必須的。進一步分析發(fā)現(xiàn)不同物種NPC4的C末端序列存在一個保守的半胱氨酸（cysteine，cys）位點，對NPC4第533位的半胱氨酸（Cys-533）進行點突變，發(fā)現(xiàn)NPC4C533A也定位于細胞質(zhì)中，表明該位點Cys決定了其細胞質(zhì)膜定位。Cys位點?；揎検菦Q定蛋白質(zhì)亞細胞定位的一種重要方式，通過?；瘷z測、體外酶活、質(zhì)譜分析等鑒定到擬南芥NPC4的Cys-533為棕櫚酸?；揎棥＿M一步分析發(fā)現(xiàn)，定位于細胞質(zhì)膜的油菜BnaC01.NPC4第531位的半胱氨酸也受到了棕櫚酸?；揎?。
為了研究NPC4的?；揎検欠袷瞧渌饧毎|(zhì)膜脂筏中鞘脂的關(guān)鍵，研究者發(fā)現(xiàn)NPC4C533A的突變不影響其酶活，通過互補實驗發(fā)現(xiàn)NPC4C533A無法回補npc4突變體在缺磷條件下鞘脂代謝的缺陷，這是由于NPC4C533A定位于細胞質(zhì)中，無法水解細胞質(zhì)膜脂筏中的鞘脂。以上結(jié)果表明，蛋白質(zhì)C末端半胱氨酸的棕櫚酸?；揎棇?dǎo)致NPC4定位于細胞質(zhì)膜，進而決定了其在缺磷條件下參與膜脂重塑的功能。
【英文摘要】
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. 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.
論文鏈接：https://doi.org/10.1111/tpj.15260
日期：2021-04-06
非特異性磷脂酶C（non-specific phospholipase C，NPC）是植物特有的一類磷脂酶家族，因其對底物沒有特異性而得名。擬南芥有6個NPC基因，研究表明，不同NPC在植物的生長發(fā)育和逆境響應(yīng)過程中發(fā)揮著完全不同的作用，NPC功能上的差異可能由于它們的亞細胞定位不同造成的。NPC4與NPC5氨基酸序列相似性達88%，NPC4和NPC5都不存在跨膜結(jié)構(gòu)域，NPC5定位于胞質(zhì)中，而NPC4則定位于細胞質(zhì)膜，NPC4的細胞質(zhì)膜定位機制仍然未知。
在該研究中，研究者分析發(fā)現(xiàn)NPC4的C末端比NPC5多了17個氨基酸，對NPC4的C末端進行截短，發(fā)現(xiàn)NPC4Δ17定位于細胞質(zhì)中，表明NPC4蛋白質(zhì)C末端17氨基酸對于其細胞質(zhì)膜定位是必須的。進一步分析發(fā)現(xiàn)不同物種NPC4的C末端序列存在一個保守的半胱氨酸（cysteine，cys）位點，對NPC4第533位的半胱氨酸（Cys-533）進行點突變，發(fā)現(xiàn)NPC4C533A也定位于細胞質(zhì)中，表明該位點Cys決定了其細胞質(zhì)膜定位。Cys位點?；揎検菦Q定蛋白質(zhì)亞細胞定位的一種重要方式，通過?；瘷z測、體外酶活、質(zhì)譜分析等鑒定到擬南芥NPC4的Cys-533為棕櫚酸?；揎棥＿M一步分析發(fā)現(xiàn)，定位于細胞質(zhì)膜的油菜BnaC01.NPC4第531位的半胱氨酸也受到了棕櫚酸?；揎?。
為了研究NPC4的?；揎検欠袷瞧渌饧毎|(zhì)膜脂筏中鞘脂的關(guān)鍵，研究者發(fā)現(xiàn)NPC4C533A的突變不影響其酶活，通過互補實驗發(fā)現(xiàn)NPC4C533A無法回補npc4突變體在缺磷條件下鞘脂代謝的缺陷，這是由于NPC4C533A定位于細胞質(zhì)中，無法水解細胞質(zhì)膜脂筏中的鞘脂。以上結(jié)果表明，蛋白質(zhì)C末端半胱氨酸的棕櫚酸?；揎棇?dǎo)致NPC4定位于細胞質(zhì)膜，進而決定了其在缺磷條件下參與膜脂重塑的功能。
【英文摘要】
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. 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.
論文鏈接：https://doi.org/10.1111/tpj.15260
日期：2021-04-06
