Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum
Increasing plasma membrane phosphatidylinositol(4,5)bisphosphate biosynthesis increases phosphoinositide metabolism in Nicotiana tabacum
复制标题
DOI：
10.1105/tpc.107.051367
复制
发表时间：
2007-05-01
期刊：
影响因子：
11.6
通讯作者：
Boss, Wendy F.
中科院分区：
文献类型：
--
作者：
Im, Yang Ju;Perera, Imara Y.;Boss, Wendy F.
采用遗传学方法增加磷脂酰肌醇（4，5）二磷酸[PtdIns（4，5）P-2]的生物合成，并验证PtdInsP激酶（PIPK）在植物磷脂酰肌醇（PI）途径中是流量限制性的假说。在烟草（Nicotiana tabacum）细胞中表达人PIPKI α使质膜PtdIns（4，5）P-2增加100倍。体内研究显示，（32）Pi掺入全细胞PtdIns（4，5）P-2的速率增加> 12倍，[H-3]PtdInSP（2）与[H-3]PtdInsP的比率增加6倍，但PtdInsP水平没有降低，表明PtdInsP生物合成不受限制。两个[H-3]肌醇三磷酸和[H-3]肌醇六磷酸增加，分别为3倍和1.5倍，在转基因株系标记后18小时。肌醇（1，4，5）三磷酸[Ins（1，4，5）P-3]结合测定显示，在转基因烟草品系中，总细胞Ins（1，4，5）P-3/g鲜重高出> 40倍;然而，即使具有这种高稳态水平的Ins（1，4，5）P-3，该途径也未饱和。用高渗胁迫刺激转基因细胞导致另一个2倍的增加，表明转基因细胞处于恒定的PI刺激状态。此外，表达Hs PIPKI α增加了糖的使用和氧的摄取。我们的研究结果表明，PIPK是流量限制，这种高速率的PI代谢增加了这些细胞的能量需求。
A genetic approach was used to increase phosphatidylincisitol(4,5)bisphosphate [PtdIns(4,5)P-2] biosynthesis and test the hypothesis that PtdInsP kinase (PIPK) is flux limiting in the plant phosphoinositide (PI) pathway. Expressing human PIPKI alpha in tobacco (Nicotiana tabacum) cells increased plasma membrane PtdIns(4,5)P-2 100-fold. In vivo studies revealed that the rate of (32)Pi incorporation into whole-cell PtdIns(4,5)P-2 increased > 12-fold, and the ratio of [H-3]PtdInSP(2) to [H-3]PtdInsP increased 6-fold, but PtdInsP levels did not decrease, indicating that PtdInsP biosynthesis was not limiting. Both [H-3]inositol trisphosphate and [H-3]inositol hexakisphosphate increased 3-and 1.5-fold, respectively, in the transgenic lines after 18 h of labeling. The inositol(1,4,5)trisphosphate [Ins(1,4,5)P-3] binding assay showed that total cellular Ins(1,4,5)P-3/g fresh weight was > 40-fold higher in transgenic tobacco lines; however, even with this high steady state level of Ins(1,4,5)P-3, the pathway was not saturated. Stimulating transgenic cells with hyperosmotic stress led to another 2-fold increase, suggesting that the transgenic cells were in a constant state of PI stimulation. Furthermore, expressing Hs PIPKI alpha increased sugar use and oxygen uptake. Our results demonstrate that PIPK is flux limiting and that this high rate of PI metabolism increased the energy demands in these cells.