Balla Andras, Tuymetova Galina, Tsiomenko Arnold, Várnai Péter, Balla Tamas
Endocrinology and Reproduction Research Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA.
Mol Biol Cell. 2005 Mar;16(3):1282-95. doi: 10.1091/mbc.e04-07-0578. Epub 2005 Jan 5.
The PH domains of OSBP and FAPP1 fused to GFP were used to monitor PI(4)P distribution in COS-7 cells during manipulations of PI 4-kinase (PI4K) activities. Both domains were associated with the Golgi and small cytoplasmic vesicles, and a small fraction of OSBP-PH was found at the plasma membrane (PM). Inhibition of type-III PI4Ks with 10 microM wortmannin (Wm) significantly reduced but did not abolish Golgi localization of either PH domains. Downregulation of PI4KIIalpha or PI4KIIIbeta by siRNA reduced the localization of the PH domains to the Golgi and in the former case any remaining Golgi localization was eliminated by Wm treatment. PLC activation by Ca2+ ionophores dissociated the domains from all membranes, but after Ca2+ chelation, they rapidly reassociated with the Golgi, the intracellular vesicles and with the PM. PM association of the domains was significantly higher after the Ca2+ transient and was abolished by Wm pretreatment. PM relocalization was not affected by down-regulation of PI4KIIIbeta or -IIalpha, but was inhibited by down-regulation of PI4KIIIalpha, or by 10 microM PAO, which also inhibits PI4KIIIalpha. Our data suggest that these PH domains detect PI(4)P formation in extra-Golgi compartments under dynamic conditions and that various PI4Ks regulate PI(4)P synthesis in distinct cellular compartments.
将与绿色荧光蛋白（GFP）融合的OSBP和FAPP1的PH结构域用于在操纵磷脂酰肌醇4-激酶（PI4K）活性期间监测COS-7细胞中PI(4)P的分布。这两个结构域都与高尔基体和小的细胞质囊泡相关，并且在质膜（PM）上发现一小部分OSBP-PH。用10微摩尔渥曼青霉素（Wm）抑制III型PI4K可显著降低但并未消除任何一个PH结构域在高尔基体的定位。通过小干扰RNA（siRNA）下调PI4KIIα或PI4KIIIβ可减少PH结构域在高尔基体的定位，并且在前一种情况下，任何剩余的高尔基体定位都可通过Wm处理消除。钙离子载体激活磷脂酶C（PLC）使这些结构域从所有膜上解离，但在钙离子螯合后，它们迅速重新与高尔基体、细胞内囊泡和质膜结合。钙离子瞬变后这些结构域与质膜的结合显著增加，并被Wm预处理消除。质膜重新定位不受PI4KIIIβ或-IIα下调的影响，但受PI4KIIIα下调或10微摩尔PAO（其也抑制PI4KIIIα）的抑制。我们的数据表明，这些PH结构域在动态条件下检测高尔基体以外的区室中PI(4)P的形成，并且各种PI4K在不同的细胞区室中调节PI(4)P的合成。
