Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization
Identification of mammalian Vps24p as an effector of phosphatidylinositol 3,5-bisphosphate-dependent endosome compartmentalization
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DOI：
10.1074/jbc.m306864200
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发表时间：
2003-10-03
影响因子：
4.8
通讯作者：
Holman, GD
中科院分区：
文献类型：
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作者：
Whitley, P;Reaves, BJ;Holman, GD
磷脂酰肌醇3,5-二磷酸是迄今为止研究的所有真核生物中发现的膜脂，但该脂质的下游效应蛋白尚未确定。本文报道了利用cDNA噬菌体文库与合成的磷脂酰肌醇3,5-二磷酸生物素化衍生物一起鉴定哺乳动物磷脂酰肌醇3,5-二磷酸结合蛋白mVps24p。该蛋白与酿酒酵母蛋白Vps24p同源，Vps24p是e类空泡蛋白分选蛋白。通过体外脂质体结合和竞争实验，我们证明mVps24p选择性地结合磷脂酰肌醇3,5-二磷酸和磷脂酰肌醇3,4-二磷酸，优于其他测试的磷脂肌醇。当在培养的哺乳动物细胞中表达时，全长mVps24p是细胞质的。然而，当表达全长mVps24p的细胞与突变形式的mVps4p （ATP水解缺陷）共转染时，或者当表达mVps24p的n端结构时，诱导具有肿胀液泡的e类细胞表型，mVps24p与膜相关。此外，当磷脂酰肌醇3,5-二磷酸合成被wortmannin阻断时，n端mVps24p结构体在肿胀的内体膜上的积累被取消。这些数据首次提供了磷脂酰肌醇3,5-二磷酸和参与e类细胞表型产生的蛋白质机制之间的直接联系。我们假设，当膜结合（依赖于磷脂酰肌醇3,5-二磷酸与蛋白n端结构域的相互作用）与膜解耦（由Vps4p驱动）分离时，Vps24在膜上的积累就会发生。
Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but down-stream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).