Nicot Anne-Sophie, Fares Hanna, Payrastre Bernard, Chisholm Andrew D, Labouesse Michel, Laporte Jocelyn
Department of Molecular Pathology, Institut National de la Santé et de la Recherche Médicale U596, Centre National de la Recherche Scientifique Unité Mixte de Recherche 7104, Université Louis Pasteur de Strasbourg, Illkirch, France.
Mol Biol Cell. 2006 Jul;17(7):3062-74. doi: 10.1091/mbc.e05-12-1120.
Membrane dynamics is necessary for cell homeostasis and signal transduction and is in part regulated by phosphoinositides. Pikfyve/Fab1p is a phosphoinositide kinase that phosphorylates phosphatidylinositol 3-monophosphate into phosphatidylinositol-3,5-bisphosphate [PtdIns(3,5)P2] and is implicated in membrane homeostasis in yeast and in mammalian cells. These two phosphoinositides are substrates of myotubularin phosphatases found mutated in neuromuscular diseases. We studied the roles of phosphatidylinositol phosphate kinase 3 (PPK-3), the orthologue of PIKfyve/Fab1p, in a multicellular organism, Caenorhabditis elegans. Complete loss of ppk-3 function induces developmental defects characterized by embryonic lethality, whereas partial loss of function leads to growth retardation. At the cellular level, ppk-3 mutants display a striking enlargement of vacuoles positive for lysosome-associated membrane protein 1 in different tissues. In the intestine, RAB-7-positive late endosomes are also enlarged. Membranes of the enlarged lysosomes originate at least in part from smaller lysosomes, and functional and genetic analyses show that the terminal maturation of lysosomes is defective. Protein degradation is not affected in the hypomorphic ppk-3 mutant and is thus uncoupled from membrane retrieval. We measured the level of PtdIns(3,5)P2 and showed that its production is impaired in this mutant. This work strongly suggests that the main function of PPK-3 is to mediate membrane retrieval from matured lysosomes through regulation of PtdIns(3,5)P2.
膜动力学对于细胞稳态和信号转导是必需的，并且部分受磷酸肌醇调节。Pikfyve/Fab1p是一种磷酸肌醇激酶，可将磷脂酰肌醇3-单磷酸磷酸化为磷脂酰肌醇-3,5-二磷酸[PtdIns(3,5)P2]，并参与酵母和哺乳动物细胞中的膜稳态。这两种磷酸肌醇是在神经肌肉疾病中发现发生突变的肌管蛋白磷酸酶的底物。我们研究了磷脂酰肌醇磷酸激酶3（PPK-3），即Pikfyve/Fab1p的直系同源物，在多细胞生物秀丽隐杆线虫中的作用。ppk-3功能的完全丧失会导致以胚胎致死率为特征的发育缺陷，而部分功能丧失则会导致生长迟缓。在细胞水平上，ppk-3突变体在不同组织中显示出溶酶体相关膜蛋白1阳性的液泡显著增大。在肠道中，RAB-7阳性的晚期内体也增大。增大的溶酶体的膜至少部分源自较小的溶酶体，功能和遗传分析表明溶酶体的终末成熟存在缺陷。蛋白降解在低表达的ppk-3突变体中不受影响，因此与膜回收解偶联。我们测量了PtdIns(3,5)P2的水平，并表明其在该突变体中的产生受损。这项工作强烈表明PPK-3的主要功能是通过调节PtdIns(3,5)P2介导从成熟溶酶体的膜回收。
