Obara Keisuke, Noda Takeshi, Niimi Kaori, Ohsumi Yoshinori
Department of Cell Biology, National Institute for Basic Biology, Okazaki 444-8585, Japan.
Genes Cells. 2008 Jun;13(6):537-47. doi: 10.1111/j.1365-2443.2008.01188.x.
Vps34, the sole PtdIns 3-kinase in yeast, is essential for autophagy. Here, we show that the lipid-kinase activity of Vps34 is required for autophagy, implying an essential role of its product PtdIns(3)P. The protein-kinase activity of Vps15, a regulatory subunit of the PtdIns 3-kinase complex, is also required for efficient autophagy. We monitored the distribution of PtdIns(3)P in living cells using a specific indicator, the 2xFYVE domain derived from mammalian Hrs. PtdIns(3)P was abundant at endosomes and on the vacuolar membrane during logarithmic growth phase. Under starvation conditions, we observed massive transport of PtdIns(3)P into the vacuole. This accumulation was dependent on the membrane dynamics of autophagy. Notably, PtdIns(3)P was highly enriched and delivered into the vacuole as a component of autophagosome membranes but not as a cargo enclosed within them, implying direct involvement of this phosphoinositide in autophagosome formation. We also found a possible enrichment of PtdIns(3)P on the inner autophagosomal membrane compared to the outer membrane. Based on these results we discuss the function of PtdIns(3)P in autophagy.
Vps34是酵母中唯一的磷脂酰肌醇3激酶，对自噬至关重要。在此，我们表明Vps34的脂质激酶活性是自噬所必需的，这意味着其产物磷脂酰肌醇-3-磷酸[PtdIns(3)P]具有重要作用。磷脂酰肌醇3激酶复合物的调节亚基Vps15的蛋白激酶活性对于高效自噬也是必需的。我们使用源自哺乳动物肝细胞生长因子（Hrs）的特异性指示剂2xFYVE结构域监测了活细胞中PtdIns(3)P的分布。在对数生长期，PtdIns(3)P在内体和液泡膜上大量存在。在饥饿条件下，我们观察到PtdIns(3)P大量转运至液泡中。这种积累依赖于自噬的膜动态变化。值得注意的是，PtdIns(3)P作为自噬体膜的一个组成部分高度富集并被转运至液泡中，而不是作为包裹在自噬体中的货物，这意味着这种磷酸肌醇直接参与了自噬体的形成。我们还发现，与自噬体外膜相比，PtdIns(3)P可能在自噬体内膜上富集。基于这些结果，我们讨论了PtdIns(3)P在自噬中的功能。
