Vac14 controls PtdIns(3,5)P2 synthesis and Fab1-dependent protein trafficking to the multivesicular body
Vac14 controls PtdIns(3,5)P2 synthesis and Fab1-dependent protein trafficking to the multivesicular body
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DOI：
10.1016/s0960-9822(02)00891-6
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发表时间：
2002-06-04
期刊：
影响因子：
9.2
通讯作者：
Michell, RH
中科院分区：
文献类型：
--
作者：
Dove, SK;McEwen, RK;Michell, RH
背景资料：PtdIns 3 P 5-激酶Fab 1使PtdIns（3，5）P-2成为一种磷酸肌醇，其对于液泡/溶酶体和晚期内体之间的逆行运输以及一些蛋白质通过多泡体（MVB）运输到液泡中是必需的。直到最近才确定Fab 1的调节剂。结果：用肉眼筛选出的Eurofan Ⅱ型沙门氏菌。酿酒酵母缺失突变体鉴定YLR 386 w为液泡功能的新调节子。其他人最近发现这个ORF编码液泡遗传基因VAC 14。像fab 1突变体一样，缺乏Vac 14的酵母具有不能正确酸化的扩大的空泡。FAB 1过表达纠正了这些缺陷。vac 14 δ细胞产生很少的PtdIns（3，5）P-2，高渗休克不能以正常方式刺激PtdIns（3，5）P-2的合成，表明Vac 14参与Fab 1的调节。我们还表明，像fab 1Delta突变体，vac 14 Delta细胞无法将GFP-Phm 5分选到MVB，从而分选到液泡：GFP-Phm 5的不可逆泛素化克服了这一缺陷。在BY 4742的遗传背景下，Vac 14的缺失对磷酸肌醇代谢和液泡运输的影响比Fab 1的另一个调节因子Vac 7的缺失大得多。Vac 14含有提示在蛋白质运输中起作用的基序，并与参与网格蛋白介导的膜分选和磷酸肌醇代谢的几种蛋白质相互作用。结论：Vac 14和Vac 7都是Fab 1催化的PtdIns（3，5）P-2合成的上游活化剂，Vac 14是控制层级的主要贡献者。Vac 14对于PtdIns（3，5）P-2的调节合成、控制某些蛋白质通过MVB运输到液泡腔以及维持液泡大小和酸度是必不可少的。
Background: The PtdIns3P 5-kinase Fab1 makes PtdIns(3,5)P-2, a phosphoinositide essential for retrograde trafficking between the vacuole/lysosome and the late endosome and also for trafficking of some proteins into the vacuole via multivesicular bodies (MVB). No regulators of Fab1 were identified until recently. Results: Visual screening of the Eurofan II panel of S. cerevisiae deletion mutants identified YLR386w as a novel regulator of vacuolar function. Others recently identified this ORF as encoding the vacuolar inheritance gene VAC14. Like fab1 mutants, yeast lacking Vac14 have enlarged vacuoles that do not acidify correctly. FAB1 overexpression corrects these defects. vac14Delta cells make very little PtdIns(3,5)P-2, and hyperosmotic shock does not stimulate PtdIns(3,5)P-2 synthesis in the normal manner, implicating Vac14 in Fab1 regulation. We also show that, like fab1Delta mutants, vac14Delta cells fail to sort GFP-Phm5 to the MVB and thence to the vacuole: irreversible ubiquitination of GFP-Phm5 overcomes this defect. In the BY4742 genetic background, loss of Vac14 causes much more penetrant effects on phosphoinositide metabolism and vacuolar trafficking than does loss of Vac7, another regulator of Fab1. Vac14 contains motifs suggestive of a role in protein trafficking and interacts with several proteins involved in clathrin-mediated membrane sorting and phosphoinositide metabolism. Conclusions: Vac14 and Vac7 are both upstream activators of Fab1-catalysed PtdIns(3,5)P-2 synthesis, with Vac14 the dominant contributor to the hierarchy of control. Vac14 is essential for the regulated synthesis of PtdIns(3,5)P-2, for control of trafficking of some proteins to the vacuole lumen via the MVB, and for maintenance of vacuole size and acidity.