Genetic evidence for phospholipid-mediated regulation of the Rab GDP-dissociation inhibitor in fission yeast
Genetic evidence for phospholipid-mediated regulation of the Rab GDP-dissociation inhibitor in fission yeast
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DOI：
10.1534/genetics.106.064709
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发表时间：
2006-11-01
期刊：
影响因子：
3.3
通讯作者：
Sugiura, Reiko
中科院分区：
文献类型：
--
作者：
Ma, Yan;Kuno, Takayoshi;Sugiura, Reiko
我们以前已经确定了突变等位基因的基因编码两个Rab蛋白，Ypt 3和Ryh 1，通过遗传筛选使用免疫抑制剂药物FK 506在裂殖酵母。在相同的筛选中，我们分离了gdi 1-i11，一种编码Rab GDP解离抑制剂的必需gdi 1（+）基因的突变等位基因。在gdi 1-i11中，保守的Gly 267被Asp取代。Gdil（G267 D）蛋白不能从膜中提取Rab，并且在gdil-i11突变体细胞中Rab从胞质级分中耗尽。一致地，Gdi 1（G267 D）蛋白主要被发现在膜部分，而野生型Gdi 1被发现在胞质和膜部分。值得注意的是，编码磷脂酰胆碱/磷脂酰肌醇转移蛋白的spo 20（+）的过表达挽救了gdi 1-i11突变，但不能挽救ypt 3-i5或ryh 1-i6。gdi 1-i11和spo 20-KC 104突变是合成致死的，野生型Gdi 1不能从spo 20-KC 104突变体的膜中提取Rab。Spo 20的磷脂酰肌醇转移活性对于抑制gdi 1-i11突变是不确定的，这表明磷脂酰胆碱转移活性对于抑制是重要的。此外，敲除编码磷酸胆碱胞苷转移酶的pct 1（+）基因挽救了gdi 1-i11突变。总之，我们的研究结果表明，Spo 20通过调节膜的磷脂代谢来调节Gdi 1功能。
We have previously identified mutant alleles of genes encoding two Rab proteins, Ypt3 and Ryh1, through a genetic screen using the immunosuppressant drug FK506 in fission yeast. In the same screen, we isolated gdi1-i11, a mutant allele of the essential gdi1(+) gene encoding Rab GDP-dissociation inhibitor. In gdi1-i11, a conserved Gly267 was substituted by Asp. The Gdil(G267D) protein failed to extract Rabs from membrane and Rabs were depleted from the cytosolic fraction in the gdi1-i11 mutant cells. Consistently, the Gdi1(G267D) protein was found mostly in the membrane fraction, whereas wild-type Gdi1 was found in both the cytosolic and the membrane fraction. Notably, overexpression of spo20(+), encoding a phosphatidylcholine/ phosphatidylinositol transfer protein, rescued gdi1-i11 mutation, but not ypt3-i5 or ryh1-i6 The gdi1-i11 and spo20-KC104 mutations are synthetically lethal, and the wild-type Gdi1 failed to extract Rabs from the membrane in the spo20-KC104 mutant. The phosphatidylinositol-transfer activity of Spo20 is dispensable for the suppression of the gdi1-i11 mutation, suggesting that the phosphatidylcholine-transfer activity is important for the suppression. Furthermore, knockout of the pct1(+) gene encoding a choline phosphate cytidyltransferase rescued the gdi1-i11 mutation. Together, our findings suggest that Spo20 modulates Gdi1 function via regulation of phospholipid metabolism of the membranes.