Identification of immunosuppressant-sensitive genes: a study using model organisms

1.在分裂酵母中，钙调神经磷酸酶去磷酸化并激活Prz1转录因子。在这里，我们鉴定了przl(+)基因启动子区域的钙调神经磷酸酶依赖的反应元件(CDRE)，并使用融合到cdre的三个串联重复序列的不稳定的荧光素酶报告基因来监测活细胞中钙调神经磷酸酶的活性。通过Yam8/Cchl介导的Ca(2+)内流刺激钙调神经磷酸酶(CaN)需要Pck 2蛋白激酶C-Pmk1丝裂原激活蛋白激酶途径，而细胞外Ca(2+)升高的刺激不需要Pck 2蛋白激酶C-Pmk1丝裂原激活蛋白激酶途径，提示钙调神经磷酸酶有两条不同的激活途径。在这里，我们分离了Vic(在免疫抑制剂和氯离子存在下仍能存活)突变体。其中一个突变体vicl-1在编码蛋白法尼基转移酶的β亚基的cppl+基因上带有错义突变。对突变株的分析表明，Rho2是Cppl的一个新靶点。此外，Cpp1和Rho2作用于Pock2-Pmk1 MAPK…的上游更多的信号通路，从而导致其突变后的Vic表型。为了进一步了解丙戊酸作用(VPA)的分子机制，对对VPA敏感的分裂酵母突变株进行了遗传筛选。我们鉴定的其中一个基因是vps45+，它编码与膜运输有关的Sec1/Munc18家族的一个成员。我们的结果表明，VPA影响细胞膜的转运，从而导致分裂酵母对细胞壁损伤的敏感性增强。在同一筛选中，使用免疫抑制药物FK506在裂解酵母中，我们分离到编码Rab GDP解离抑制因子的必需gdil+基因的突变等位基因gdil-ill。值得注意的是，编码磷脂酰胆碱/磷脂酰肌醇转移蛋白的sp20+的过表达挽救了gdil-ill突变。Gdil-ill和sp20-KC104突变在合成上是致命的。我们的发现表明，Spo20通过调节细胞膜的磷脂代谢来调节Gdil的功能。较少

1. In fission yeast, calcineurin dephosphorylates and activates the Przl transcription factor. Here, we identified the calcineurin-dependent response element (CDRE) in the promoter region of przl(+) gene and monitored the calcineurin activity in living cells using a destabilized luciferase reporter gene fused to three tandem repeats of CDRE. The Pck2 protein kinase C-Pmkl mitogen-activate protein kinase pathway was required for the stimulation of calcineurin via Yam8/Cchl-mediated Ca(2+) influx, but it was not required for the stimulation by elevated extracellular Ca(2+), suggesting two distinct pathways for calcineurin activation.2. Here we isolated vic (viable in the presence of immunosuppressant and chloride ion) mutants. One of the mutants, vicl-1, carried a missense mutation in the cppl+ gene encoding a beta subunit of the protein farnesyltransferase. Analysis of the mutant strain revealed that Rho2 is a novel target of Cppl. Moreover, Cppl and Rho2 act upstream of Pck2-Pmk1 MAPK … More signaling pathway, thereby resulting in the vic phenotype upon their mutations.3. To gain further insights into the molecular mechanisms of valproic acid action (VPA), a genetic screen for fission yeast mutants that show hypersensitivity to VPA was performed. One of the genes we identified was vps45+ which encodes a member of the Sec1/Munc18 family that is implicated in membrane trafficking. Our results suggest that VPA affects membrane trafficking, which leads to the enhanced sensitivity to cell wall damage in fission yeast.4. In the same screen using the immunosuppressant drug FK506 in fission yeast, we isolated gdil-ill, a mutant allele of the essential gdil+ gene encoding Rab GDP-dissociation inhibitor. Notably, overexpression of spo20+, encoding a phosphatidylcholine/phosphatidylinositol transfer protein, rescued gdil-ill mutation. The gdil-ill and spo20-KC104 mutations are synthetically lethal. Our findings suggest that Spo20 modulates Gdil function via regulation of phospholipid metabolism of the membranes. Less