The Ras/cAMP/Protein Kinase A Pathway Regulates Glucose-dependent Assembly of the Vacuolar (H+)-ATPase in Yeast
The Ras/cAMP/Protein Kinase A Pathway Regulates Glucose-dependent Assembly of the Vacuolar (H+)-ATPase in Yeast
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DOI：
10.1074/jbc.m805232200
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发表时间：
2008-12-26
影响因子：
4.8
通讯作者：
Forgac, Michael
中科院分区：
文献类型：
--
作者：
Bond, Sarah;Forgac, Michael
极性（H+）-ATP酶（V-ATP酶）是普遍存在的ATP驱动的质子泵，其酸化细胞器或细胞外空间。调节V-ATP酶活性的快速有效机制涉及泵的两个功能域V-1和V-0的可逆解离。该过程在酵母中得到最好的表征，其中V-ATP酶响应于葡萄糖消耗而可逆地分解。为了鉴定在体内控制这一过程的调节剂，在酵母中进行遗传筛选以寻找在不存在葡萄糖的情况下生长时不能分解其V-ATP酶的突变体。该筛选将IRA 1（Ras/cAMP途径1的抑制性调节剂）和IRA 2鉴定为调节体内V-ATP酶解离的必需基因。IRA 1和IRA 2编码GTP酶激活蛋白，在营养不良的条件下负调节Ras。Ras的下调通过降低腺苷酸环化酶活性来降低cAMP水平。cAMP水平降低反过来导致蛋白激酶A（PKA）活性降低。我们的研究结果表明，IRA 2的靶向缺失导致葡萄糖耗竭时V-ATP酶的分解缺陷，基因的重新表达挽救了这种表型。葡萄糖依赖性解离也在表达显性活性RAS 2（val 19）等位基因的菌株或PKA调节亚基缺陷的菌株中被阻断，这两者都导致组成型活性PKA。这些结果揭示了PKA在控制酵母中葡萄糖依赖性V-ATP酶组装中的作用。
Vacuolar (H+)-ATPases (V-ATPases) are ubiquitous, ATP-driven proton pumps that acidify organelles or the extracellular space. A rapid and effective mechanism for regulating V-ATPase activity involves reversible dissociation of the two functional domains of the pump, V-1 and V-0. This process is best characterized in yeast, where V-ATPases are reversibly disassembled in response to glucose depletion. To identify regulators that control this process in vivo, a genetic screen was performed in yeast to search for mutants that cannot disassemble their V-ATPases when grown in the absence of glucose. This screen identified IRA1 (inhibitory regulator of the Ras/cAMP pathway 1) and IRA2 as essential genes for regulating V-ATPase dissociation in vivo. IRA1 and IRA2 encode GTPase-activating proteins that negatively regulate Ras in nutrient-poor conditions. Downregulation of Ras lowers cAMP levels by reducing adenylate cyclase activity. Decreased cAMP levels in turn lead to reduced activity of protein kinase A (PKA). Our results show that targeted deletion of IRA2 results in defective disassembly of the V-ATPase in response to glucose depletion, and reexpression of the gene rescues this phenotype. Glucose-dependent dissociation is also blocked in strains expressing the dominant active RAS2(val19) allele or in strains deficient for the regulatory subunit of PKA, both of which lead to constitutively active PKA. These results reveal a role for PKA in controlling glucose-dependent V-ATPase assembly in yeast.