Zaremberg V, Moreno S
Departamento de Química Biológica, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires, Argentina.
Eur J Biochem. 1996 Apr 1;237(1):136-42. doi: 10.1111/j.1432-1033.1996.0136n.x.
Spontaneous mutations in the gene which encodes the regulatory subunit of cAMP-dependent protein kinase (PKA) of Saccharomyces cerevisiae (BCY1) have been isolated previously [Cannon, J. F., Gibbs, J. B. & Tatchell, K. (1986) Genetics 113, 247-264] by selection of ras2::LEU2 revertants that grew on non-fermentable carbon sources. The revertants were placed into groups of increasing severity based on the number of PKA-dependent traits affected [Cannon, J. F., Gitan, R. & Tatchell, K. (1990) J. Biol. Chem. 265, 11897-11904]. In this work the ras2 mutation has been crossed out in each bcy1 allele and the phenotypes of these mutants have been assessed. The order of severity of the mutants in both genetic backgrounds is maintained but the severity of each mutant in the normal background is higher than in the ras2::LEU2 background. Total catalytic-subunit and regulatory-subunit activities were measured in crude extracts of the bcy1 ras2::LEU2 mutants. With one exception (bcy1-6) the calculated regulatory subunit/catalytic subunit ratios of the bcy1 mutants relative to that of wild-type cells were greater than one. The dependence of PKA activity on cAMP was measured in permeabilized cells. The strains show an activity ratio in the absence and presence of cAMP in the range 0.5-1 for Kemptide phosphorylation. Overexpression of the high-affinity cAMP phosphodiesterase gene (PDE2) in the bcy1 ras2::LEU2 strains did not alter their PKA-dependent phenotypes. However, transformants were not observed from the parental ras2::LEU2 strain and the bcy1-6 ras2::LEU2 strain. The results are discussed with respect to a hypothesis for the molecular mechanism of the differential reversal of ras2 phenotypes by the bcy1 alleles. Mutations in the regulatory subunit are predicted to affect the structure of the holoenzyme such that the catalytic subunit is capable of maintaining an active catalytic state, without the need to dissociate from the regulatory subunit.
先前已通过筛选能在非发酵性碳源上生长的ras2::LEU2回复子，分离出了酿酒酵母（Saccharomyces cerevisiae）中编码cAMP依赖性蛋白激酶（PKA）调节亚基的基因（BCY1）的自发突变体[坎农，J.F.，吉布斯，J.B.和塔切尔，K.（1986年）遗传学113，247 - 264]。根据受PKA依赖性性状影响的数量，将回复子分为严重程度递增的组[坎农，J.F.，吉坦，R.和塔切尔，K.（1990年）生物化学杂志265，11897 - 11904]。在这项工作中，已在每个bcy1等位基因中去除了ras2突变，并评估了这些突变体的表型。在两种遗传背景下，突变体的严重程度顺序得以维持，但在正常背景下每个突变体的严重程度高于在ras2::LEU2背景下。在bcy1 ras2::LEU2突变体的粗提物中测量了总催化亚基和调节亚基活性。除了一个例外（bcy1 - 6），相对于野生型细胞，bcy1突变体计算出的调节亚基/催化亚基比率大于1。在通透细胞中测量了PKA活性对cAMP的依赖性。对于Kemptide磷酸化，这些菌株在有无cAMP时的活性比率在0.5 - 1范围内。在bcy1 ras2::LEU2菌株中高亲和力cAMP磷酸二酯酶基因（PDE2）的过表达并未改变它们的PKA依赖性表型。然而，未从亲本ras2::LEU2菌株和bcy1 - 6 ras2::LEU2菌株中观察到转化体。针对bcy1等位基因对ras2表型差异逆转的分子机制的假设，对结果进行了讨论。预计调节亚基中的突变会影响全酶的结构，使得催化亚基能够维持活性催化状态，而无需与调节亚基解离。
