Cervantes-Chávez José A, Ruiz-Herrera José
Departamento de Ingeniería Genética, Unidad Irapuato, Centro de Investigación y de Estudios Avanzados del IPN, Irapuato, Gto. México.
FEMS Yeast Res. 2007 Sep;7(6):929-40. doi: 10.1111/j.1567-1364.2007.00265.x. Epub 2007 Jun 30.
The gene encoding the regulatory subunit (RKA1) of the cAMP-dependent protein kinase (PKA) of Yarrowia lipolytica was isolated to analyze the role of the PKA pathway in the dimorphic transition of the fungus. The gene encoded a protein of 397 amino acids that exhibits significant homology to fungal PKA regulatory subunits. Attempts to disrupt the gene by double homologous recombination, or the Pop-in Pop-out technique, were unsuccessful. The gene could be mutated only in merodiploids constructed with an autonomous replicating plasmid. Loss of the plasmid occurred with growth under nonselective conditions in the whole population of merodiploids carrying the mutation in the plasmid, but in merodiploids with the mutation at the chromosome, a resistant population prevailed. These data suggest that RKA1 is essential in Y. lipolytica. cAMP addition inhibited the dimorphic transition of the parental strain, but merodiploids carrying several copies of RKA1 were more resistant to cAMP. These results, and the observation that RKA1 was upregulated in mycelial cells, indicate that an active PKA pathway promotes yeast-like growth and opposes mycelial development. This behavior is in contrast to that of Candida albicans, where the PKA pathway favors hyphal growth.
为了分析蛋白激酶A（PKA）途径在解脂耶氏酵母双态转变中的作用，分离了编码解脂耶氏酵母cAMP依赖性蛋白激酶（PKA）调节亚基（RKA1）的基因。该基因编码一个由397个氨基酸组成的蛋白质，与真菌PKA调节亚基具有显著同源性。通过双同源重组或Pop-in Pop-out技术破坏该基因的尝试均未成功。该基因只能在用自主复制质粒构建的部分二倍体中发生突变。在携带质粒突变的部分二倍体群体中，在非选择性条件下生长时质粒会丢失，但在染色体上有突变的部分二倍体中，会出现一个抗性群体。这些数据表明RKA1在解脂耶氏酵母中是必需的。添加cAMP可抑制亲本菌株的双态转变，但携带多个RKA1拷贝的部分二倍体对cAMP更具抗性。这些结果，以及RKA1在菌丝体细胞中上调的观察结果，表明活跃的PKA途径促进酵母样生长并抑制菌丝体发育。这种行为与白色念珠菌相反，在白色念珠菌中PKA途径有利于菌丝生长。
