Stoyan Tanja, Carbon John
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106, USA.
Eukaryot Cell. 2004 Oct;3(5):1154-63. doi: 10.1128/EC.3.5.1154-1163.2004.
The human pathogenic yeast Candida glabrata is the second most common Candida pathogen after Candida albicans, causing both bloodstream and mucosal infections. The centromere (CEN) DNA of C. glabrata (CgCEN), although structurally very similar to that of Saccharomyces cerevisiae, is not functional in S. cerevisiae. To further examine the structure of the C. glabrata inner kinetochore, we isolated several C. glabrata homologs of S. cerevisiae inner kinetochore protein genes, namely, genes for components of the CBF3 complex (Ndc10p, Cep3p, and Ctf13p) and genes for the proteins Mif2p and Cse4p. The amino acid sequence identities of these proteins were 32 to 49% relative to S. cerevisiae. CgNDC10, CgCEP3, and CgCTF13 are required for growth in C. glabrata and are specifically found at CgCEN, as demonstrated by chromatin immunoprecipitation experiments. Cross-complementation experiments revealed that the isolated genes, with the exception of CgCSE4, are species specific and cannot functionally substitute for the corresponding genes in S. cerevisiae deletion strains. Likewise, the S. cerevisiae CBF3 genes NDC10, CEP3, and CTF13 cannot functionally replace their homologs in C. glabrata CBF3 deletion strains. Two-hybrid analysis revealed several interactions between these proteins, all of which were previously reported for the inner kinetochore proteins of S. cerevisiae. Our findings indicate that although many of the inner kinetochore components have evolved considerably between the two closely related species, the organization of the C. glabrata inner kinetochore is similar to that in S. cerevisiae.
人类致病酵母光滑念珠菌是仅次于白色念珠菌的第二常见念珠菌病原体，可引起血流感染和黏膜感染。光滑念珠菌的着丝粒（CEN）DNA（CgCEN）虽然在结构上与酿酒酵母的着丝粒DNA非常相似，但在酿酒酵母中无功能。为了进一步研究光滑念珠菌内着丝粒的结构，我们分离了酿酒酵母内着丝粒蛋白基因的几个光滑念珠菌同源物，即CBF3复合体（Ndc10p、Cep3p和Ctf13p）组分的基因以及Mif2p和Cse4p蛋白的基因。这些蛋白的氨基酸序列与酿酒酵母相比，同一性为32%至49%。染色质免疫沉淀实验表明，CgNDC10、CgCEP3和CgCTF13是光滑念珠菌生长所必需的，且特异性地存在于CgCEN处。交叉互补实验表明，除CgCSE4外，分离出的基因具有物种特异性，不能在功能上替代酿酒酵母缺失菌株中的相应基因。同样，酿酒酵母的CBF3基因NDC10、CEP3和CTF13也不能在功能上替代光滑念珠菌CBF3缺失菌株中的同源物。双杂交分析揭示了这些蛋白之间的几种相互作用，所有这些相互作用先前都在酿酒酵母的内着丝粒蛋白中报道过。我们的研究结果表明，尽管在这两个密切相关的物种之间，许多内着丝粒组分已经发生了相当大的进化，但光滑念珠菌内着丝粒的组织与酿酒酵母中的相似。
