The opportunist Candida albicans: yeast proliferation and nutritional signaling

描述(申请人提供)：这项建议的目的是确定白念珠菌酵母和菌丝之间的不同机制，以控制增殖，并通过雷帕霉素途径的靶标对营养信号做出反应。在以前的工作中，我们确定了菌丝到酵母形态发生的调节因子PES1。虽然这个基因的同源物在所有其他被研究的真核生物中都是必不可少的，但白念珠菌菌丝在其枯竭期间继续旺盛生长，而酵母则停止生长。我们的初步数据显示，PES1缺失的菌丝对Tor营养信号的抑制做出了类似于野生型的反应，而酵母未能适当地阻止生长。因此，对于正常的酵母增殖和酵母对Tor抑制的正常反应，PES1都是必需的。我们发现Pes1是正常水平的翻译活性标记物所必需的，我们将其适应于在白色念珠菌中使用。我们还发现它是细胞周期从G1期进入S期所必需的。由于Pes1的定位在不同的细胞类型以及激活和抑制的Tor信号状态之间不同，我们推测它的一些蛋白质相互作用伙伴在这些不同的条件下是不同的，初步的亲和纯化实验支持这一观点。我们的目标是确定 通过亲和纯化，从不同的细胞类型和营养信号状态中分离出与PES1相互作用的蛋白质。为了测试特定于这些条件的Pes1相互作用伙伴的生物学相关性，将进行具有互易表位和亲和标签的免疫共沉淀实验，并将确认这些蛋白质与Pes1在同源条件下的共同定位。这些Pes1相互作用伙伴在细胞类型特异性增殖控制中的作用 将通过反向遗传方法进行研究。Pes1在酵母和菌丝中的定位，以及在Tor活性和抑制细胞中的定位将使用特定细胞器的已知标记的共定位来确定。对Tor信号通路中心节点的遗传分析将回答这样一个问题，即细胞类型特异性信号是该通路的所有分支所固有的，还是对一个分支特有的，并将确定PES1与Tor营养信号的这些关键元件之间的上位性和功能关系。这项工作的结果将促进我们对普遍存在的白念珠菌细胞类型酵母的特定增殖控制的理解。

DESCRIPTION (provided by applicant): The objective of this proposal is to identify the mechanisms which differ between C. albicans yeast and hyphae, for control of proliferation, and for the response to nutritional signaling through the target of rapamycin pathway. In previous work we identified a regulator of hypha-to-yeast morphogenesis, Pes1. Whereas homologs of this gene are essential in all other eukaryotes studied, C. albicans hyphae continue to grow robustly during its depletion, while yeast arrest growth. Our preliminary data show that PES1-depleted hyphae respond to inhibition of Tor nutritional signaling similarly to the wild type, whil yeast fail to arrest growth appropriately. Pes1 is therefore required both for normal yeast proliferation, and for the normal response of yeast to Tor inhibition. We found Pes1 to be required for normal levels of a marker of translational activity which we adapted for use in C. albicans. We also found it to be required for progression of the cell cycle from G1 to S phase. As Pes1 localization differs between cell types and between active and inhibited Tor signaling states, we reason that some of its protein interaction partners differ between these distinct conditions, and preliminary affinity purification experiments support this idea. We aim to identify Pes1 interacting proteins by affinity purification from the distinct cell types and nutritional signaling states. To test the biological relevance of Pes1 interaction partners specific to these conditions, co- immunoprecipitation experiments with reciprocal epitope and affinity tags will be performed, and co- localization of these proteins with Pes1 in the cognate conditions will be confirmed. The role of these Pes1 interaction partners for cell-type specific proliferation control will be investigated by reverse genetic approaches. Localization of Pes1 in yeast and hyphae, and in Tor-active versus -inhibited cells will be determined using co-localization with known markers of specific organelles. Genetic analysis of central nodes of the Tor signaling pathway will answer the question whether cell-type specific signaling is intrinsic to all branches of the pathway or is specific to one branch, and will determine the epistatic and functional relationship of PES1 to these critical elements of Tor nutritional signaling. The results of this work will promote our understanding of specific proliferation control of the ubiquitous C. albicans cell type, the yeast.