The opportunist Candida albicans: yeast proliferation and nutritional signaling

描述（由申请人提供）：本提案的目的是确定白色念珠菌酵母和菌丝之间的不同机制，增殖控制以及通过雷帕霉素途径对营养信号的反应。在之前的工作中，我们确定了菌丝到酵母形态发生的调节因子Pes1。尽管该基因的同系物在研究的所有其他真核生物中都是必不可少的，但白色念珠菌菌丝在其耗尽期间继续强劲生长，而酵母则停止生长。我们的初步数据表明，pes1缺失的菌丝对Tor营养信号的抑制反应与野生型相似，而酵母不能适当地阻止生长。因此，酵母正常增殖和酵母对Tor抑制的正常反应都需要Pes1。我们发现Pes1是正常水平翻译活性标记所必需的，我们将其用于白色念珠菌。我们还发现它是细胞周期从G1期到S期进展所必需的。由于Pes1的定位在不同的细胞类型以及活跃和抑制的Tor信号状态之间是不同的，我们推断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

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.