A proteomics study on establishment and functionality of chimeric protein complexes in hybrid yeast species and their effect on reproductive isolation

属于不同物种的生物通常不能相互交配。然而，在自然界中，如果两个物种的亲缘关系足够密切，它们有时可以繁殖出可存活的、尽管不育的杂交后代。这种情况的一个常见例子是“骡子”，一头公驴和一匹母马的杂交，它是可以存活的，但没有生育能力。该项目旨在研究杂交物种中发生的蛋白质相互作用，并着手了解在这种杂交物种中存在的两个遗传信息是作为单独的单位发挥作用，还是它们之间是否存在自然相互作用。如果这两组亲本蛋白质确实是相互作用的，我们想从杂种的适应性和适合性的角度来量化这种交流的影响(即特征骡子的固执和坏脾气是否是由于马和驴的蛋白质组之间的复杂相互作用造成的？难道是因为这样的互动，骡子比他们的父母更健康，更有活力吗？)我建议使用属于酵母菌属的酵母杂交来解决这个问题，酵母菌是真菌世界中的骡子。酿酒酵母、奇异葡萄球菌、米卡特氏葡萄球菌和巴耶氏葡萄球菌可以交配，由此产生的杂交细胞虽然能够通过萌发进行分裂，但不能产生可存活的生殖细胞，也称为孢子。所有这些物种的完整遗传蓝图的可获得性和它们的遗传多样性为科学家研究这些生物的分子进化和建立遗传隔离屏障提供了一个很好的机会。目前，还没有关于蛋白质网络相互作用及其对物种间杂交适合度的影响的实验研究。我们建议在杂交酵母细胞中建立参与不同细胞功能的蛋白质复合体。一个分子标签将被贴在表达所选复合体的亚单位A的基因上。然后，可以使用生化和化学策略来分离和注释带有所有相互作用伙伴的标记的亚基。鉴定出的相互作用蛋白可能都属于一个亲本物种(基因已被标记的那个物种)，或者同时属于两个亲本类型。因此，这个方法将告诉我们，蛋白质复合体的成员是否能够与来自这两个酵母亲本物种的伙伴自然地相互作用。我们还建议强制在酵母杂交种中组装嵌合蛋白复合体。这将使我们能够研究所产生的杂交在营养条件差和营养丰富的条件下的适合性，并评估同源蛋白的不同程度的互补能力(即，物种1的蛋白A是否能够在功能上取代杂交1，2细胞中物种2的蛋白A‘？)。这个项目是第一次尝试研究来自两个物种的同源蛋白质之间的联系，这些蛋白质同时存在于杂交细胞中，以及它们对所考虑的两个物种的适合性和交配后障碍的影响。

Organisms belonging to different species usually cannot mate with each other. However, in nature, if two species are sufficiently closely related, they can sometimes breed producing viable, although infertile, hybrids. A common place example of this condition is the 'mule', a cross of a male donkey and a female horse, which is viable but sterile. This Project aims to study the protein interactions which occur in hybrid species, and sets out to understand whether the two genetic information present in such hybrids acts and function as separate units or if there is a natural interaction between them. If the two set of parental proteins indeed cross-talk, we would like to quantify the effect of such communication in term of adaptation and fitness of the hybrid (i.e. Are the characteristic mule's stubbornness and bad-temper due to complex interactions between the horse's and donkey's proteomes? Is it because of such interactions that mules are fitter and more vigorous than their parents?). I propose to tackle this problem using hybrids of yeast species belonging to the genus Saccharomyces, the mules of the fungal world. S. cerevisiae, S. paradoxus, S. mikatae and S. bayanus can mate and the resulting hybrid cells, although abler to divide by budding, are unable to produce viable germs cells, also called spores. The availability of the complete genetic blueprint for all these species and their genetic versatility provide a great opportunity for the scientist to study molecular evolution and the establishment of genetic isolation barriers in these organisms. At this time no experimental studies looking at the protein network interaction and its effect on fitness in hybrids between species is available. We propose to look at the establishment of protein complexes, involved in different cell functions in hybrid yeast cell. A molecular tag will be attached to genes expressing the subunit A of the chosen complexes. The tagged subunit with all the interacting partners can then be isolated and annotated using biochemical and chemical strategies. The interacting proteins identified could either all belong to one parental species (the one in which the gene has been tagged), or to both parental types. So, this approach will tell us whether members of protein complexes, are able to interact naturally with the partners derived from both yeast parental species. We also propose to force the assembly of chimeric protein complexes in yeast hybrids. This will allow us to study the fitness of the resulting hybrids in poor and rich nutrition conditions, and to assess the different degree of complementation ability of the homologous protein (i.e. is protein A of species 1 able to functionally substitute protein A' from species 2 in the hybrid 1,2 cell?). This project represents the first attempt to investigate the connections of homologous proteins, deriving from two species, that are present simultaneously in the hybrid cell, and their impact on fitness and post-mating barriers of the two species considered.