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

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.

Thanks to its versatile nature, Saccharomyces cerevisiae is an excellent experimental model for biological, biotechnological and evolutionary studies. In fact, the availability of vast amounts of sequence information from the genomes of closely related yeast species (1) provide a unique opportunity for an in-depth analysis at all level of comparative and functional genomics. In the past ten years several studies on genetic redundancy, conservation of synteny and gene order, and polyploid origin of the yeast genome have been carried out(2,3). Experimental evolution studies also have shown that different mechanisms of speciation may have taken place during the evolutionary history of these eukaryotes: the mismatch repair system(4) as well as chromosomal rearrangements(5,6) may play a role in hybrid sterility and fitness, and dominant genic incompatibilities are absent in these species (7). At the preoteome level, recent 'in silico' studies attempted to measure, respectively, the rate of protein evolution in these yeasts (8), and to assess the importance of having a balance stoichiometry within subunits of a protein complex(9). While we are witnessing a flourishing of bioinformatics studies on evolution of protein networks in several organisms, experimental proteomics studies has not yet been widely applied in the evolutionary field. We proposed to carry out 'wet laboratory' studies to understand the nature of the protein-protein interactions present in hybrids of yeast species. In fact, these hybrids, although sterile, are viable and possess two homeologous copies of the proteome derived from the parental species (i.e. one set of proteins from species 1: a,b,c,...;and one from species 2: a',b',c'...). Assessing experimentally whether functional protein complexes of hybrid nature can be established (i.e. a/b'/c, or a'/b/c'...) and how these chimeric interactions can affect the fitness of the hybrids are the main objectives of this Project. To carry out these tasks two approaches are proposed. In the first, one subunit of a protein complex (i.e. subunit a) is molecularly tagged in the hybrid, and isolated, together with all the interacting partners, on an affinity column (10). The interacting subunits, identified by mass-spectrometry (11) could either all belong to one parental species (the one in which the gene has been tagged), or to both parental types (i.e. isolation of b, c, b', c' rather than only b, c), confirming in this way that chimeric protein complexes can be established in the hybrid. In the second approach, different members of the homeologous protein complex abc and a'b'c' are deleted in the yeast hybrids (i.e. the subunits a and b' derived from parental species 1 and 2, respectively). In this way, the cell is forced to create chimeric complexes (a'bc' and a'bc) to survive. This approach will allow us to study the efficiency of the functional compensation between homeologous proteins in relation to the phylogenetic distance of the parental species. The degree of complementation will be assessed by measuring their relative fitness on rich and minimal nutrition media. Overall, this Project intends to shed new lights on the nature of protein-protein interactions and incompatibilities in yeast hybrid species, and on how evolutionary divergence affects the complementation capacity of genes. References 1. Kellis et al. (2003), Nature 423: 241-254 2. Dujon et al. (2004), Nature 430: 35-44 3. Wolfe & Shields (1997), Nature 387: 798-713 4. Hunter et al, EMBO J 15:1726-1733. 5. Delneri et al. (2003), Nature 422: 68-72 6. Colson et al. (2004), EMBO rep. 5: 392-398 7. Greig et al. (2002), Proc. R. Soc. Lond. B 269: 1167-1171 8. Wall et al. (2005), Proc. Natl. Acad. Sci. USA, 102: 5483-5488 9. Papp et al. (2003), Nature 424, 194-197 10. Rigaut et al (1999), Nature Biotech. 17: 1030-1032 11. Brancia et al. (2001), Electrophoresis 22:552-559.