Yeast Proteomics Milestone

Courtesy of Won-Ki Huh LOCAL COLOR: Researchers used librar-ies of tagged proteins to determine where those proteins accumulate, and to what levels. Shown are representative localization (top) and co-localization (bottom) data. Researchers from the University of California, San Francisco, have probed the yeast proteome in the highest level of detail to date. Using sets of fusion-tagged proteins, the scientists, led by Howard Hughes Medical Institute investigators Erin O'Shea and Jonathan Weissman, obtained abundance and cellular localization data for more than 75% of the proteins expressed in Saccharomyces cerevisiae during log-phase growth.1,2 Their results represent the first truly global view of the yeast proteome. "This is a landmark in that it's making the leap from a purely descriptive cataloging of protein expression to one that describes a genome-wide quantitative analysis," says Charlie Boone of the University of Toronto. The researchers used homologous recombination to introduce engineered genes directly into the genome in frame with the gene of interest. This ensured that each gene was expressed by its own promoter, making it more likely that the resulting full-length fusion proteins were expressed in the same levels and localized to the same cellular compartments as their native counterparts. Two sets of fusion strains were generated: one encoding tandem affinity purification (TAP)-tagged constructs to analyze protein abundance, and another encoding green fluorescent protein (GFP) to interrogate protein localization. "We can now look at the localization [and] abundance, watch changes, [and] do biochemical experiments with a level of comprehensiveness that just was never possible before," says Weissman. NOT JUST A METHODS PAPER The work generated an important set of data for researchers who use yeast as a model organism to test new methods for probing proteomes; now, for instance, scientists have a good idea of how many genes should be expressed in yeast under normal laboratory conditions. "It's going to make our life a lot easier to try to sort out how we're progressing as we develop mass spectrometry-based methods to look at yeast proteins," says John Yates of the Scripps Research Institute, La Jolla, Calif. The main significance of the research lies in several important findings about the yeast proteome itself. Prior efforts to probe protein abundance using DNA microarrays, for example, linked mRNA expression levels with those of corresponding proteins. "The hope [was] that watching changes in message [would] be a proxy for changes in protein function," says Weissman. The current study, although it did find a surprisingly constant ratio of 4,800 mRNA messages per expressed protein, nevertheless demonstrated substantial variability among individual genes. "I think it's clear that the microarray data is only capturing only a small fraction of the real regulation that is going on in a cell," says Weissman. Further, the study generated localization data for 70% of previously unlocalized yeast proteins, and many genes that were coexpressed also localized into similar subcellular compartments. The results could help researchers tease out protein functions. "Once you have a large-scale data set of localization, then you can integrate that with protein-protein interaction data, genetic interaction data, [and] microarray information, to develop a glimpse of how numerous pathways coordinate their action[s]. Knowing where sets of proteins are localized provides a real visual sense of how the pathway might work," says Boone. TECHNOLOGY TRANSFER? O'Shea emphasizes that the methods used to generate the fusion strains will not necessarily work in other systems. "This strategy of modifying the chromosome really takes advantage of the fact that in budding or bakers' yeast, the cells have a very high efficiency of homologous recombination that allows you to target segments of DNA to essentially any place in the genome," says O'Shea. Marc Vidal of Harvard University explains that introducing genes directly into the genomes of multicellular organisms can be accomplished, but not as precisely as in yeast. Vidal is generating fusion vectors for the complete set of open reading frames in Caenorhabditis elegans using Gateway technology from Invitrogen of Carlsbad, Calif. He plans eventually to study the expression and localization of these constructs. "It's not as precise and as beautiful technically as what you could get in yeast, but once you have those constructs integrated, then you can follow their expression against many conditions like you would do with yeast cells," Vidal says. While others view the yeast proteome research as a stepping-stone to more complex organisms, O'Shea and Weissman emphasize that work still needs to be done in yeast. Speeding up the robotics is one immediate goal; a broader aim is to measure dynamic changes in protein abundance. "A big next step is to look at changes in the abundance and regulation and localization of proteins in response to different environmental perturbations and mutations," says O'Shea. Weissman notes that the TAP-tagged constructs can now be purchased for a nominal fee from Open Biosystems of Huntsville, Ala. (www.openbiosystems.com); the GFP fusion library will soon be available. --Aileen Constans References 1. S. Ghaemmaghami et al., "Global analysis of protein expression in yeast," Nature, 425:737-41, Oct. 16, 2003. 2. W.-K. Huh, "Global analysis of protein localization in budding yeast," Nature, 425:686-91, Oct. 16, 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

Yeast Proteomics Milestone

Interested in reading more?

Become a Member of

Courtesy of Won-Ki Huh
LOCAL COLOR: Researchers used librar-ies of tagged proteins to determine where those proteins accumulate, and to what levels. Shown are representative localization (top) and co-localization (bottom) data.