Where do proteins go when they move around the cell? Interested scientists will now be able to look up the answer. Dr. Maya Schuldiner
and graduate student Michal Breker of the Molecular Genetics Department recently produced a comprehensive atlas of changes
in yeast proteins’ localization and abundance under stress that presents a wealth of new information. This atlas, which they are making available online, is likely to become an important tool for the many scientists who use this model research organism to investigate the workings of living cells.
Schuldiner and Breker began with a different kind of reference work: a strain library. Strain libraries hold sets of cells with specific genetic alterations. Bakers’ yeast cells contain some 6000 different genes, each encoding a protein. In each “volume” of their library, one “bookmark” has been created – a gene has been modified such that a particular protein is tagged with a fluorescent “highlighter” molecule that glows under a special microscope. In Schuldiner’s lab, using a state-of-the-art, automated microscopy system, researchers can examine an entire strain library at once. Other types of libraries exist, as well, including those in which each gene, in turn, has been removed. Libraries can even be mixed and matched to create new combinations. A robotic search then scans the entire set to find out which proteins are active in any given experimental situation.
This unique set-up enabled Schuldiner and Breker to track protein movement – one of the “big” questions in cell biology. Knowing where proteins go, says Schuldiner, can help to answer a number of important research questions: “How many of the cells’ proteins are mobile? Under what conditions? How does the cell use this protein mobility to remain healthy and divide under a variety of different conditions?”
Subjecting the yeast cell libraries to various conditions and putting them through the robotic system, the researchers were able to trace the movements of each protein in the yeast cell. The result: a complete, detailed map describing protein routes, as well as a record of the amounts of each protein produced in the different situations.
A bird's-eye view of the data presents a picture of constant bustle in the cells. At any one time, hundreds of proteins are in transit. But the numbers the researchers collected on protein amounts held some real surprises. In much of today’s research, protein levels are inferred from experiments that actually check the production of messenger RNA (the instructions sent to the cell’s protein factories) – something like using building plans rather than actual buildings to map a town, when those plans can be shelved or reused for the next housing development. In tracing the proteins themselves, Schuldiner and Breker revealed that messenger RNA and true protein quantities don’t always match. Schuldiner: “Many beautiful works have already shown that protein production is regulated in many different ways after the RNA leaves the nucleus. Our findings hint that some of the later stages may be more significant than we thought in determining protein levels.”
The online atlas is named Loqate (LOcalzation and Quantization Atlas of the yeast proteome). Schuldiner: “The atlas can be used by those seeking answers to such specific questions as: Which proteins are involved in a particular cellular activity, and when and where do they act? In addition, those who want to integrate different kinds of information to attain a more comprehensive picture of the cell’s life will find the atlas an indispensible aid.”
Checking all possibilities
The latest research methods have advanced molecular biology beyond the standard ABCs of research – formulating a hypothesis and then testing it through experiments. Using fast, powerful, completely automated equipment, much of it assembled according to Schuldiner’s specifications, she and her research team can now check all of the possibilities and extract the significant data. “With these tools, our research can be totally unbiased,” she says. “If we once started with an educated guess – say, ‘A affects B’ – and then tried to confirm our conjecture experimentally, we can now ask: ‘Which proteins are involved in B’s activity?’ In this way, we might find that G, L and M also act on B. And if a doctoral student formerly spent the whole of his or her studies checking that hypothesis, he or she can now get answers in a matter of weeks.”
Dr. Maya Schuldiner's research is supported by the European Research Council; the Berlin Family Foundation; James and Ilene Nathan, Beverly Hills, CA; the Minna James Heineman Stiftung; the Enoch Foundation; Roberto and Renata Ruhman, Brazil; Karen Siem, UK; and the Kahn Family Research Center for Systems Biology of the Human Cell.