July 01, 2022
Yeast are keenly sensitive to internal pH. Several membrane proteins pump H+ ions out of the cell to keep the internal pH near neutral. When carbon becomes scarce, however, it is essential for survival that these pumps get inactivated so the internal space is rapidly acidified. This acidification is postulated to conserve energy and trigger a number of subsequent pathways to combat starvation. Key among these adaptive responses is the derepression of the glucose-repressed genes. The well-studied SWI/SNF complex has been established as a key mediator for this, but the details of how the transcriptional boost is effected have not been known.
A recent study in eLife by Gutierrez et al. has shown the pivotal subunit of the SWI/SNF complex to be Snf5p, which performs a regulatory role by sensing pH. But how would a protein sense pH?
In studying the sequence and structure of the eleven subunits of the SWI/SNF complex, the authors noted that ten of the eleven subunits had large intrinsically disordered regions and that four of the eleven contained glutamine-rich low-complexity sequences (QLCs) that contain multiple histidine residues. QLCs were previously identified as important for binding transcription factors.
In looking for a link between pH and activation, the authors postulated that the histidine residues might be important because the histidine sidechain has an intrinsic pKa of 6.9, and thus might change conformation when pH drops.
Detailed comparative analysis of QLCs from yeast and other organisms led the authors to conclude that, in yeast, the histidines are salient features of QLCs that have been evolutionarily conserved. Given this, they noted that the N terminus of Snf5p has one of the largest QLCs in the whole yeast proteome and is in the top three for number of histidines.
Naturally, given the tools of the yeast model, the next step was to mutate the protein, for which they compared a full deletion against an N-terminal deletion of the QLC and a targeted allele with four histidines within the QLC mutated to alanine.
They found that total loss of the gene was phenotypically distinct from either of the QLC-targeted mutants. Total absence of the protein caused disruption of the SWI/SNF architecture, while QLC-mutants maintained an intact complex but showed disruptions in transcriptional reprogramming in response to starvation, as specifically measured by derepression of the ADH2 gene.
By a subsequent series of elegant biochemical experiments—conducted both in vivo and in vitro—the authors show with great precision how the Snf5p QLC specifically senses pH to trigger widespread reprogramming of genes that will help yeast metabolize non-preferred carbon sources. Even more specifically, they show how acidification leads to protonation of the histidines in the QLC, causing that region of the protein to expand and change conformation, thereby affecting the binding properties of the whole SWI/SNF complex.
The ability to do these experiments and develop a model of how the cell accomplishes delicate regulation once more astounds us with the awesome power of yeast genetics.
Categories: Research Spotlight
Tags: glucose metabolism, glucose repression, glucose starvation, glucose-repressed genes, pH sensing, Saccharomyces cerevisiae, signal transduction
November 29, 2016
One of the best parts about doing outreach with a museum is creating a successful hands on activity for the visitors. This is not an easy thing to do.
You first create something that you think will appeal to and educate visitors. When that falls flat on its face, you then do a series of tweaks until it is working smoothly. In the end you have smiling kids who understand DNA better (or at all)!
Genetic engineering can be similar. You can import the genes for a complex pathway into your beast of choice but it may not work first try. A bit of tinkering, evolution style, is often needed to get the engineering working well enough to be useful.
This is exactly what happened when a group of researchers tried to get our favorite beast, the yeast Saccharomyces cerevisiae, to turn the sugar xylose into ethanol. They added the right genes from either fungi or bacteria, but S. cerevisiae couldn’t convert enough xylose into ethanol to be useful.
And it is important for all of us that some beast be able to do this well. A yeast that can turn xylose into ethanol means a yeast that can turn a higher percent of agricultural waste into a biofuel. Which, of course, means lots of low carbon fuel to run our cars so that we have a better shot of limiting the Earth’s heating up by 1.5-2 degrees Celsius.
To get their engineered yeast to better utilize xylose, Sato and coworkers forced it to grow with xylose as its only carbon source. Ten months and hundreds of generations later, this yeast had evolved into two new strains that were much better at turning xylose into ethanol. One strain did its magic with oxygen, the other without it.
In a new study in PLOS Genetics, Sato and coworkers set out to figure out which of the mutations that came up in their evolution experiments mattered and why.
Two genes were common in both the aerobic and anaerobic strains – HOG1 and ISU1. Both needed to be nonfunctional in order to maximize ethanol yields from xylose. They confirmed this by deleting each individually and together from the parental strain.
HOG1 encodes a MAP kinase, and ISU1 encodes a mitochondrial iron-sulfur cluster chaperone. These probably would not have been the first genes to go after with a more biased approach. The benefits of evolution and natural selection!
Further experiments showed deleting each gene individually was not as good as deleting both at once when oxygen was around. In fact, while deleting only ISU1 had a small effect on the ability of this yeast to convert xylose into ethanol, deleting HOG1 alone had no effect at all. Its deletion can only help a strain already deleted for ISU1.
In the absence of oxygen, yeast needs a couple of additional genes mutated – GRE3 and IRA2. GRE3 is an aldose reductase and IRA2 is an inhibitor of RAS. Again, not very obvious genes!
Still, once you find the genes you can come up with reasonable hypotheses for why they are important.
Some are easier than others. Hog1p, for example, is known to enhance a cell’s ability to turn xylose into xylitol, which shunts the xylose away from the ethanol conversion pathway. GRE3 is involved in this as well. Deleting either should make more xylose available to the yeast.
This doesn’t mean this is Hog1p’s only role in boosting this yeast’s ability to turn xylose into ethanol of course. It also probably “…relieves growth inhibition and restores glycolytic activity in response to non-glucose carbon sources.” Consistent with this, the authors found that the xylose-utilizing strain deleted for HOG1 was also better at using glycerol and acetate as carbon sources.
Other genes were less obvious. For example, perhaps mutating ISU1 frees up some iron so extra heme can be made. Or alternatively, it may have increased the mass of mitochondria available. Again, probably would not have been the first gene to go after to improve yeast’s ability to convert xylose to ethanol.
Which again underlines the importance of letting natural selection improve an engineered organism as opposed to only trying to pick and tweak the genes you think are important. Biology is simply too complicated and our understanding too limited to be able to know which are the best genes to go after. This is reminiscent of prototyping museum activities.
Some tweaks are obvious but others you would never have guessed would be needed. For example, we had visitors spreading bacteria on a plate and found that if they labeled their plate first, they almost always put their transformation mixture on the lid instead of on the LB agar. This problem was solved by having them add their mixture first and then labeling their plate.
It would be very hard to predict something like this from the get-go. The activity needed to evolve on the museum floor to work optimally. Much like the yeast engineered to utilize xylose needed to evolve in the presence of xylose to work optimally. And to perhaps take a big step towards saving the Earth from warming up too much.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: biofuel, ethanol, evolution, fermentation, glucose metabolism, xylose