New & Noteworthy

Snf5p Senses pH to Combat Starvation

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 pK_a 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

Yeast Gets By With a Little Help From its Friends

October 09, 2014

If you spend any time looking at social media, you’ve seen the viral videos about interspecies “friendships” – heartwarming scenes of elephants playing with dogs, or lions cuddling with antelopes. These animal relationships strike a chord with most people. Maybe they make us feel there’s hope for harmony within the human species, if such different creatures can get along with each other.

It may not give you quite as warm and fuzzy a feeling, but in a recent Cell paper, Jarosz and colleagues have shown that yeast and bacteria enjoy a friendship too.  However, these microbes have taken it a step further than the larger animals.

Not only do the yeast and bacteria get something good out of the relationship, but the yeast also get a permanent change that they can pass down to their daughters. It is as if being friends with an elephant could give a dog (and her puppies!) the ability to survive on grasses and fruit.

Like koalas with their eucalyptus leaves and pandas with their bamboo, yeast is a nutritional specialist. It is very good at consuming glucose, and will eat nothing else if glucose is available. All the genes necessary to metabolize other carbon sources are tightly turned off in the presence of glucose, a phenomenon termed glucose repression.

As Jarosz and coworkers studied this glucose repression, they stumbled upon the finding that contaminating bacteria could short circuit this process in yeast.  In other words, when yeast and these bacteria grew together, the yeast gained the ability to metabolize other carbon sources in the presence of glucose!  And even more surprisingly, that trait was passed on to the yeast’s future generations.

Here’s how this discovery unfolded.  The authors had plated yeast on medium containing glycerol as a carbon source, plus a small amount of glucosamine, which is a nonmetabolizable glucose analog. Wild-type cells cannot grow on this medium because the presence of the glucose analog makes it seem like glucose is present, causing glucose repression and preventing utilization of the glycerol.

However, there happened to be a contaminating bacterial colony on one plate, and the yeast cells immediately around this colony were able to grow on the glycerol + glucosamine medium.  When those yeast cells were re-streaked onto a fresh glycerol + glucosamine plate, with no bacteria present, they were still able to grow: they had undergone a heritable change. The ability to utilize glycerol in the presence of glucosamine was stably inherited for many generations, even without any selective pressure.

Although the first observation was serendipitous, this proved not to be an isolated phenomenon. The researchers were able not only to reconfirm it, but also to show that it could happen in 15 diverse S. cerevisiae strains. They identified the original bacterial contaminant as Staphylococcus hominis, but showed that some other bacterial species could also give yeast the ability to bypass glucose repression.

This group had previously found a way that yeast could become a nutritional generalist: by acquiring the [GAR+] prion. Prions are proteins that take on an altered conformation and can be inherited from generation to generation.  They usually confer certain phenotypes; one of the best known is bovine spongiform encephalopathy, or mad cow disease.

Luckily for the yeast, the [GAR+] prion is not nearly so devastating. Instead of a deteriorating brain, S. cerevisiae cells carrying the [GAR+] prion can grow on multiple carbon sources even in the presence of glucose.

Since this phenotype was suspiciously similar to that of the yeast that had been exposed to bacteria, Jarosz and colleagues tested them for the presence of the [GAR+] prion, and found by several different criteria that the cells had indeed acquired it. They looked to see if the yeast got other prions as well, but found that bacterial contact specifically induced only the [GAR+] prion.

The next step was to find out how the bacteria were communicating with the yeast.  Since active extracts could be boiled, frozen and thawed, digested with RNAse, DNAse, or proteases, or filtered through a 3 kDa filter without losing activity, the signaling molecule(s) was probably small.  But the researchers ended up with a complex mixture of small molecules, and more work will be needed to find which compound(s) are responsible for this effect.

In the case of animal friendships, it’s believable that intelligent animals are getting some emotional reward from their relationships (If you don’t believe it, the story of Tarra the elephant and Bella the dog in the video below may convince you!). We can’t exactly invoke this for microbes, so why would these organisms have evolved to affect each other in this way?  It seems there must be a “reward” of some kind.

The benefit to yeast cells from their bacterial friendship is that when they carry the [GAR+] prion, they can grow much better in mixed carbon sources and have better viability during aging.

Conversely, the bacteria benefit because [GAR+] yeast cells produce less ethanol than do cells without the prion. This makes a better environment for bacteria to grow, since too much ethanol is toxic. Interestingly, although the bacterial species that were the best inducers of [GAR+] are not phylogenetically closely related to each other, several of them share an ecological niche. They are often found in arrested wine fermentations, which are unsuccessful fermentations in which the yeast stop growing and bacteria take over.

So interspecies “friendships” can have more profound effects than just tugging at the heartstrings of viewers.  One example is the cat that acts as the eyes for that blind dog.  Another is this case, where bacteria can do yeast some permanent good and make a more hospitable environment for themselves in the process.

And this study reminds scientists of two important things. First, that the laboratory environment cannot tell us everything about biology. How often do yeast cells in nature grow as a monoculture on pure glucose, anyway?  And second, that sometimes accidental occurrences in the laboratory, in this case “contamination,” can broaden our findings…if we pay attention to them.  Just ask Alexander Fleming!

by Maria Costanzo, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: bacteria, glucose repression, prions, Saccharomyces cerevisiae

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