New & Noteworthy
Yeast, the Spam Filter
November 11, 2015
If you don’t have a good spam filter for your email, you may be overwhelmed—just as the sheer number of variants of human genes can be overwhelming. Luckily, yeast can help us filter out the variants that matter. Image by Jean Pierre Gallot via Flickr
Imagine what our email inboxes would look like if we didn’t have spam filters! To find the meaningful emails, we’d have to wade through hundreds of messages about winning lottery tickets, discount medications, and other things that don’t interest us.
When it comes to sorting out meaningful mutations from meaningless variation in human genes, it turns out that our friend S. cerevisiae makes a pretty good spam filter. And as more and more human genomic sequence data are becoming available every day, this is becoming more and more important.
For example, when you look at the sequence of a gene from, say, a cancer cell, you may see many differences from the wild-type gene. How can you tell which changes are significant and which are not?
SuperBud to the rescue! Because many human proteins can work in yeast, simple phenotypes like viability or growth rate can be assayed to test whether variations in human genes affect the function of their gene products. This may be one answer to the increasingly thorny problem of variants of uncertain significance—those dreaded VUS’s.
In a new paper in GENETICS, Hamza and colleagues systematically screened for human genes that can replace their yeast equivalents, and went on to test the function of tumor-specific variants in several selected genes that maintain chromosome stability in S. cerevisiae. This work extends the growing catalog of human genes that can replace yeast genes.
More importantly, it also provides compelling evidence that yeast can help us tell which mutations in a cancer cell are driver mutations, the ones that are involved in tumorigenesis, and which are the passenger mutations, those that are just the consequence of a seriously messed up cell. Talk about a useful filter!
The researchers started by testing systematically for human genes that could complement yeast mutations. Other groups have done similar large-scale screens, but this study had a couple of different twists.
Previous work from the Hieter lab had identified genes in yeast that, when mutated, made chromosomes unstable: the CIN (Chromosome INstability) phenotype. Reduction-of-function alleles of a significant fraction (29%) of essential genes confer a CIN phenotype. The human orthologs of these genes could be important in cancer, since tumor cells often show chromosome rearrangements or loss.
So in one experiment, Hamza and colleagues focused specifically on the set of CIN genes, starting with a set of 322 pairs of yeast CIN genes and their human homologs. They tested functional complementation by transforming plasmids expressing the human cDNAs into diploid yeast strains that were heterozygous null mutant for the corresponding CIN genes. Since all of the CIN genes were essential, sporulating those diploids would generate inviable spores—unless the human gene could step in and provide the missing function.
In addition to this one-to-one test, the researchers cast a wider net by doing a pool-to-pool transformation. They mixed cultures of diploid heterozygous null mutants in 621 essential yeast genes, and transformed the pooled strains with a mixture of 1010 human cDNAs. This unbiased strategy could identify unrecognized orthologs, or demonstrate complementation between non-orthologous genes.
In combination, these two screens found 65 human cDNAs that complemented null mutations in 58 essential yeast genes. Twenty of these yeast-human gene pairs were previously undiscovered.
The investigators looked at this group of “replaceable” yeast genes as a whole to see whether they shared any characteristics. Most of their gene products localized to the cytoplasm or cytoplasmic organelles rather than to the nucleus. They also tended to have enzymatic activity rather than, for example, regulatory roles. And they had relatively few physical interactions.
So yeast could “receive messages” from human genes, allowing us to see their function in yeast. But could it filter out the meaningful messages—variations that actually affect function—from the spam?
The authors chose three CIN genes that were functionally complemented by their human orthologs and screened 35 missense mutations that are found in those orthologs in colorectal cancer cells. Four of the human missense variants failed to support the life of the corresponding yeast null mutant, pointing to these mutations as potentially the most significant of the set.
Despite the fact that these mutations block the function of the human proteins, a mutation in one of the yeast orthologs that is analogous to one of these mutations, changing the same conserved residue, doesn’t destroy the yeast protein’s function. This underscores that whenever possible, testing mutations in the context of the entire human protein is preferable to creating disease-analogous mutations in the yeast ortholog.
Another 19 of the missense mutations allowed the yeast mutants to grow, but at a different rate from the wild-type human gene. (Eighteen conferred slower growth, but one actually made the yeast grow faster!)
For those 19 human variants that did support life for the yeast mutants, Hamza and colleagues tested the sensitivity of the complemented strains to MMS and HU, two agents that cause DNA damage. Most of the alleles altered resistance to these chemicals, making the yeast either more or less resistant than did the wild-type human gene. This is consistent with the idea that the cancer-associated mutations in these human CIN gene orthologs affect chromosome dynamics.
As researchers are inundated by a tsunami of genomic data, they may be able to turn to yeast to help discover the mutations that matter for human disease. They can help us separate those emails touting the virtues of Viagra from those not-to-be-missed kitten videos. And when we know which mutations are likely to be important for disease, we’re one step closer to finding ways to alleviate their effects.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight, Yeast and Human Disease
Tags: chromosome instability, functional complementation, Saccharomyces cerevisiae, yeast model for human disease
New SGD Help Video: Variant Viewer
November 05, 2015
Using SGD’s Variant Viewer, you can compare the nucleotide and protein sequences of your favorite genes in twelve widely-used S. cerevisiae genomes. This tool shows alignments, similarity scores, and sequence variants for open reading frames (ORFs) from the different strains relative to the S288C reference genome. Sequence data are derived from Song et al., 2015.
Take a look at our new video tutorial to get started with the Variant Viewer, and let us know if you have questions or suggestions.
Categories: Sequence, Tutorial
SGD Help Video: Mutant Phenotypes
November 04, 2015
SGD’s Phenotype pages present detailed information about single mutant phenotypes for a particular gene, along with references for each observation. Phenotype pages are accessible from the ‘Phenotype’ tab of the Locus Summary and is also linked from the Mutant Phenotypes section of the Locus Summary, where the phenotype data are presented in summary form. Data are presented in tabular form on the Phenotype page.
This brief video will give you an overview of the contents and organization of SGD’s Phenotype pages.
Categories: Tutorial
SGD Help Video: Literature Page
October 29, 2015
If you’re interested in finding all the published literature about a gene or protein, there’s no need to wade through long lists of PubMed results. SGD curators have already done that for you! We review PubMed weekly for new papers about S. cerevisiae. You can find papers about a specific gene or protein on its Literature tab page (see an example).
Articles on the Literature page are categorized by several topics. The Primary Literature section lists papers in which the gene of interest is a primary focus of the study, while the Additional Literature section lists papers in which the gene is mentioned but is more peripheral to the research. There are other categories of references, and also a cool interactive graphic that shows the relationships between papers that are about the same set, or overlapping sets, of genes. You can get to the Literature page for a gene or protein via the Literature tab, located at the top of its Locus Summary page and all of its other tab pages.
Categories: Tutorial
Tags: video
New SGD Help Video: GO Term Finder
October 26, 2015
Our GO Term Finder tool lets you start with a list of genes—perhaps a set of genes that are co-regulated, or a group of genes that can all mutate to the same phenotype—and analyze their Gene Ontology (GO) annotations to find out what else they might have in common. GO Term Finder searches for significantly shared terms within the GO annotations associated with the genes in your list. It takes advantage of the tree structure of GO to find terms that are related to each other within the ontology.
Finding shared terms within a gene set can bring meaning to experimental results and suggest new avenues to explore. For example, if the GO Term Finder results show that most of the genes in your co-regulated set mediate steps in a pathway, this might be a hint that the uncharacterized genes in the set also participate in that pathway. Or perhaps GO Term Finder will show that a group of genes that can mutate to confer resistance to a certain drug are all annotated to a certain cellular location, suggesting a mechanism for the effects of that drug. Give it a try and see what interesting results your gene list has in store!
Our new SGD Help video gives you a quick overview of how to use the GO Term Finder. You can find all the details on our GO Term Finder help page.
Categories: Tutorial
Tags: Gene Ontology, GO Term Finder, video
Life Needs to be More Like a 1950’s Chevy
October 21, 2015
Stripped of modern bells and whistles, cars last a lot longer. The same may be true of life. It may last longer when some extra, nonessential genes are removed. Image via Wikimedia Commons
In the old days, a car came with the bare minimum of features to get from point A to point B. The windows rolled down with a crank and it usually had a radio. That was about it.
As the car has evolved, it has gained a huge number of bells and whistles. There are power windows and power brakes, a baffling number of computer-based bonus features, personal wifi hotspots, and so on. All of these have undoubtedly made cars more fun and comfortable to drive. But they have come at a cost. Many cars simply do not last as long as their predecessors because these extras break easily.
Turns out life may be like a modern car. It has lots of nice features that help it to do better in the world. But a lot of these features may shorten its life span.
This point was reinforced in a recent study by McCormick and coworkers. They painstakingly searched through a library of 4,698 single gene deletion strains in S. cerevisiae and found that 238 of these strains were able to produce significantly more buds over their lifetime. Many nonessential genes seem to shorten a yeast’s life.
And boy was it painstaking! Believe it or not, they manually dissected over 2.2 million individual yeast daughter cells to generate these results. Luckily it was worth it, as they found so many interesting things.
First off, many of the genes they found fall into a set of five pathways that includes cytosolic and mitochondrial translation, the SAGA complex, protein mannosylation, the TCA cycle, and proteasomal activity. So there are certain pathways we can target to extend the lifespan of our friend yeast. And even better, yeast may not be the only beneficiary of these studies.
Two of the pathways, cytosolic and mitochondrial translation and the TCA cycle, have also been found to be significant in extending the life of the roundworm C. elegans. These pathways are also shared with humans.
And just because the authors found no overlap with the other three pathways in other beasts doesn’t mean they may not be targets for life extension in them too. It could be that previous screens in C. elegans simply missed genes from these pathways.
It could also be that what is found in yeast may turn out to be important in people but not in C. elegans. For example, the authors failed to find any equivalent to the SAGA complex in C. elegans. Either the roundworm lost this complex during evolution, or the homologs between yeast and C. elegans are so different that they’re unrecognizable. In any event, humans at least do have an equivalent to SAGA, called STAGA.
All of this suggests that there may be common ways to make organisms, including people, live longer, healthier lives. Here’s hoping!
And these five pathways are certainly not the whole story. The majority of the genes McCormick and coworkers identified were not in these five, which means there are probably lots of other ways to get at living longer.
One fascinating example that the authors decided to look at in depth was LOS1. Deleting it had one of the biggest effects on a yeast’s reproductive life span.
At first this seems a little weird, as Los1p exports tRNAs out of the nucleus. As expected, deleting LOS1 led to a buildup in tRNAs in the nucleus. The authors confirmed that this buildup is important by showing that overexpressing MTR10, a gene involved in transporting tRNAs from the cytoplasm to the nucleus, led to a longer lived yeast with a buildup of tRNAs in its nucleus.
The next step was to figure out why having a lot of tRNA in the nucleus makes yeast live longer. It was known previously that Los1p is kept out of the nucleus under glucose starvation conditions. The authors confirmed this result.
Most everyone knows that restricting calorie intake (also called dietary restriction or DR) can extend the lives of most every beast tested so far, including yeast. The authors found that growing a los1 deletion strain at low glucose did not increase the lifespan of this strain any further. It thus appears that an important consequence of DR is keeping Los1p out of the nucleus and thereby increasing the amount of tRNA in the nucleus.
While we don’t know yet exactly why keeping tRNAs in the nucleus helps yeast live longer, it is interesting that the increased lifespan associated with the loss of LOS1 is linked to caloric restriction. Finding a way to inhibit Los1p has to be better than starving yourself!
This study has identified 238 genes to follow up on for future studies. And of course there is a whole class of genes that haven’t yet been investigated—the essential genes! Many of these may be important for extending life too.
Stripping life down to its bare essentials may help individuals live longer at the expense of being the most fit in terms of survival in the hurly burly world of nature. After all, those “nonessential” genes undoubtedly have a function in helping yeast outcompete their less well-endowed yeast neighbors. Just like those power sliding doors are way better than the manual ones on a minivan.
But if you want a long-lived minivan, get the one with the manual doors. And if you want a long-lived yeast (or person), get rid of some of those nonessential genes that cause you to break down.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: aging, lifespan, Saccharomyces cerevisiae
You Can Take Yeast Off of the Grapevine, But…
October 14, 2015
All over the world and through the ages, people have moved from the country into the big city to look for a better life. These folks often find that even though they can adapt to city life and city ways, they still hang on to their core country values. As the old saying goes, “You can take the boy out of the country, but you can’t take the country out of the boy.”
Even when he goes to the city, the country mouse hangs on to his country ways. The same is true for S. cerevisiae—even though it entered the lab, it still clung to genes that were most useful out in the vineyard. Illustration by Arthur Rackham (1912) via Wikimedia Commons
Our friend Saccharomyces cerevisiae didn’t migrate voluntarily into the lab. But it ended up there, and has been as lonely as a new migrant in a big city.
Which is of course how we need it to be. One of the basic tenets of classical microbiology is that you can’t begin to study an organism until you’ve isolated it in a pure culture.
And studying pure S. cerevisiae has yielded a huge body of knowledge about molecular biology, cell biology, and genetics. But by not studying yeast in the context of its old country home, we may have missed a few things.
In a new article in PLOS ONE, Rossouw and colleagues uncover one of them. S. cerevisiae has a family of FLO genes that promote flocculation, the adherence of yeast cells to each other. It has always been a bit puzzling why a whole family of genes that are pretty much redundant with each other would be maintained through evolution.
When the researchers took S. cerevisiae out of its lab isolation by mixing it with other yeast species, they found that the different flocculation genes actually determine which species it can co-flocculate with. Different Flo proteins prefer different partners.
This discovery helps us understand the evolution of this gene family and also opens the door to further study of inter-species interactions in the vineyard. And since flocculation is an important property in winemaking and brewing, there could even be tasty practical applications of this knowledge.
The researchers started by surveying 18 non-Saccharomyces yeast strains that are found in vineyards. They looked at the ability of the yeasts to flocculate both as pure strains and when mixed with either of two S. cerevisiae wine strains.
Intriguingly, certain species showed a synergistic effect when mixed with S. cerevisiae, flocculating more than either species on its own. Rossouw and colleagues used microscopy to confirm that the “flocs” did indeed contain both yeast species—a simple observation, since the cells of different species have slightly different shapes.
To test the effects of different FLO genes on co-flocculation, the authors assayed the co-flocculation ability of flo1, flo10, and flo11 deletion mutants as well as Flo1, Flo5, and Flo11 overproducers in individual combinations with six of the non-Saccharomyces yeasts.
The results showed that Flo1 has general effects on flocculation. Overproduction increased co-flocculation across the board with all the species tested, while deletion of FLO1 consistently decreased it. In contrast, deletion of FLO10 didn’t have much effect on co-flocculation.
It was a different story for Flo5 and Flo11, though. Overproduction of each of these not only affected co-flocculation, but had species-specific or even strain-specific effects. Flo5 overproduction caused a relative increase in co-flocculation with Metchnikowia fructicola and a substantial decrease in co-flocculation with two different strains of Hanseniaspora opuntiae. Flo11 overproduction reduced co-flocculation with one of the Hanseniaspora opuntiae strains but not with the other.
All of these experiments were done on mixtures of two species at a time. To get S. cerevisiae even further out of the lab, Rossouw and colleagues created a “consortium” of wine yeasts, a mixture of six species that are found in wine must (freshly pressed grapes) at the start of fermentation. They then added the FLO overproducer strains individually to the consortium, to see their effects in a more natural situation.
They let the yeast consortium flocculate, extracted total DNA from the flocculated or supernatant parts of the culture, and then used automated ribosomal intergenic spacer analysis (ARISA) to see which strains had co-flocculated. This technique can determine the relative abundance of different yeast species in a sample by sequencing a particular region of ribosomal DNA.
In this experiment, overexpression of each of the three FLO genes had significant effects on at least one of the species in the consortium. The species composition of the flocculated yeasts was uniquely different, depending on which gene was overexpressed.
The discovery that the flocculation genes have individual effects on association with other species goes a long way towards explaining why S. cerevisiae has maintained this gene family with so many members that apparently have the same function—at least, when you study a pure culture. Differential regulation of the FLO genes could affect the spectrum of other species that our favorite yeast interacts with.
So, our friend S. cerevisiae didn’t actually get out of the lab in these experiments, but at least it got to rub shoulders with some of its old friends (buds?) from the vineyard. These experiments are a good reminder for researchers to think outside the lab.
And when S. cerevisiae and its friends get together outside the lab, beautiful things can happen. We’ll drink a toast to that!
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
If yeast could sing about its forced migration to the lab, it might sound like this.
Categories: Research Spotlight
Tags: flocculation, Saccharomyces cerevisiae, vineyard
Unleashing the Awesome Power of Yeast Transcription
October 07, 2015
With the right mutations, yeast can activate transcription over long distances—just as with the right social media, distance is meaningless for finding and organizing like-minded people. Image via Pixabay.com
In the old days, before the internet, planes or even mass publishing, it was hard to spark a quick, worldwide movement. You simply couldn’t reach out to likeminded people who lived far away.
Nowadays things are very different. With the advent of social media, it is now trivially easy to spread the word. Using Twitter and Facebook, organizers can easily and effectively organize people who live on the other side of the world.
In terms of transcription activation, our friend Saccharomyces cerevisiae seems to be stuck in the old world. Its transcription factors can only turn up nearby genes. This is different from most other eukaryotic beasts, where activation at a distance is routine.
Except maybe yeast isn’t as backward as we think. It may be that yeast has the potential to activate transcription at a distance, but keeps that potential locked away.
This idea is supported in a new study out in GENETICS by Reavey and coworkers. These authors found that they could mutate away yeast’s inability to activate transcription at a distance.
In other words, this ability is there but is just prevented from being used. Yeast is keeping social media out of the hands of its genes.
Getting around social media/internet controls is not easy. Pressure might need to be applied at multiple points before people power is finally released through social media. And even when it does happen, it can sometimes be hard to figure out exactly why certain events tipped the balance.
Turns out that both of these are also true for long range transcription activation in yeast. Mutating a single gene was not enough—it took mutations in multiple genes to see any significant effect.
As you can imagine, it would be very tricky to hit all of the right mutations for a polygenic trait in one fell swoop. This is why Reavey and coworkers started their mutant hunt with a strain that could already weakly activate transcription from a distance, a strain in which the SIN4 gene was deleted. Now they just needed additional mutations to make the effect stronger.
In their screen they used a reporter in which the GAL4 upstream activating sequence (UAS) was placed 799 base pairs upstream of the HIS3 reporter. This reporter gives very low levels of activity in a wild type strain. They included a second reporter, the URA3 gene under control of the same upstream sequences, because Reavey and Winston had discovered in previous work using a single reporter that cis acting mutations and chromosomal rearrangements were a frequent source of false positive results.
The researchers put the reporter strain through multiple rounds of mutation with UV light and selection with increasing levels of 3AT, a competitive inhibitor of His3. After each round of selection, they measured mRNA levels for HIS3 and URA3 and chose strains that not only had higher 3AT resistance but also showed more transcription of the reporter genes. In the end they found three strains that survived in the presence of galactose (to turn on the activator) and 10 mM 3AT.
As expected, each strain had multiple mutations. One strain had acquired mutations in the GRR1 and MOT3 genes. To confirm that these were the most important mutations, Reavey and colleagues engineered a fresh strain with just the original sin4 null mutation and the selected grr1-1 and mot3-1 mutations. The fresh strain completely recapitulated the selected strain, showing that these three mutations could unlock yeast’s potential for long-range transcriptional activation.
It makes sense that a grr1 mutation could affect transcriptional activation. Grr1 is a ubiquitin ligase that destabilizes Med3 (also known as Pdg1), a key component of the Mediator complex involved in transcription activation. The researchers provided evidence that this is how the grr1-1 mutation affects the process, by showing that mutating MED3 mimicked the effects of mutating GRR1.
It’s also not too hard to imagine how a mutation in Mot3, a sequence-specific transcriptional activator, could affect transcriptional activation, presumably by changing the expression of a gene under its control.
If you stay away from social media, you’ll lose opportunities for impromptu pillow fights. Who knows what yeast is missing without long-range transcription activation? Image via Wikimedia Commons
The results were not so clear-cut for two other strains that were selected. They arose from the same lineage, and each had acquired the ptr3-1 mutation on top of the original sin4 null mutation. One strain went on to further pick up the mit1-1 mutation, while the other got an msn2-1 mutation.
Again it isn’t too surprising that mutations in genes that encode sequence-specific transcriptional activators like Mit1 and Msn2 arose in these strains. But the selection of the ptr3 mutation in these lineages is something of a mystery.
It is hard to imagine how the usual job of Ptr3 in nutrient sensing and transport would be involved in keeping long range transcription activation down. Perhaps the researchers have uncovered a novel function for this gene.
And re-creating these two strains only partially restored the levels of transcription activation at a distance that were seen in the original strains. A little genetic detective work showed that a big reason for this was that both of the selected strains had acquired an extra copy of the chromosome that had the HIS3 reporter, chromosome III.
Reavey and colleagues deleted each of their identified genes to see which ones caused their effect through a loss of function mutation. Deleting GRR1, PTR3, and MSN2 all had the same effect as the original isolated mutations.
The same was not true for MOT3 and MIT1. Deleting either gene actually weakens long range transcription activation, suggesting that these two had their effect through gain of function mutations.
Finally, the researchers showed that the increase in long-distance transcriptional activation was not simply due to a general increase in transcription activation in the selected strains, by showing that their mutants did not have increased activity of a reporter with the GAL4 UAS placed 280 base pairs upstream of HIS3. In fact, if anything, the strains showed decreased activation with this reporter.
So this experimental strategy allowed Reavey and coworkers to identify some of the key genes involved in keeping transcription activation at a distance under control in yeast. In particular, they found compelling evidence that the Mediator complex is an important player. But there is still plenty of work to do. For example, which of the genes regulated by Mit1, Msn2, and Mot3 are important in long range activation? And what on Earth is Ptr3 doing in all of this?
The success of this approach also confirms that doing repeated rounds of selection in yeast is a viable way to select multiple mutants and study polygenic traits. This strategy may prove a boon for studying the many human diseases that are the result of polygenic traits.
Not only can we use yeast to uncover its activation potential, but we can also now potentially use it to uncover new treatments for human disease. Unleashing another awesome yeast power…
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: polygenic trait, Saccharomyces cerevisiae, transcription
New SGD Help Video: GO Slim Mapper
October 05, 2015
The GO Slim Mapper is a very useful tool that maps specific Gene Ontology (GO) annotations to more general GO terms. This allows you to take a group of genes and bin them into broad categories of function, process, or localization by mapping their GO annotations to broader terms.
Watch our new video to get an overview of how the GO Slim Mapper works:
Categories: Tutorial
Tags: Gene Ontology, GO Slim Mapper, video
The Latest Buzz on Stressed-Out Mitochondria
September 30, 2015
Stinging wasps get our attention, and with good reason—getting stung hurts a lot! If you see wasps going into a nest within the walls of your house, you’ll likely try to block their access.
Just like wasps who can’t get into their nest, excess mitochondrial precursor proteins that can’t be imported into mitochondria are bad news for the cell—but it’s developed ways to deal with them. Image via Pixabay.com
But this could backfire: instead of being able to peacefully go into their nest, a swarm of angry wasps could be buzzing around looking for trouble. It might get so bad that you’ll need to call in an exterminator to take care of the problem.
Mitochondrial proteins might seem a lot less scary than wasps, but it turns out that they can also cause trouble if they can’t get into mitochondria. In two new letters to Nature, Wrobel and colleagues and Wang and Chen used complementary approaches to ask what happens when dysfunctional mitochondria aren’t able to import all of the proteins that are waiting to get in.
What they both found was that these piled up proteins cause real problems for the cells. In desperation, the cells slow down protein synthesis to reduce the excess, and also turn to their own exterminator, the proteasome, to keep these proteins under control.
This is a paradigm shift in thinking about how poorly functioning mitochondria cause disease. In the past, almost everyone focused on how damaged mitochondria couldn’t make enough energy for a cell. Now it looks like there are other ways for a nonworking mitochondria to do a cell in. And new targets for scientists to go after in treating mitochondrial disease.
As we all know, mitochondria are the powerhouses of the cell, where energy is generated, and they’re also the site of many other essential biochemical reactions. They’re composed of about 1,000 proteins, and nearly all of those are synthesized in the cytoplasm and then imported into mitochondria by an intricate system of transporters.
In order to find out what happens when these 1,000 proteins don’t get into mitochondria as efficiently as they should, both groups created strains whose mitochondrial import was impaired.
Wrobel and colleagues used the temperature-sensitive mia40-4int mutation, affecting an essential component of the mitochondrial import system. Wang and Chen started with the aac2-A128P mutation in PET9 (which is also known as AAC2), an ADP/ATP carrier of the mitochondrial inner membrane. Overexpression of the aac2-A128P allele causes mitochondrial dysfunction and eventual cell death.
Wrobel and colleagues decided to get a comprehensive look at what happens in the mia40-4int mutant by assaying its transcriptome and proteome, using RNA-seq and stable isotope labelling by amino acids in cell culture (SILAC), respectively. Surprisingly, one of the biggest differences from wild type that they saw in the import-defective mutant was a decrease in cytoplasmic translation. Whether they looked at the mRNAs encoding ribosomal proteins, the proteins themselves, or the polysome content and translational activity of the cells, everything pointed to down-regulation of translation. And at the same time, the proteasome—the molecular machine that breaks down unwanted proteins—was activated.
To verify that what they were seeing wasn’t peculiar to the mia40-4int mutant, Wrobel and colleagues slowed down mitochondrial import in several other ways: using different mia40 mutant alleles, other import mutants, or treatment with a chemical that destroys the mitochondrial membrane potential required for import. Under these different conditions causing the accumulation of mitochondrial precursor proteins in the cytoplasm, they still saw decreased cytoplasmic translation and increased proteasome activity.
“Quick, Henry, the Flit!” When mitochondrial precursors start to swarm around the cytoplasm, the cell keeps them under control by activating the proteasome. Upper image, Flit insecticide, by Bullenwächter, lower image, structure of the yeast 26S proteasome by FridoFoe; both via Wikimedia Commons
Wang and Chen took a different approach, looking to see whether over-expression of any other genes could compensate for the lethality of overexpressing the aac2-A128P allele. The researchers transformed the mutant with a library of yeast genes on a multicopy plasmid, and found 40 genes whose expression could keep it alive.
The suppressor genes found by Wang and Chen were all involved in some aspect of synthesis or degradation of cytoplasmic proteins, just like the genes found by Wrobel and colleagues whose expression was altered in the mia40 mutant. And Wang and Chen also verified that these suppressors weren’t specific to the aac2-A128P mutation: they suppressed a variety of other mutations that decreased import.
Both groups observed precursors of mitochondrial proteins accumulating in the cytosol of the mutant strains they studied. Wang and Chen saw a couple other very interesting proteins increase in abundance: Gis2 and Nog2. These proteins are involved in regulating ribosome function, and the researchers speculate that their stabilization during this stress response contributes to the translational down-regulation. Intriguingly, their human orthologs are implicated in neuromuscular degenerative disease.
So, using orthogonal approaches, the two groups converged on the same model: a newly discovered cellular pathway that regulates cytosolic translation and protein degradation in order to deal with the stress of inefficient mitochondrial import. Wrobel and colleagues have named it UPRam, for Unfolded Protein Response activated by mistargeting of proteins, while Wang and Chen call it mPOS, mitochondrial Precursor Over-accumulation Stress.
Before this work, it was unknown whether cytosolic pathways were even affected by mitochondrial dysfunction. Now we know that the cell has a specific response when mitochondrial precursor proteins begin swarming in the cytosol, unable to get into their home: it slows down the production of those proteins and calls in the proteasome exterminator to take care of them.
We usually think of mitochondrial disease symptoms as being caused by the reduced energy generation of sick mitochondria, or by the lack of other key events that happen in mitochondria—for example, the synthesis of the iron-sulfur clusters that some vitally important enzymes need. Now, these findings raise the possibility that proteostatic stress on the cell caused by the accumulation of mitochondrial precursors could also lead to impaired cell function and disease.
Perhaps drugs that inhibited cytoplasmic translation, or activated the proteasome exterminator, would be helpful in reducing the buzzing swarm of mitochondrial precursor proteins. Wouldn’t it be wonderful if this knowledge suggested new avenues of treatment to take some of the sting out of human mitochondrial disease?
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
Tags: mitochondria, proteasome, proteostatic stress, Saccharomyces cerevisiae