New & Noteworthy

Message in a Bubble

February 18, 2015

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If you’re shipwrecked on a desert island, writing a message on a scrap of paper, sealing it in a bottle, and flinging it into the ocean could be your only chance at communication. As the song goes (see below), the message in a bottle is an S.O.S. to the world.

It turns out that cells may do something very similar. But instead of using a bottle, they enclose their messages in membrane-bound bubbles.

Many different mammalian cell types have been seen to form these extracellular vesicles (EVs). In mammalian cells, it’s known that EVs are used for cell-to-cell communication. They contain signals that allow cells to influence their neighbors, both for good (for example, regulating the immune response) and for bad (transmitting viruses or toxic peptides). In most cases these signals aren’t well-characterized, but the EVs may include DNAs and proteins, and they’re rich in RNAs.

Fungi have been found to produce EVs too, but they’ve been much less studied. In a new paper in Scientific Reports, da Silva and colleagues looked at the extracellular vesicles (EVs) produced by four different fungal species, including S. cerevisiae, and found that among other things, the vesicles actually include at least parts of many RNAs, both protein-coding and non-coding.

The scientists decided to look at S. cerevisiae and three species of fungal pathogens that infect humans: Cryptococcus neoformans, Paracoccidioides brasiliensis, and Candida albicans. (S. cerevisiae can be pathogenic too, but isn’t as virulent as any of those species.) They isolated EVs from each and treated the unbroken EVs with RNase to get rid of any RNA that might be contaminating their surfaces.

Then they broke open the vesicles to see what was inside. They found that the EVs contained many small RNAs, most less than 250 nucleotides in length. The scientists used RNA-seq analysis to determine the sequences of these small RNAs, and compared them to the genomic sequences that were already known for these organisms.

Many of the sequences corresponded to noncoding RNAs. For S. cerevisiae, the RNA sequences identified included the mitochondrial small and large ribosomal RNAs, RNA components of RNase enzyme complexes, a variety of small nuclear and small nucleolar RNAs, and tRNAs.

Sequences corresponding to several dozen S. cerevisiae mRNAs were also detected. There wasn’t much rhyme or reason to the kinds of proteins they encoded.  But the set of mRNA fragments didn’t correspond simply to the set of most abundant mRNAs in the cell. So it seemed like the vesicles didn’t just contain random samples of the cytoplasm, but instead had been loaded selectively with particular mRNAs.

This study raises as many questions as it answers, and there is a lot of work to be done before fungal EVs will be understood. The intriguing discovery of RNA in EVs suggests the possibility that the RNAs could influence gene expression in cells that take up the EVs, either by regulating processes like splicing or translation, or even by encoding a protein that gets translated in the recipient cell.

Being able to influence neighboring fungal cells via EVs could be an advantage for fungi in the fierce competition for biological resources. Or perhaps EVs are used to subvert gene expression in host tissues during a fungal infection. Far from being an S.O.S., these messages could be threatening.

These speculations all need much more research. Fungi are an integral part of our world, and we need to pay careful attention to the messages that they send us!

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

“Message in a Bottle,” The Police, 1979

Categories: Research Spotlight

Tags: cell-cell communication, extracellular vesicles, Saccharomyces cerevisiae

Yeast Finds Needles in a Haystack to Combat Malaria

February 11, 2015

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Finding a needle in a haystack would take a long time and would be very tedious (although it’s been done!) Finding a specific drug to fight malaria by testing the effect of each drug, one at a time, on a purified protein in vitro would be at least as tedious and maybe even more so.

Luckily, we don’t have to sift through a haystack. In a new study in ACS Chemical Biology, Frame and colleagues used our friend S. cerevisiae to find nine drugs out of a collection of more than 64,000 that are promising candidates for stopping the malaria parasite in its tracks. It is as if yeast allowed them to set fire to the haystack and see nine needles gleaming in the ashes.

Malaria is a huge problem for global health. Plasmodium falciparum, the organism that causes malaria, is fast developing resistance to the few effective drugs that we have left.

But P. falciparum has an Achilles heel—it can’t make its own purine nucleotides! Since these are the building blocks of DNA and, obviously, essential for life, if we can keep P. falciparum from being able to take them up, we can kill it. 

P. falciparum imports purines via a major transporter protein, called PfENT1, located in the plasma membrane. So a drug that specifically inhibited this transporter could be a good way to attack the pathogen.

It’s possible to assay the activity of the transporter in vitro, adding different drugs one at a time and seeing which inhibits transport. But doing this for thousands of drugs might make you wish you were looking for a needle in a haystack. Frame and colleagues decided to harness the awesome power of yeast genetics to test a very large set of drugs more quickly.

The toxic nucleoside analog 5-fluorouridine (5-FUrd) is taken into yeast cells by the high-affinity uridine transporter Fui1. It kills normal yeast cells, but fui1 null mutant yeast can survive in the presence of 5-FUrd.

The researchers engineered a yeast codon-optimized version of pfENT1 and expressed it in the mutant, restoring 5-FUrd uptake. The nucleoside analog was again toxic to this strain, and the only way the yeast could survive was if the transporter activity of pfENT1 was inhibited.

This system allowed a simple and powerful screen for pfENT1 inhibitors. The yeast strain expressing pfENT1 would be able to grow in the presence of 5-FUrd only if pfENT1 transporter activity was blocked by the drug that was being tested.

Setting up the screen on a large scale, the scientists were able to test 64,560 compounds. They initially found 171 compounds that allowed the yeast to grow. They narrowed these down to 9 compounds that worked well and belonged to different structural classes of chemicals.

Because of the way the study was designed, it was likely that these compounds allowed yeast to grow because they prevented PfENT1 from pumping the toxic 5-FUrd into the cell. But what if the compounds were actually doing something different, and unexpected? To rule out this possibility, the researchers designed a secondary screen for the 9 top candidate drugs.

They used ade2 mutant yeast, which can’t make their own adenine and need to be fed it in order to survive.  These mutants can make do with the related compound adenosine, but it can’t normally get inside the cell; yeast doesn’t have a transporter that will take it up. However, PfENT1 can transport adenosine, so ade2 mutants can grow on it if they are expressing PfENT1.

With this system, if the candidate drugs are working as expected, they should prevent yeast growth. And that is exactly what the researchers found. This confirmed that the drugs are working because they specifically inhibit PfENT1 and do not allow growth by some other, indirect mechanism.

To be completely sure of the mechanism, the scientists did a direct test. They found that the nine drugs prevented PfENT1-expressing cells from taking up radiolabeled adenosine.

This was all fine, but the ultimate goal of the study was to affect growth of the malaria parasite. So Frame and colleagues tested the drugs on P. falciparum. 

In the presence of any of the nine drugs, the parasite couldn’t take up adenosine and also failed to grow. This even happened when the parasites were grown in medium containing  much higher purine concentrations than found in human blood.

Even though PfENT1 was targeted by the drugs, all nine of the drugs also killed Pfent1 null mutants. This suggested that the drugs have a secondary target or targets in addition to PfENT1. This could be a real advantage, because it could help prevent the parasites from developing resistance to the drugs.

All nine of these diverse drugs are promising candidates for the treatment of malaria. And the same approach could be used to find chemicals that affect the function of other transporters from various organisms. 

As usual, yeast is providing scientists with streamlined ways to find new treatments for serious human diseases. Instead of tediously rummaging about in a haystack, yeast lets us quickly and easily find the needles we need. 

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

Categories: Research Spotlight

Tags: high throughput screen, malaria, Saccharomyces cerevisiae, transporters

Ticket to Transcribe

February 04, 2015

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tRNA_CUG may not just be for translation anymore:

Back before airplanes and cars, when times got tough people would often take trains to what they hoped were greener pastures.  And to hitch a ride on a train, they’d usually need to have a ticket. Turns out the same is true for Gln3, a transcription factor in yeast.

Basically, Gln3 stays in the cytoplasm as long as there are good sources of nitrogen available to the cell.  When these sources run out, Gln3 moves from the cytoplasm to the nucleus where it can turn on genes that can help the yeast cope with its new situation.

In a new study in GENETICS, Tate and coworkers have identified one of the tickets that lets Gln3 take the trip to the nucleus. And it was totally unexpected. To get to the nucleus, Gln3 needs a fully functional glutamine tRNA_CUG. No, really.

To get this evidence, Tate and coworkers used a reporter in which Gln3 was linked to GFP (green fluorescent protein). They tracked the location of Gln3 in the cell using fluorescence microscopy.

Using a temperature-sensitive mutant of tRNA_CUG, sup70-65, the authors showed that at the nonpermissive temperature of 30 degrees C, Gln3 could not translocate to the nucleus under a wide variety of conditions in which nitrogen was limiting. Gln3 had no problems translocating at the permissive temperature of 22 degrees C, and in wild-type cells Gln3 translocated at both temperatures. Clearly tRNA_CUG is doing something important in this process!

The next experiment showed that tRNA_CUG was more like a one-way ticket. Once Gln3 entered the nucleus under nitrogen starvation conditions at the permissive temperature, switching to the nonpermissive temperature had little effect. Gln3 stayed put.

A possible wrinkle in these experiments was that cells harboring sup70-65 formed chains reminiscent of pseudohyphae at the nonpermissive temperature no matter what the nitrogen conditions. One possible explanation for the results seen here was that many of these cells lacked nuclei. In this case, they might not see nuclear translocation because there was no nucleus to translocate to.

In the course of these studies, Tate and coworkers showed that adding rapamycin mimicked the effects of nitrogen starvation with one big difference—nuclear localization happened much more rapidly than with nitrogen starvation. This fast response allowed the authors to look at Gln3 localization while visualizing nuclei by staining DNA with DAPI (which gives a short-lived signal). They were able to use the DAPI to see that these cells did indeed have nuclei and that when they raised the temperature, Gln3 did not colocalize with the DAPI stained nuclei.  Gln3 was being kept out of nuclei at the nonpermissive temperature.

So it really looks like Gln3 needs a working tRNA_CUG to get into the nucleus. There are a couple of possible ways that this tRNA could be needed for Gln3 to make the trip.

In the first model, the tRNA is part of a complex that allows Gln3 to make the trip to the nucleus. In this model, it is almost as if Gln3 (or one of its compatriots) is clutching its ticket, tRNA_CUG. In the second, less fun model, the tRNA is required to translate a protein involved in Gln3’s transit. Which model is the correct one is still up in the air, but it will be interesting to see which is the right one.

This was the most astonishing finding in the article, but it was by no means the only one. We don’t have the time to go into the other experiments, which, among other things, teased apart differences in the four or five distinguishable pathways that work to turn on the cell’s nitrogen response.

This work highlights a recurring theme in basic research: we may think we know everything that’s going on (tRNAs just help to translate proteins, right?) but just about every time we look more closely, there is much more to see than first meets the eye. Being in the right place at the right time is essential, whether you’re escaping the Dust Bowl in The Grapes of Wrath or a transcription factor responding to the lack of a nutrient. It’s not so surprising that the cell has drafted every possible player into this process, even a lowly tRNA. 

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Since the title has this song stuck in our heads, we thought you might want to hear it too. Enjoy!

Categories: Research Spotlight

Tags: nitrogen utilization, Saccharomyces cerevisiae, transcription factor localization, tRNA

Species Can’t Risk the New Coke

January 29, 2015

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Genome organization may protect key genes from the ravages of increased mutation rate during meiosis:

Back in 1985, Coca Cola decided to completely rejigger the flavor of their flagship soft drink, calling it the New Coke. This radical change to the product was a colossal failure. Toying with such an essential part of a key product was simply too risky a move. If only they had learned from our favorite beast, Saccharomyces cerevisiae.

In a new study in PLOS Genetics, Rattray and coworkers show that the mutation rate is higher during meiosis in yeast because of the double-strand breaks associated with recombination. This makes sense, because any new mutations need to be passed on to the next generation for evolution to happen, and germ cells are made by meiosis. But their results also bring up the possibility that key genes might be protected from too many mutations by being in recombination cold spots. Unlike the Coca Cola company, yeast (and everything else) may protect essential genes from radical change.

Previous work in the Strathern lab had suggested that when double strand breaks (DSBs) in the DNA are repaired, one result is an increased mutation rate in the vicinity. The major culprit responsible for the mutations appeared to be DNA polymerase zeta (Rev3p and Rev7p).

To test whether the same is true for the DSBs that happen during the first meiotic prophase, Rattray and coworkers created a strain that contained the CAN1 gene linked to the HIS3 gene. The idea is that mutants in the CAN1 gene can be identified as they will be resistant to canavanine. The HIS3 gene is included as a way to rule out yeast that have become canavanine resistant through a loss of the CAN1 gene. So the authors were looking for strains that were both resistant to canavanine and could grow in the absence of histidine.

The first things the authors found was that the mutation rate during meiosis was indeed increased as compared to mitosis in diploids. For example, when the reporter cassette was inserted into the BUD5 gene, the mitotic mutation rate was 5.7 X 10^-8 while the meiotic mutation rate was 3.7 X 10^-7, a difference of around 6.5 fold.

This effect was dependent on the DSBs associated with recombination, since the increased mutation rate wasn’t seen in a spo11 mutant; the SPO11 gene is required for these breaks. Using a rev3 mutant, the authors could also conclude that at least half of the increased mutation rate is due to DNA polymerase zeta. This all strongly suggests that the act of recombination increases the local mutation rate.

If recombination is associated with the mutation rate, then areas on the genome that recombine more frequently should have a higher rate of mutation during meiosis. And they do. The authors inserted their cassette into a known recombination hotspot between the BUD23 and the ARE1 genes and saw a meiotic mutation rate of 1.77 X 10^-6  as compared to a rate of 4.9 X 10^-7 when inserted into a recombination coldspot. This 3.6 fold increase provides additional evidence that recombination is an important factor in meiotic recombination.

This may be more than just an unavoidable side effect of recombination. It could be that yeast and perhaps other beasts end up with their genes arrayed in such a way as to protect important genes by placing them in recombination dead zones.

And perhaps genes where lots of variation is tolerated or even helpful are placed in active recombination areas. In keeping with this, recent studies have shown that essential S. cerevisiae genes tend to be located in recombination cold spots, and that this arrangement is conserved in other yeasts.

It is too early to tell yet how pervasive this sort of gene placement is.  But if this turns out to be a good way to protect essential genes, Coca Cola should definitely have left the Coke formula in a part of its genome with little or no recombination. Mutating that set of instructions was as disastrous as mutating an essential gene!

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: evolution, meiosis, recombination, Saccharomyces cerevisiae

Not Lost Without Translation

January 22, 2015

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We all learned in biology class that nucleotides get added to an mRNA using a DNA template. And that amino acids get added to proteins using an mRNA template. But as with most everything in biology, there are exceptions.

For example, a long string of A’s gets added to mRNAs in eukaryotes without a DNA template of T’s. And now, in a new study published in Science, Shen and co-workers have shown that in certain cases amino acids can be added to proteins without translating an mRNA template.

Specifically, these authors showed that threonines and alanines that are not encoded by mRNA can be added to polypeptide chains stalled on the ribosome and that a key protein in this process is Rqc2p. It makes sense that Rqcp is on the spot to do this job, as this protein is part of the ribosome quality control (RQC) complex whose job it is to ubiquinate proteins stalled at the ribosome to target them for destruction.    

These added amino acids aren’t the result of some glitch of a misbehaving cell. They appear to be critical for the cells to mount a response to a situation where ribosomes are failing to complete normal translation.

Working with our favorite model organism, S. cerevisiae, the researchers started out to investigate the ribosome quality control (RQC) complex. This complex is like a cleanup crew for stalled ribosomes. If something goes wrong during translation and the ribosome stops elongating the nascent protein chain, the RQC complex steps in and tags the partially synthesized protein with ubiquitin, marking it for degradation.

The scientists were hoping to figure out how the RQC complex finds and recognizes stalled ribosomes. So they immunoprecipitated RQC and used cryo-electron microscopy to look at the structure of the complex bound to stalled ribosomes.

After a ribosome stops translating, it falls apart into its large and small subunits. Shen and colleagues found that one component of the RQC complex, Rqc2p, binds to the large (60S) ribosomal subunit after dissociation. They also found something unanticipated: tRNAs were present in the 60S ribosomal subunit at the A and P sites. This is where the tRNAs normally reside during translation, but translation obviously couldn’t be happening, since there was no mRNA present.

The presence of a tRNA at the ribosomal A site was especially surprising because tRNAs don’t bind there stably; they need mRNA and elongation factors to stabilize the interaction. It turned out that what was keeping the tRNAs on the ribosomal large subunit was Rqc2p, which bound to both of them and stabilized them. The researchers used a new thermostable reverse transcriptase and deep sequencing to find that the bound tRNAs were two specific alanine and threonine tRNA species. Why these specific tRNAs?

Pursuing this question, Shen and colleagues made another unexpected discovery: nascent polypeptide chains from stalled ribosomes were smaller in the rqc2 null mutant than in wild type.  This suggested that Rqc2p was adding something extra to the unfinished, stalled proteins.

Putting these observations together, the researchers formulated the hypothesis that Rqc2p mediates the addition of extra alanine and threonine residues to the C termini of proteins whose translation has been stalled. They created an ingenious set of reporter constructs to test this hypothesis.

The basic reporter contained the green fluorescent protein (GFP) gene fused to a coding sequence containing multiple “difficult” codons that would cause the ribosome to stall. The researchers found that most of the strains that were mutant for different subunits of the RQC complex contained a smear of variably sized protein products, the size of GFP and larger. But the rqc2 mutant only contained unmodified GFP; it failed to add anything. This confirmed the earlier suggestion that Rqc2p was responsible for adding the mysterious extra mass.

Next, they added a protease cleavage site at various locations in the reporter gene, and found that the extra mass was added at or downstream of the stalling sequence. In other words, it was added to the C terminus of the nascent polypeptide.

To be completely sure that translation was not involved, the researchers put stop codons in every frame after the stalling sequence. They had no effect, so the extra mass couldn’t be attributed to translation in any frame.

Finally, the scientists analyzed the C-terminal extensions by total amino acid analysis, Edman degradation, and mass spectrometry. They found that the extensions consisted of between 5 and 19 alanine and threonine residues, in no defined sequence. They named them Carboxy-terminal Ala and Thr extensions, or CAT tails.

This is all very cool, but do the tails actually do anything? Yes, it looks like they do!

When translation stalls, the cell responds to this stress using the transcription factor Hsf1p. By mutating three conserved residues in the Rqc2p NFACT nucleotide-binding domain, Shen and colleagues were able to generate a protein that could still recognize the plugged-up ribosome, but couldn’t add the alanines or threonines. It still worked fine to clean up stalled proteins, but the stalled proteins had no CAT tails. And sure enough, there was no Hsf1p-mediated heat shock response either.

So the CAT tails are part of the signal that tells the cell it had better start a stress response because things aren’t looking too good at the ribosome. It’s still not obvious exactly how the CAT tails participate in this process. But this isn’t some peculiarity of yeast: the genes are conserved, and mutations in homologs of some of the RQC genes and other genes involved in translation quality control cause neurodegeneration in mice.

The ribosome is one of the best-studied molecular machines, and you might have thought we already knew just about everything there was to know about it. This work reminds us that no matter how familiar something seems, there is always more to learn when we pay attention to unexpected results.

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

Categories: Research Spotlight

Tags: ribosome quality control, Saccharomyces cerevisiae, stress response

How to Make A Safe and Fun Mitochondrion

January 13, 2015

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Influencing mitochondrial import to treat disease:

Bounce houses are a great way for kids to burn off their excess energy. They can bounce off the floor and walls and scream to their hearts’ content.

Of course, adults need to keep an eye on how many kids are in the house at any one time, to keep things safe. And if one child starts to push and kick the others, it might be easier to restore calm if the adults are careful about how many kids, and which ones, they allow inside.

The yeast mitochondrion is actually a lot like a bounce house. It’s full of energy, and it has multiple gatekeepers—protein complexes in the mitochondrial membrane that imported proteins must pass through on their way in.

And, just like a bounce house, things can go very wrong inside the mitochondrion if its proteins don’t behave properly. The end result isn’t just an upset child with a black eye, either. Genetic diseases that affect mitochondrial function are among the most severe and the hardest to treat.

Now, described in a new paper in Nature Communications, Aiyar and colleagues have used a yeast model of human mitochondrial disease to discover both a drug and a genetic means to regulate a mitochondrial import complex. Surprisingly, tweaking mitochondrial import slightly by either of these methods mitigated the disease symptoms in both yeast and human cells.  They found a gatekeeper who can make sure there is the right number of kids in the bounce house and that they’re all behaving properly (at least, as well as they can!).

The researchers were interested in mitochondrial disorders that affected ATP synthase. This huge molecular machine in the mitochondrial inner membrane is responsible for generating most of the cell’s energy, so if it doesn’t work properly it can be a disaster for both yeast and human cells.

Aiyar and coworkers used a genetic trick to create a yeast model that had lower amounts of functional ATP synthase. This mimics many mitochondrial disorders.

They were able to reduce the amount of functional ATP synthase by using an fmc1 null mutant. Fmc1p is involved in assembly of the complex, so the fmc1 null mutant has lower amounts of functional ATP synthase and a reduced respiration rate.

First, they looked for a drug that would mitigate the effects of the fmc1 mutation. They tested the drugs in a collection that had already been FDA approved—a drug repurposing library—to see if any would improve the mutant’s respiratory growth.

The one candidate drug that emerged from the screen was sodium pyrithione (NaPT), which is used as an antiseptic. Not only did it improve the respiration of the yeast fmc1 mutant, it also improved the respiratory growth of a human cell line carrying the atp6-T8993G mutation found in patients with neuropathy, ataxia and retinitis pigmentosa (NARP, one type of ATP synthase disorder).

Aiyar and colleagues wondered exactly what was being affected by the NaPT. To figure this out, they used the S. cerevisiae genome-wide heterozygous deletion mutant collection. This is a set of diploid strains, each heterozygous for a null mutation of a different gene, that has been an incredibly useful resource for all kinds of studies in yeast.

They tested the effect of NaPT on each of the mutant strains and found that strains with mutations in the TIM17 and TIM23 genes were among the most sensitive. And, when they checked the data from previous chemogenomic screens, they saw that these two mutants were much more sensitive to NaPT than to any other drug, showing that the effect was specific.

TIM17 and TIM23 are both subunits of the Tim23 complex in the mitochondrial inner membrane that acts as a gate for many of the proteins that end up in mitochondria. The researchers found that NaPT specifically inhibited the function of this mitochondrial gatekeeper complex in an in vitro mitochondrial import assay, confirming its selectivity.

So, Aiyar and coworkers had found a drug that alleviates the effects of an ATP synthase disorder by modulating the function of a mitochondrial gatekeeper. This in itself was a huge advance: the discovery that a potentially useful, already-approved drug has a specific effect on this disease phenotype.

However, the scientists took things a step further by looking to see whether a genetic therapy could accomplish the same thing as the drug.  It was already known that overexpressing Tim21p, a regulatory subunit of the Tim23 complex, could modulate the function of the complex similarly to the effects they had seen for NaPT.

So the researchers tested whether overexpressing Tim21p would improve respiratory growth of the fmc1 mutant. Sure enough, it did. Consistent with this, assembly of the respiratory enzyme complexes of the mitochondrial inner membrane was more efficient when Tim21p was overexpressed.

Most importantly, overexpression of Tim21p in the fmc1 mutant cells caused their total ATP synthesis to more than double.  And even more exciting was the discovery that overexpressing TIMM21, the human ortholog of TIM21, in the NARP disease human cell line improved survival of those cells.

So, just like a parent deciding how many kids should be in a bounce house so that everyone has a good time, the Tim23 complex can be made to “decide” which proteins, or perhaps how many proteins, get into mitochondria, with the end result that ATP synthesis happens as efficiently as possible. The exact mechanism of this effect is still unclear, but it is clear that modulating import in this way can improve mitochondrial health even when disease mutant proteins are present.

The next step will be to translate this discovery into therapies that will help mitochondrial disease patients. People with various mitochondrial disorders may finally be able to turn their mitochondria into safe, fun places.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: ATP synthase, mitochondria, respiration, Saccharomyces cerevisiae, yeast model for human disease

Yeast Genetics Makes Awesome Sauce

January 08, 2015

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What’s for dinner tonight? For many of us the answer will be “pasta with tomato sauce”, even if we don’t have Italian roots.

But as you know, there isn’t any single recipe for homemade tomato sauce. Onions and garlic, or just garlic? Pork, beef, or no meat at all? How many bay leaves go in the pot? Every cook will use a slightly different combination of ingredients, but all will end up with tomato sauce.

When it comes to combining different allele variants to survive a lethal challenge, yeast is a lot like those cooks. In a new paper in GENETICS, Sirr and colleagues used divergent yeast strains to generate a wide range of allelic backgrounds and found that there is more than one way to survive a deadly mutation in the GAL7 gene.  Just as there is more than one way to make a delicious tomato sauce…

This isn’t just an academic exercise either.  GAL7 is the ortholog of the human GALT gene, which when mutated leads to the disease galactosemia. And just like in yeast, people with different genetic backgrounds may do better or worse when both copies of their GALT gene are mutated.  

GAL7 and GALT encode an enzyme, galactose-1-phosphate uridyl transferase, that breaks down the sugar galactose. If people with this mutation eat galactose, the toxic compound galactose-1-phosphate accumulates; this can cause serious symptoms or even death. The same is often true for yeast with a mutated GAL7 gene.

Ideally we would want to be able to predict how severe the symptoms of galactosemia would be, based on a patient’s genetic background. So far, though, it’s been a challenge to identify comprehensively the whole set of genes that affect a given human phenotype. Which is why Sirr and colleagues turned to our friend S. cerevisiae to study alleles affecting the highly conserved galactose utilization pathway.

The researchers started with two very divergent yeast strains, one isolated from a canyon in Israel and the other from an oak tree in Pennsylvania. Both were able to utilize galactose normally, but the scientists made them “galactosemic” by knocking out the GAL7 gene in each.

After mating the strains to create a galactosemic diploid, the researchers needed to let the strain sporulate and isolate haploid progeny. But its sporulation efficiency wasn’t very good, only about 20%. And they needed to have millions of progeny to get a comprehensive look at genetic backgrounds.

To isolate a virtually pure population of haploid progeny, Sirr and coworkers came up with a neat trick. They added a green fluorescent protein gene to the strain and put it under control of a sporulation-specific promoter.

Cells that were undergoing sporulation would fluoresce and could be separated from the others by fluorescence-activated cell sorting (FACS). The FACS technique also allowed sorting by size, so they could select complete tetrads containing four haploid spores and discard incomplete products of meiosis such as dyads containing only two spores.

After using this step to isolate tetrads, the researchers broke them open to free the spores and put them on Petri dishes containing galactose—an amount that was enough to kill either of the parent strains. One in a thousand spores was able to survive the galactose toxicity. Recombination between the divergent alleles from the two parent strains somehow came up with the right combination of alleles for a survival sauce.

Sirr and colleagues individually genotyped 247 of the surviving progeny, using partial genome sequencing. They mapped QTLs (quantitative trait loci) to identify genomic regions associated with survival. If they found a particular allele in the survivors more often than would be expected by chance, that was a clue that a gene in that region had a role in survival.

We don’t have the space here to do justice to the details of the results, but we can summarize by saying that a whole variety of factors contributed to the galactose tolerance of the surviving progeny. They had three major QTLs, regions where multiple alleles were over- or under-represented. The QTLs were centered on genes involved in sugar metabolism: GAL3 and GAL80, both involved in transcriptional regulation of galactose utilization genes, and three hexose transporter genes (HXT3, HXT6, and HXT7) that are located very close to each other.

It makes sense that all of these genes could affect galactosemia. Gal3p and Gal80p are regulators of the pathway, so alleles of these genes that make galactose catabolism less active would result in less production of toxic intermediates. And although the hexose transporters don’t transport galactose as their preferred substrates, they may induce the pathway by allowing a little galactose into the cell. So less active alleles of these transporters would also result in less galactose catabolism.

Another event that occurred in over half of the surviving progeny was aneuploidy (altered chromosome number), most often an extra copy of chromosome XIII where the GAL80 gene is located. The same three QTL peaks were also seen in the disomic strains, though, leading the authors to conclude that the extra chromosome alone was not sufficient for survival of galactosemia.

And finally, some rare non-genetic events contributed to survival of the progeny. The authors discovered this when they found that the galactose tolerance of some of the progeny wasn’t stably inherited. This could result from differences in protein levels between individual cells. For example, if one cell happened to have lower levels of a galactose transporter than other cells, it might be more resistant to galactose.

The take-home message here is that there are many different ways to get to the same phenotype. The new method that they developed allowed the researchers to see rare combinations of alleles in large numbers of individual progeny, in contrast to other genotyping methods where progeny are pooled and only the average can be detected.

For any disease or trait the ultimate goal is to identify all the alleles of all the genes that influence it. Imagine the impact on human health, if we could look at a person’s genotype and accurately predict their phenotype!

So far, it’s been a challenge to identify these large sets of human genes in a comprehensive way. But this approach using yeast could provide a feast of data to help us understand monogenic diseases like galactosemia, cystic fibrosis, porphyria, and many more, and maybe even more complex traits and diseases. Now that’s an appetizing prospect for human disease researchers. Buon appetito!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: galactosemia, Saccharomyces cerevisiae, yeast model for human disease

Mother Yeast Keeps Daughters Tidy

December 18, 2014

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Yeast need translation and organelle tethering to sequester misfolded proteins away from the cytoplasm:

Sometimes parents feel like they’re constantly nagging their kids to clean things up. Eventually, some parents just give in and do it themselves. It turns out that yeast cells aren’t all that different.

And it is even more important for cells to clean up their cellular trash than it is for that sullen teenager. If trash like misfolded or damaged proteins is not sequestered from the rest of the cytoplasm, it can cause other proteins to change their conformation or interfere with metabolic processes. This is obviously much worse than having a smelly room!

One way that cells bin their trash is by compacting it into globs, or aggregates. While this works in the short run, as cells age these aggregates build up, and in human cells, they’re hallmarks of diseases like ALS and Alzheimer’s.

In a new study published in Cell, Zhou and colleagues looked at the process of protein aggregation in S. cerevisiae. A number of other studies had already suggested that just like human parents, mother yeast cells clean up trash for their daughters. The researchers confirmed this and also made a couple of very surprising findings.

Zhou and coworkers discovered that although aggregates are mostly composed of previously translated proteins, they don’t form without new translation. And they found that aggregates aren’t littered around the cytoplasm, but instead are collected in very specific trash bins located on the surface of cellular organelles.

To do these studies, the scientists assembled a toolkit of ways to induce and visualize protein aggregation.  They used stresses like heat shock or various chemicals to stimulate aggregate formation.

They visualized the aggregates by using GFP-labeled Hsp104p, which binds to them specifically. They also had some thermally unstable reporter proteins fused to different colored fluorescent markers that helped them track aggregation. And they created time-lapse videos to watch the whole process.

They first looked in detail at aggregate formation, creating two different kinds of cellular trash (aggregates of distinct sets of marker proteins) at different times to ask whether they would all end up in the same aggregates or in separate ones. These experiments showed that in general, newly aggregated proteins will join existing aggregates rather than creating new ones. And intriguingly, these results also hinted that new translation was needed to start the aggregation process.

Zhou and coworkers tested this directly by adding cycloheximide, an inhibitor of translation, to their aggregation experiments. Sure enough, cycloheximide prevented all of the treatments from causing aggregation, and other treatments and conditions that blocked translation did the same thing.

So just as the threat of taking away the car keys may get that sullen teenager off the couch to start cleaning up, newly synthesized polypeptides are the inducer for the cellular clean-up. Without them, all the trash just stays littered around the cell.

The researchers guessed that if aggregation starts at sites where translation is occurring, it might be concentrated at the surface of the endoplasmic reticulum (ER), where a large proportion of ribosomes are bound. They used several different sophisticated microscopy techniques to confirm that most aggregates were in fact associated with the ER.

But they also got a surprise: in addition to the ER, many aggregates were associated with the mitochondrial surface and some even formed directly on it.* The authors also observed aggregates that formed at the ER but migrated along it to ER-mitochondrial contact points and eventually to mitochondria. So, cells don’t litter their trash just anywhere; they start specific collection points on the surfaces of organelles. And much of the trash seems to end up attached to mitochondria.

The researchers noticed something else when they looked at aggregates in cells that were dividing. When a bud forms, mitochondria are actively transported into it. However, the aggregates that were on mitochondria didn’t go into buds. They stayed on mitochondria that remained in the mother cell. Mom protects her daughter from any trash that mom created!

To figure out what controls this asymmetric segregation, Zhou and colleagues tested a panel of 72 mutant strains, each with a deletion in a mitochondrial outer membrane protein. One strain, the fis1 null mutant, was markedly defective: aggregates often went into the bud. Fis1p is known to be involved in mitochondrial fission, but this result suggests it may have an entirely separate role in making sure that trash-bearing mitochondria stay in the mother cell.

And finally, the authors saw that as mother cells got older, they got less and less able to keep aggregates out of their daughters’ cytoplasm. Towards the end of the mothers’ lives, aggregates were distributed more or less randomly between mother and daughter.

Trash build up is a big problem in teenagers’ rooms and in cells. Just like mom, the cell packs the trash away into bins where it will do less harm. Unfortunately, as the cell (and mom) get older, this gets harder and harder to do. In both cases, the daughter is saddled with more and more trash as mom struggles to keep up. And this is bad for the daughter as well as for the mom.

So, there’s actually a very good reason behind all that nagging to clean up your room. The secret to a long life is to always pick up your trash!

*New data from the Weissman lab, described here in a recent blog post, dovetail nicely with this finding since they establish that a lot of translation takes place on the mitochondrial surface.

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

Categories: Research Spotlight

Tags: mitochondria, protein aggregation, Saccharomyces cerevisiae

Telomerator is the Chromosomal Terminator

December 10, 2014

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A simple tool for adding telomeres to linear DNA:

First there was the Terminator. Now, in a new study published in PNAS by Mitchell and Boeke, we have the telomerator.

Instead of being a homicidal robot from the future bent on killing Sarah Connor, the telomerator is a tool that lets scientists easily turn circular DNA into stable chromosomes in the yeast Saccharomyces cerevisiae. While less splashy, this bit of synthetic biology is definitely cool in its own way (and much less dangerous!).

The system that Mitchell and Boeke created is very clever. They first inserted the intron from the ACT1 gene into the middle of the URA3 gene. The URA3 gene was still functional, as ura3 mutants could use it to grow on medium lacking uracil.  

They next inserted a sequence into the middle of the intron that consisted of an 18 base pair I-SceI cleavage site flanked on each side by around 40 base pairs of yeast telomere repeats (called Telomere Seed Sequences or TeSSs). This construct still allowed ura3 mutants to grow in the absence of added uracil.

The final step was to introduce the homing endonuclease I-SceI to the cell so that it cut the circular DNA precisely between the two TeSSs. The idea is that when you add the homing endonuclease, the newly linearized piece of DNA ends up with the telomere seeds on each end. Telomerase adds more repeats to the seeds until the DNA has proper telomeres. Voilà, a chromosome is born.

The URA3 gene part of the plasmid is important for selecting cells with the linearized DNA. Basically a circularized DNA will grow on medium lacking uracil but fail to grow on medium with 5-FOA, while the linearized DNA will do the opposite. In other words, the process of linearization should destroy the URA3 gene. And that’s just what they found.

Previous work had shown that to be stable in yeast, a chromosome needs to be at least 90 kilobases (kb) or so long. This is why they tested their new telomerator in synIXR, a synthetic yeast chromosome that is about 100 kb in length. This chromosome has 52 genes from the right arm of chromosome 9, two genes from the left arm, around 10 kb of nonessential BAC DNA, the native centromere CEN9, and a LEU2 marker. 

Mitchell and Boeke inserted the telomerator sequence into two different locations in the BAC part of the circularized synIXR and found that adding I-SceI appeared to linearize the DNA. In both cases they found that around 100 out of 200 cells were resistant to 5-FOA and unable to grow in the absence of uracil but could still grow in the absence of leucine. This is just what we would predict if we cut the DNA in the middle of the URA3 gene and created a stable piece of linear DNA.

They next wanted to use this tool to study the effects of telomeric DNA on nearby genes. We would predict that because of telomeric silencing, genes near a telomere will be downregulated. Any genes that affect growth when turned down should quickly become evident.

To accomplish this they inserted the telomerator three base pairs downstream of each of the 54 genes on synIXR, generating 54 new plasmids. After activating the telomerator by expressing the I-SceI nuclease, they used pulsed field gel electrophoresis to confirm that 51 of the 54 synthetic chromosomes had indeed been linearized.

As expected, they found that putting a telomere near a gene sometimes has profound effects. For example, when they linearized DNA where the telomerator was 3’ of either YIR014W, MRS1, or YIR020C-B, they got no growth. They also found many more effects on the growth rate at both 30° C and 37° C at many different, “telomerized” genes. The implication is that when these genes are near telomeric DNA, they no longer function at a high enough level for the yeast to grow well or in some cases to even survive.

To confirm that the effects they saw were due to telomeric silencing, Mitchell and Boeke tested each linearized DNA in a sir2 mutant, a key player in this form of silencing. Mutating sir2 reversed the effects of placing a telomere near the gene, further supporting the idea that the newly created chromosome ends are like normal telomeres because they undergo the same Sir2-mediated silencing.

Finally, the researchers tested the stability of the newly created chromosomes by selecting for Ura⁺ revertants from six individual cultures with different linearized molecules. They failed to select any revertants in which the DNA had recircularized, showing that the linear chromosomes are stable.

So in contrast to the Terminator, who sliced and diced his victims randomly, the telomerator will allow synthetic biologists to create linear chromosomes with precisely positioned telomeres. This study proved the concept, and this tool will be incredibly useful in the future, both in yeast and potentially in other eukaryotes. Both the Terminator and the telomerator can say, “I’ll be back”!

by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, synthetic biology, telomere

A Lot Hinges on Ndc80

December 04, 2014

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A collapsible protein complex makes sure that dividing cells get the right number of chromosomes:

The invention of collapsible tent poles was a boon to backpackers everywhere. These long, rigid poles provide strong support for a tent, but when it’s time to pack up and go, they fold up into a short package. The secret? Flexible regions in between the rigid sections.

Although the inventors of these poles couldn’t have known it, something similar already existed in nature. In a new paper in GENETICS, Tien and colleagues used the awesome power of yeast genetics, along with some cool biochemistry, to look at the shape of the S. cerevisiae Ndc80 complex in vivo. They found that, just like a tent pole, it bends sharply at a flexible region to fold two rigid sections next to each other. And this ability to fold is critical for accurate chromosome segregation.

During cell division, chromosomes must be correctly attached to the mitotic spindle so that the mother and daughter cells each get one, and only one, copy of each. If a yeast cell doesn’t get this process right, it can become sick or even die. And if it happens in an animal cell, it can lead to cancer.

The Ndc80 complex, which is conserved from yeast to humans, is an integral part of this process because it connects the chromosomes to the spindle during mitosis. It consists of four subunits and has an elongated middle and globular parts on each end, sort of like a dumbbell. The Ndc80 protein, one of the subunits, has an unstructured  “loop” region in the middle of its elongated section.

Previous work had shown that the loop region of Ndc80 is flexible in vitro, and in vivo experiments had shown that the whole complex can change its conformation. Tien and colleagues wanted to know whether the Ndc80 loop region was important for the shape of the complex during mitosis, and whether flexibility in this region was important for function.

They started with a genetic approach, and isolated mutations in NDC80 that caused heat sensitivity. One particular allele, ndc80-121, was especially interesting. The mutant protein had two amino acid changes, near each other and near the loop region. The Ndc80 complex containing the mutant protein was just as stable, and bound to microtubules just as tightly, as the wild-type complex. So why did the cells die at higher temperatures?

Tien and colleagues visualized mitosis in the mutant cells using fluorescence microscopy. They could see that when they raised the temperature, dividing mutant cells had lots of aberrant attachments between chromosomes and the spindle. Because of these attachments, proceeding through mitosis caused their spindles to break—a lethal event.

However, if they timed the temperature shift to happen later in the cell cycle, the ndc80-121 mutant cells were fine. If chromosomes had already been lined up correctly on the spindle before the temperature was raised, then the rest of mitosis could go on without a problem.

Tien and coworkers wondered whether the mutation might disrupt the binding of some other protein to the complex at high temperatures. To look for interactions, they selected mutations that suppressed the heat-sensitive phenotype of ndc80-121. But they didn’t find any suppressor mutations in other genes. However, they did find an intragenic suppressor mutation within the ndc80-121 gene.

Interestingly, this mutation affected a residue that was on the other side of the loop relative to the original two changes. If the Ndc80 complex is a dumbell, imagine that the dumbell is collapsible like a two-segment tent pole, with the loop region of Ndc80 as the elastic between the sections. If you folded the complex in this way, the amino acids changed in the ndc80-121 mutant protein would be positioned close to the amino acid that the suppressor mutation affected—an intriguing explanation for how these mutations might affect each other.

Of course, genetic interactions don’t prove a direct physical interaction. So the researchers looked to see whether they could detect physical interactions between these regions. They treated the complex with a reagent that would permanently cross-link amino acids that were close to each other. Then they chopped the complex into smaller peptides using a protease, and analyzed the cross-linked peptides using mass spectrometry to locate the linked residues.

Sure enough, they were able to detect multiple cross-links within the complex, and their locations confirmed that the complex folds much like a tent pole. Based on their mutant phenotypes, the researchers think it’s likely that the original ndc80-121mutation destabilizes folding of the complex and that the intragenic suppressor mutation makes folding tighter. Consistent with this idea, the intragenic suppressor mutation alone confers a slow-growth phenotype, as if it makes the complex fold just a little too tightly to support vigorous growth.

These experiments as a whole establish that the Ndc80 complex folds tightly early in mitosis. So, creative inventors and Mother Nature have arrived at similar solutions for the tent pole and for this important complex. And just as collapsible tent poles have become ubiquitous in the backpacking world, so too has the collapsible Ndc80 complex been conserved throughout evolution: even the specific residues that mediate the folding are highly conserved. Since this work has shown that correct folding of the yeast complex is necessary for its role in helping chromosomes to line up accurately on the spindle, the same is almost certainly true in mammalian cells.

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

Categories: Research Spotlight

Tags: mitosis, Ndc80 complex, protein structure, Saccharomyces cerevisiae

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