New & Noteworthy

Ribosomes Caught in the Act

November 24, 2014

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If you want to see what animals really do out in the wild, first you need to hide a camera and a trip-wire so well that the jungle seems totally undisturbed. Then, if you’re lucky, you’ll be able to catch them in the middle of the night as they pass by. Now you can surprise that tiger and find out what he is doing at that specific spot.

In two companion Science articles from the Weissman group at UCSF, Jan et al. and Williams et al. did essentially the same thing to S. cerevisiae ribosomes. They hid a molecular tag and the enzyme that recognizes it at various interesting places within yeast cells, so cleverly that the cells had no idea anything was different. Instead of a flash of light, they used a pulse of the small molecule biotin to find out which mRNAs were being translated at specific locations in the cell.

What they found was that when ribosomes are translating proteins that are targeted to a particular organelle, they hang around the surface of that organelle—way more frequently than was previously thought. And the exquisite specificity of this technique, allowing them to pinpoint one particular mRNA within the cell, uncovered a fascinating case of dual protein localization.

Pinpointing Translation Locations

The researchers needed to develop a technique for catching ribosomes in the act of translation. One part of this had already been worked out in the same group: ribosomal profiling, a method that allows you to map very precisely the positions of ribosomes on mRNAs.

Briefly, cells are lysed and translating ribosomes are treated with nucleases that nibble away mRNAs, except for the 30 nucleotides or so that are protected within the ribosome. Then those protected fragments are analyzed by deep sequencing. This shows, at the single nucleotide level, where ribosomes are sitting on each individual mRNA.

Ribosomal profiling tells us where translating ribosomes are in relation to mRNAs, but not where they are in relation to the rest of the cell. To get this location information, the researchers came up with a clever tagging strategy.

They started with a bacterial gene, E. coli BirA, that encodes a biotin ligase—an enzyme that can attach biotin to specific acceptor peptides. They fused BirA to various yeast genes in order to target biotin ligase to different places in the cell.

Next they tagged ribosomes by putting a biotin acceptor, called the AviTag, on ribosomal proteins such that the tag would be sticking out on the ribosomal outer surface. They tested both the BirA and AviTag fusions to make sure that they didn’t interfere with the functions of any proteins. Just like the camera hidden in the jungle, the tags didn’t perturb yeast cells in the least.

Now the researchers were set to surprise ribosomes with a pulse of biotin. Any ribosomes that were close to BirA would become biotinylated. The tagged ribosomes could then be isolated, and the mRNA sequences being translated in those ribosomes could be identified. The method as a whole is termed proximity-specific ribosomal profiling.

Jan and coworkers set up and validated this method in their paper, and used it to look at translation of secretory proteins at the surface of the endoplasmic reticulum (ER), while Williams and colleagues used the method to look closely at translation at the mitochondrial surface. Import into both of those organelles has previously been studied intensively, but often in vitro and mostly for just a few model protein substrates. In contrast, proximity-specific ribosomal profiling gives us the ability to look at translation of the entire proteome in vivo.

While it was known before that proteins targeted towards a certain organelle tended to be translated near that organelle, these researchers found that it was much more common than previously believed. For example, they found that most mitochondrial inner membrane proteins were translated at the mitochondrial surface and imported cotranslationally, in contrast to the previous view that mitochondrial import is predominantly posttranslational.

Both studies discovered many more details than we can summarize here. But the comparison between the ER and mitochondrial studies led to a special insight about one protein.

Osm1p Goes Both Ways

Osm1p, fumarate reductase, was thought to be a mitochondrial protein (although results from a few high-throughput studies had hinted at a link to the ER). But proximity-specific ribosomal profiling showed very clearly that it was translated at both the ER and mitochondrial surfaces. Williams and coworkers went on to confirm by fluorescence microscopy of an Osm1p-GFP fusion that Osm1p is indeed present in both ER and mitochondria.

Both of these organelles have pretty strict criteria for the signal sequences of proteins they import, so how could it be possible that the same protein goes to both locations? The researchers found that in fact, it’s not! They repeated the ribosomal profiling on the OSM1 mRNA, this time adding the drug lactimidomycin which makes ribosomes pile up at translational start sites. This showed that OSM1 actually has two start codons and produces two different proteins targeted to the two locations.

The OSM1 methionine codon currently annotated as the start would produce a protein with an ER targeting signal. Ribosomes piled up there, but also at another methionine codon 32 codons downstream. Starting translation at this codon would produce a protein with a mitochondrial targeting signal. Williams and colleagues confirmed this idea by showing that mutating the first Met codon made all of the Osm1p go to mitochondria, while mutating the Met codon at position 32 sent all of it to the ER.

The mutant form of Osm1p that couldn’t go to the ER conferred an intriguing phenotype: the inability to grow in the absence of oxygen. Osm1p generates oxidized FAD, which is necessary for oxidative protein folding, and it also interacts genetically with ERO1, which is involved in this process. Taken together, this all suggests that Osm1p activity drives oxidative protein folding in the ER.

The traditional ways of determining where a protein is in the cell, microscopic visualization or physical fractionation, can both be difficult and imprecise. Proximity-specific ribosomal profiling gets around those challenges, and gives a very precise picture of exactly where proteins are being created and how ribosomes are oriented with respect to organelles.

The example of Osm1p localization gives just a hint of the insights that are waiting for scientists who exploit this technique further. And we’re not just talking about yeast: the authors tested and validated the method in mammalian cells. Just like that tiger, surprised ribosomes in many different cell types will be giving up their secrets about where they roam and what they do.

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

Categories: Research Spotlight

Tags: endoplasmic reticulum, mitochondria, protein targeting, Saccharomyces cerevisiae, translation

Lots of Ways to Get to the Same Place

November 13, 2014

 

How Bad Mutations Can Help Yeast Thrive in New Environments:

If you’ve ever asked for directions from more than one person, you know there are many ways to get to the same place. But not all routes are created equal. You might be trying to get to the post office, but on some routes you’ll pass by the zoo, while on others you might pass the museum or the bus station.

Turns out that something like this may be happening with individuals in a population too. Each may be well adapted for its environment, but each may have arrived there in different ways. And although they may seem similar, they might actually be more different than they look on the surface.

In a new study in PLOS biology, Szamecz and coworkers show that a lot of these different routes to the same place happen in yeast because of bad mutations. They found that when a yeast gets a deleterious mutation, it is sometimes able to mutate its way back to being competitive with wild type again. But the evolved strain is genetically distinct from the original wild type strain. Similar phenotype, distinct genotype.

And this isn’t just an interesting academic exercise either. As any biologist knows, bad mutations are much more common than are good ones. This means that populations may often be evolving to overcome the effects of these bad mutations. This process may help to explain the wide range of diverse genotypes seen in any wild population. 

The authors started out by focusing on 187 yeast strains in which a single gene had been deleted. Each strain grew more poorly than wild type under the tested conditions.

They then took 4 replicates of each mutant along with the wild type strain and grew them for 400 or so generations. They looked for strains that had evolved to overcome the growth defect caused by the mutation. 

To take into account the fact that every strain would probably evolve a bit to grow better in the environment, they only looked for those that had gained more growth advantage than the wild type had. Around 68% of the strains showed at least one replicate that met this criterion.

So as we might expect, it is possible for a strain that grows poorly to mutate its way closer to a wild type growth rate. The next question was whether these mutant strains had mutated back to something close to wild type or to something new.

The authors decided to answer this question by doing a gene expression analysis of the wild type, the eight mutant strains, and a corresponding evolved line from each of these eight. After doing transcriptome analysis, they found that for the most part the evolved lines did not simply revert back to the original gene expression pattern of the wild type strain. Instead, they generated a novel gene expression pattern to deal with the consequences of having lost the original gene. And in the next set of experiments, the authors showed that this matters when the evolved strains are put in a new environment.

The researchers took 237 evolved lines that grew nearly as well as the original wild type strain and tested how well they each did in 14 different environments. In other words, they tested genetically distinct, phenotypically similar strains in new environments.

They found that even though the original mutant strains grew poorly in all the environments tested, the evolved ones sometimes did better. Fitness improved in 52% of the strains and declined in 8%. What is even more interesting is that a few stumbled upon genotypes that were significantly better than the evolved wild type in a particular environment.

A couple of great examples are the rpl6b or atp11 deletion mutant strains. Strains evolved from either mutant did around 25% better than the evolved wild type strain in high salt, even though both of the original mutants did significantly worse than the wild type strain. By suffering a bad mutation, the evolved strain had been rerouted so that it now grew better than wild type. 

So it looks like getting a bad mutation may not be all bad after all. It might just give you that competitive edge you need when things change. Sometimes the best way to get from point A to point B is not a straight line. 

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

Categories: Research Spotlight

Tags: evolution, Saccharomyces cerevisiae

Finding the Right Tools for the Job

November 06, 2014

 

Researchers Create a New Gene Editing Tool for Prototrophs and Diploids:

If you like spending your weekends tinkering with your beautiful 1957 Ford Thunderbird that only leaves the garage on sunny days, then you probably own a set of “standard” wrenches, sized in inches.  But if you wanted to tune up the trusty 2008 Subaru that gets you to work every day, you’d be out of luck. The standard wrenches are no use and you’d need a set of metric wrenches to fit the Subaru parts.

Molecular tools can be like that too. The genetic engineering toolkit that has been used on Saccharomyces cerevisiae for many years has been terrifically handy, but it only works for strains that have specific nutritional requirements, termed auxotrophs. It’s not so useful for so-called prototrophic strains that are already able to make all the compounds that they need. And some of the non-cerevisiae Saccharomyces strains that are being studied more and more these days happen to be prototrophic.

But in a new GENETICS paper, Alexander and colleagues describe a new toolkit that works on the prototrophic wild and industrial Saccharomyces species and even works efficiently on diploid strains. This sets the stage for faster and more versatile modification of these strains that are becoming useful models for molecular evolution, as well as helping us to make wine, brew beer, and produce chemicals. 

The “standard” toolkit for S. cerevisiae is based on nutritional markers that can be selected. For example, a ura3 mutant strain can’t grow without added uracil in the medium, but when it is transformed with the wild-type URA3 gene it doesn’t need uracil any more.

Importantly, URA3 can also be selected against: ura3 mutants can survive in the presence of 5-fluoroorotic acid (5-FOA), but wild-type URA3 strains cannot. So, starting with a ura3 mutant you can replace any desired sequence with the URA3 gene, selecting for growth in the absence of uracil; then you can tinker with a gene of interest and add the modified version back into the cell to replace URA3, now selecting for 5-FOA resistance. 

Another tool in the classic toolkit is a site-specific nuclease like SceI that can encourage any added fragments to end up in the right spot in the yeast genome. Free DNA ends at a chromosomal break stimulate integration of a transformed fragment if its ends are homologous to the chromosome near the break.

To do this kind of tinkering with prototrophic strains, the researchers needed a marker that could be selected both positively and negatively like URA3. Using a gene that has no equivalent in yeast would be an added plus, since it would be easy to detect and follow. They turned to thymidine kinase (TK), which was lost from the fungal lineage a billion years ago.

TK from Herpes simplex virus had already been expressed in yeast and was known to confer resistance to antifolate drugs. And it had already been shown in other organisms that TK makes cells sensitive to 5-fluorodeoxyuridine (FUdR). The researchers tried it in yeast, and sure enough, only cells that had lost the added TK gene were able to grow in the presence of FUdR.

Alexander and colleagues next created a gene cassette, which they named HERP (Haploid Engineering and Replacement Protocol). The cassette contained the TK gene, a galactose-inducible version of the SCEI gene, and an SceI cleavage site.

As a test case, they decided to replace the S. cerevisiae ADE2 gene with the ADE2 orthologs from seven Saccharomyces species. Transforming with a mixture of seven different sequences and retrieving all seven desired constructs would be a proof of concept showing that this procedure could be used to transform with pools of different sequences and generate a whole library of different strains. And it worked.

They first replaced the ADE2 gene in S. cerevisiae with the HERP cassette, selecting for resistance to antifolates. Next they transformed the HERP-containing strain with a mixture of seven fragments, in the presence of galactose (to turn on SceI and cut the chromosome at that location) and FUdR (to select against cells in which the HERP cassette was not replaced with ADE2). The strategy worked perfectly and efficiently: they got transformants carrying each of the genes, integrated at the correct location.

This work in haploids was all well and good, but most wild type and industrial strains are diploid. And these sorts of strategies tend to work badly in diploids because the cell uses the other gene copy for homologous recombination instead of the DNA fragments scientists put in. To be really useful, HERP would need to work in diploids too.

Alexander and coworkers managed to get HERP to work efficiently in diploids by setting up a situation where both homologous chromosomes had HERP cassettes with SceI recognition sites. Now, when they induced SceI with galactose, both chromosomes of the diploid strain were cut. This dramatically increased the efficiency of transformation: 14 out of 15 strains carried the transformed sequence on both chromosomes instead of the more typical 4% seen with other methods.

So now we have a versatile toolkit that can be used on many different makes and models of yeasts. With a little modification, it could also be applied to many other fungi. And although you can’t use the metric wrench set on your Thunderbird, these HERP cassettes work just as well on S. cerevisiae as on other Saccharomyces species.

The ability to work with prototrophic strains could be a big advantage even in lab strains of S. cerevisiae, since auxotrophic strains sometimes grow slower than wild type even when supplemented with the nutrients they need. So now we have an even larger set of tools to choose from when we want to take that old Thunderbird out for a spin. 

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

Categories: Research Spotlight

Tags: genetic engineering, Saccharomyces cerevisiae, selectable marker

Taking the Pulse of a Yeast Cell

October 30, 2014

Halloween is the time of year when we are all reminded of vampires. And if our favorite yeast Saccharomyces cerevisiae isn’t careful, it might be a vampire’s next target.

Vampires are drawn to living things with pulses so they can suck out their blood. And in a new study in Current Biology, Dalal and colleagues have found a pulse of sorts in yeast cells.

Now of course the yeast aren’t pumping blood. Instead, hordes of proteins are pulsing from place to place within the cell. Dracula might be attracted by these pulses, but would be very disappointed indeed to find that there’s no blood involved…

It’s a pretty recent discovery that some proteins move around in a coordinated way. They come together in one location at the same time, and then later all move to another location in response to changes in environmental conditions, or even under constant conditions.  This phenomenon of “pulsatile dynamics” has been seen in organisms ranging from bacteria to human.

Dalal and colleagues harnessed the awesome power of yeast genomics, in combination with sophisticated imaging equipment, to ask a question that was unthinkable before these resources were available.  How many yeast proteins, out of the entire proteome, show pulsatile dynamics?

This was obviously an ambitious goal. The researchers started with a library of 4159 strains, each containing a different yeast open reading frame fused to the gene for green fluorescent protein.  Rather than following the location of each protein over time in great detail, which would have been a huge amount of work, they devised an ingenious scheme to narrow down the possibilities and focus on potentially pulsing proteins.

In the first phase, they looked at the library at fairly low resolution, following individual cells by taking pictures once an hour over about 10 hours.  This improved their focus right away: most proteins just stayed put, and only 170 showed any hint of pulsing.

In the next phase, Dalal and coworkers looked more closely at those 170 strains, taking a picture every 4 minutes over a 4-hour span.  This eliminated another large group and left them with 64 proteins that still seemed to show pulsatile dynamics.

Another two steps cut the list of candidates way down. Other scientists had shown previously that some yeast proteins show cell cycle-dependent pulsatile movement. Since the researchers were less interested in these, they looked at protein movement during the cell cycle and eliminated 25 proteins whose movement followed it.

They also wondered whether some of the apparent movement they saw was simply due to small shifts in the focal plane during the experiment. To control for this, they examined their candidate proteins in three focal planes, spaced 0.5 um apart.

After all of these steps, Dalal and colleagues were left with just 9 proteins. All of the proteins showed pulsing into the nucleus; seven were known transcription factors, and two were subunits of the RPD3L histone deacetylase complex.

The researchers wondered whether they might have missed any other transcription factors because they hadn’t used the right conditions to see pulsing. So they looked specifically at 122 known transcription factors, testing each under conditions known to induce their activity. This analysis confirmed the previous 9 proteins found, and added just one more.

Interestingly, eight of the ten were members of paralog pairs. In three of these pairs, both members showed pulsing. Looking in detail at the MSN2-MSN4 paralog pair, the researchers found that the pulsing of Msn2p was correlated with that of Msn4p. It isn’t yet clear whether the pulsing phenomenon evolved before the whole-genome duplication that created the paralog pairs, or alternatively whether regulators shared between paralogs later became pulsatile.

Since all of the yeast pulsing proteins are involved in transcriptional regulation, and pulsing brings them into the nucleus where they are active, it’s very likely that the pulsing is an important part of their regulatory role. And since this regulatory mechanism is conserved across species, yeast will provide a great model for studying and understanding it. Dracula might be disappointed, but the rest of us will benefit.

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

Categories: Research Spotlight

If Yeast Can’t Stand the Heat, Our World May Be in Trouble

October 23, 2014

In case there were still any doubters, the world is definitely heating up. April to September 2014 were the warmest these months have ever been since we started keeping records in 1880. And about 35,000 walruses were forced ashore this summer in Alaska because there wasn’t enough room left for them on the sea ice where they normally hang out. Unfortunately, the trend looks to be more record breaking heat for as long as we can see in the future.

This is bad news for us, and even worse news for nature.  But all is not lost! Our hero yeast may be able to swoop in and make us biofuels to replace gasoline.  This won’t stop global warming, but it might at the very least slow it down.

But yeast in its current form probably couldn’t make enough biofuels to make a difference without using too much precious food in the process. Like the X-men, it will need a helpful mutation or two to increase its efficiency to the point where it can make a real dent in slowing down the relentless rise in global temperatures. And ironically, one new trait yeast needs to pull this off is to be able to survive better in the heat.

To efficiently turn the parts of the plants we can’t use (mostly anything to do with cellulose) into biofuels, we need for yeast to grow in cultures where enzymes are chopping cellulose into bite sized pieces at the same time.  These enzymes work best at temperatures that are a real struggle for wild type yeast.

In a new study in Science, Caspeta and coworkers isolated mutants better able to tolerate high temperatures by simply growing Saccharomyces cerevisiae at around 39.5° C for over 300 generations.  They did this in triplicate and ended up with strains that grew on average about 1.9 times faster than wild type yeast at these temperatures.

The next step was to use genome sequencing and whole-genome transcription profiling to figure out why these mutant strains were heat tolerant. This is where things got really interesting.

They found many genes that were affected, but identified ERG3 as the most important player. All of the thermotolerant strains that they selected contained a nonsense mutation in ERG3. And in fact, when they introduced an erg3 nonsense mutation into wild type yeast, they found that this engineered strain grew 86% as well as the original mutant strain at high temperatures. In other words, most of the heat tolerance of these strains came from mutations in the ERG3 gene.

This makes sense, as membrane fluidity is a key factor in dealing with higher temperatures, and ERG3 codes for a C-5 sterol desaturase important for membrane composition.  When Caspeta and coworkers looked at the membranes of the mutant strains, they found a buildup of the “bended” sterol fecosterol.  Since “bended” sterols have been shown to protect the membranes of plants and Archaea from temperature swings, this could be the reason that the mutant strains dealt with the heat so well.

So as we might predict, changing the composition of the cell membrane affects how the yeast respond to temperature. What we couldn’t predict is that in order to get to this point via artificial selection, the yeast had to have a second mutation in either the ATP2 or the ATP3 genes that actually make yeast less able to grow at higher temperatures.

Because ATP2 and ATP3 are needed for yeast to grow on nonfermentable carbon sources, the authors hypothesized that perhaps thermotolerance could not evolve while oxidative respiration worked at full speed. Consistent with this, Caspeta and coworkers found that their evolved thermotolerant strains were more susceptible to oxidative stress and could not grow on nonfermentable carbon sources. This was not true of the strain they engineered to only have the mutation in the ERG3 gene—it grew well at 40° C and could use nonfermentable carbon sources.

So not only have these authors found a mutant yeast with the superpower of growing at higher temperatures, but they also showed that sometimes engineering works better than natural selection at creating the mutant they want. Evolving sometimes requires passing through an intermediate that makes the final product less useful than it could have been. In this case it worked best to use a combination of artificial selection and engineering to build a better mutant.   

Even this super engineered yeast mutant won’t stop global warming in its tracks, but it might help us to slow it down enough so that natural systems have a chance to adapt. A superhero indeed… 

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

Categories: Research Spotlight

Tags: biofuel, evolution, Saccharomyces cerevisiae, thermotolerance

Runaway Polymerases Can Wreak Havoc in Cells

October 16, 2014

A train without working brakes can cause a lot of destruction if it careens off the tracks. And it turns out that a runaway RNA polymerase II (pol II) can cause a lot of damage too.  But it doesn’t cause destruction, so much as disease.

Unlike a train, which has its brakes built right in, pol II has to count on outside factors to stop it in its tracks. And one of these brakes in both humans and yeast is a helicase: Sen1 in yeast and Senataxin, the product of the SETX gene, in humans. 

Mutations in SETX are associated with two devastating neurological diseases: amyotrophic lateral sclerosis type 4 (ALS4) and ataxia oculomotor apraxia type 2 (AOA2), both of which strike children and adolescents.  One idea is that these mutations may short circuit the brakes on pol II, causing it to keep on transcribing after it shouldn’t. And this is just what Chen and colleagues found in a new paper in GENETICS.

The researchers used the simple yet informative yeast model system to look at some of these mutations, and found that they disrupted the helicase function of Sen1 and caused abnormal read-through of some transcriptional terminators.  Looks like bad brakes may indeed have a role in causing these devastating diseases.

Some human proteins can function perfectly well in yeast. Unfortunately, Senataxin isn’t one of those; it could not rescue a sen1 null mutant yeast, so Chen and coworkers couldn’t study Senataxin function directly in yeast. But because Senataxin and Sen1 share significant homology,  they could instead study the yeast protein and make inferences about Senataxin from it.

First, they sliced and diced the SEN1 gene to see which regions were essential to its function. They found that the most important part, needed to keep yeast cells alive, was the helicase domain. But this wasn’t the only key region.

Some flanking residues on either side were also important, but either the N-terminal flanking region or the C-terminal flanking region was sufficient. Looking into those flanking regions more closely, the researchers found that each contained a nuclear localization sequence (NLS) that directed Sen1 into the nucleus. This makes perfect sense of course…the brakes need to go where the train is!  If we don’t put the brakes on the train, it won’t matter how well they work, the train still won’t stop.

These flanking sequences appeared to do more than direct the protein to the nuclear pol II, though.  When the authors tried to use an NLS derived from the SV40 virus instead, they found that it couldn’t completely replace the function of these flanking regions even though it did efficiently direct Sen1 to the nucleus.

Next the researchers set out to study the disease mutations found in patients affected with the neurological disease AOA2.  They re-created the equivalents of 13 AOA2-associated SETX mutations, all within the helicase domain, at the homologous codons of yeast SEN1.

Six of the 13 mutations completely destroyed the function of Sen1; yeast cells could not survive when carrying only the mutant gene. When these mutant proteins were expressed from a plasmid in otherwise wild-type cells, five of them had a dominant negative effect, interfering with transcription termination at a reporter gene. This lends support to the idea that Sen1 is important for transcription termination and that the disease mutations affected this function.

The remaining 7 of the 13 mutant genes could support life as the only copy of SEN1 in yeast. However, 5 of the mutant strains displayed heat-sensitive growth, and 4 of these showed increased transcriptional readthrough.

Taken together, these results show that the helicase domains of Senataxin and Sen1 are extremely important for their function. They also show that Sen1 can be used as a model to discover the effects of individual disease mutations in SETX, as long as those mutations are within regions that are homologous between the two proteins.

It still isn’t clear exactly how helicase activity can put the brakes on that RNA polymerase train, nor why runaway RNA polymerase can have such specific effects on the human nervous system. These questions need more investigation, and the yeast model system is now in place to help with that.

So, although it might not be obvious to the lay person (or politician) that brainless yeast cells could tell us anything about neurological diseases, in fact they can. Yeast may not have brains, but they definitely have RNA polymerase. And once we learn how the brakes work for pol II in yeast cells, we may have a clue how to repair them in humans.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: ALS, helicase, RNA polymerase II, Saccharomyces cerevisiae, transcription

You Can Thank Fruit Flies for Those Yummy Beers

October 14, 2014

It is as simple as this, beer tastes good.  And if a new study in Cell Reports by Christiaens and coworkers pans out, you can thank fruit flies for some of those delicious flavors.

No, fruit flies aren’t in your beer. Instead, they have forced the evolution of our favorite beast, Saccharomyces cerevisiae, down a path towards making the aromatic compounds that make beer so darned tasty.

See, yeast can’t get around on their own and so they often rely on insects to move to new pastures. In order to have this happen, they need to attract insects. Plants have worked this out by evolving colorful flowers and sweet nectar. And one way that yeast may do this is by generating aromas that fruit flies find irresistible.

The researchers in this study first stumbled onto this possibility around fifteen years ago. Back then the P.I. was a graduate student who left his yeast flasks out on the bench over the weekend.  Over that same weekend fruit flies escaped from a neighboring Drosophila lab and invaded the yeast lab.

In a “you got peanut butter on my chocolate” moment, the yeast researchers found the fruit flies swarming around one set of flasks and ignoring some of the others. A quick look at the flasks showed that fruit flies were ignoring the yeast strains in which the ATF1 gene was knocked out.

The ATF1 gene encodes the alcohol acetyltransferase responsible for making most of a yeast’s fruity acetate esters.  So it makes perfect sense that fruit flies ignored strains deleted for ATF1 because they didn’t smell as good anymore. To confirm this hypothesis, the authors did a fun, controlled experiment.

In this experiment, the authors set up a chamber where they could use cameras to track fruit fly movement.  One corner of the chamber had the smells from a wild type yeast strain and another corner had smells from that same strain deleted for ATF1.  As you can see in the video here, the fruit flies cluster in the corner with the wild type strains.  Fruit flies definitely prefer yeast that can make flowery sorts of acetate esters.

Christiaens and coworkers took this one step further by actually looking at the effect these chemicals had on Drosophila neurons.  They used a strain of fruit fly containing a marker for neuronal response, so that the researchers could “see” how the flies were reacting to wild type and atf1 mutant yeast smells.  As expected from the previous experiments, the olfactory sensory neurons responded differently to each smell.

To confirm that the esters were responsible for this difference, the authors observed the effect of adding esters back to media in which the atf1 mutant yeast were growing. They found that as more esters were added, the activity pattern of the Drosophila neurons shifted towards that seen with the wild type yeast.

OK, so fruit flies like good smelling yeast.  The next question the researchers asked was whether this had any effect on the dispersal of the yeast – and it definitely did.

To test this, they labeled wild type and atf1 mutant yeast with two different fluorescent markers, so the strains could be distinguished from each other. They then spotted each strain opposite from one another on a specially designed yeast plate and let a fruit fly roam the plate. They then removed the fly and the original spots of the yeast cells.

After letting the plate incubate for 48 hours, so that any yeast cells that had been moved around on the plate could grow up into colonies, they washed the plate to remove the cells that had been dispersed by the fly and used flow cytometry to determine the amount of each strain. They found that wild type yeast were transported about four times more often than the aft1 mutant yeast.

These results show that fruit flies are more likely to disperse yeast if the yeast are producing fruity smells.  Given the close relationship between fruit flies and yeast, and the fact that insect vectors are very important for yeast out in the wild, it is reasonable to think that yeast may smell good in order to attract fruit flies to carry them to new places.

This research also again points to the importance of expanding studies to include more than one organism (see our last blog here). By increasing the diversity of organisms in an experiment, we can learn much more about how things work in the real world. And maybe even learn why yeast evolved to give us such delicious beer.

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

Categories: Research Spotlight

Tags: beer, Drosophila, Saccharomyces cerevisiae

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

Not too long, not too short

October 02, 2014

Have you ever noticed that the length of your hair is just right for a small window of time? Too short, and you feel a little exposed – too long, and it gets in your face.

Like your hair, the telomeres at the ends of your chromosomes have a length that is just right for them (and you). Telomeres are non-coding sequences of DNA added to the ends of chromosomes that protect the important DNA there from being lost. Telomeres that are too long may contribute to cancer; while telomeres that are too short are associated with aging.

It makes sense, then, that telomere length should be carefully regulated (just as, in a perfect world, you might want to keep your hair the perfect length all the time). The enzyme that adds the non-coding DNA sequences to the end of chromosomes, telomerase, is composed of highly conserved subunits. In yeast, this enzyme is a quaternary complex composed of the regulatory subunits Est1 and Est3, the catalytic subunit Est2, and the RNA template component, TLC1.

Previously, it was thought that telomerase was primed for action whenever it was needed in the cell. But this does not seem to be the case.

In a recent publication in Genes & Development, Tucey et al. showed that, in addition to the active quaternary telomerase complex, two subcomplexes – a preassembly complex and a disassembled complex – were also present in the cell. Each of these subcomplexes lacked one subunit compared with the active telomerase, and the missing subunit was different for each subcomplex.

So, how did the authors discover these subcomplexes? Using a special immunoprecipitation protocol, one of the first things the researchers noticed was that Est1, Est2, and the TLC1 RNA were associated with each other throughout G1 and S phase, but that Est3 was missing from the complex during G1 phase and present only at very low levels throughout S phase. They called this Est1-TLC1-Est2 complex the “preassembly complex.”

In fact, Est3 was only appreciably associated with the preassembly complex during G2/M and late in the cell cycle. And, even then, only 25% of the preassembly complex was associated with Est3 to form the active quaternary complex. So the presence of the active holoenzyme was regulated with respect to the cell cycle.

Since Est3 was not always bound to the preassembly complex, the authors set out to determine what was required for Est3 binding. They found that neither Est1 nor Est2 could bind Est3 if it was unable to bind TLC1 RNA. Thus, the RNA component of the presassembly complex was necessary for its two other subunits to form an active telomerase by binding to Est3.

In addition, they determined that both Est1 and Est2 contained binding surfaces for Est3, and that both of these surfaces were required for the full association of Est3 with the preassembly complex. So Est3 interacts directly with each of the other protein subunits to form the active telomerase.

There was still one more question that intrigued Tucey and coworkers – Est3 appeared to exist in excess compared with the other subunits, so why did it have such limited association with the rest of the telomerase subunits? This suggested some sort of inhibitory regulation of Est3.

Indeed, the authors uncovered an Est3 mutant, Est3-S113Y, which appeared to have lost the ability to be inhibited. This mutant exhibited elongated telomeres and increased association with telomerase, and was associated with the preassembly complex in G1 phase rather than being restricted to late in the cell cycle. This mutation lies directly adjacent to Est3’s binding domain, leading the authors to conclude that Est3 is regulated by a “toggle switch” that specifies whether or not it can bind to the preassembly complex.

As mentioned previously, the authors also saw evidence of a second subcomplex during their studies. When they drilled down on the components, they identified a “disassembly complex” that lacks only Est2, in contrast to the preassembly complex that lacks Est3. They determined that this subcomplex is inactive and requires the prior formation of the quaternary complex since its formation requires Est2 binding to TLC1, just as is observed for the preassembly complex.

Given the cell’s desire to have telomeres that aren’t too long and aren’t too short, it makes sense that the enzyme that lengthens them is regulated. There is a toggle switch that signals formation of the active complex and, once formed, the active complex is transient, dissociating its catalytic subunit to become inactive. This regulation ensures that telomerase is active only late in the cell cycle. Just as you might work to keep your hair just the right length, the cell regulates telomerase to keep the lengths of telomeres from getting out of control.

by Selina Dwight, Ph.D., Senior Biocurator, SGD

Categories: Research Spotlight

Tags: aging, protein complex, telomerase, telomere

Now You Don’t See It, Now You Do!

September 25, 2014

One of the great joys of teaching can be found in the questions that students ask.  Because they are unconstrained by previous knowledge, they can think outside of the box and ask questions that force the teacher to see a problem in a new light.  Their unbiased questions often uncover aspects of a problem that a teacher didn’t think to look for or even consider.

The scientific enterprise can be very similar.  Sometimes an unbiased search of a process will uncover hidden parts scientists were completely unaware of.

This is exactly what happened in a new study in Science by Foresti and coworkers.  Using an unbiased proteomics approach they found a previously hidden part of the endoplasmic reticulum-associated degradation (ERAD) pathway in the inner nuclear membrane (INM) of the yeast Saccharomyces cerevisiae.  No one knew it existed before and, frankly, no one even knew to look!  By thinking outside of the box, these authors found that a novel protein complex in the INM targets certain proteins for degradation – both misfolded proteins, and some correctly folded proteins whose functions are no longer needed. 

Scientists already knew that the ERAD pathway uses different protein complexes to target proteins for degradation, depending on where those proteins are located.  For example, misfolded cytoplasmic proteins are targeted by a complex containing Doa10 (also known as Ssm4), while those in the membrane are targeted by the Hrd1 complex.  However, degradation of both sets of proteins requires ubiquitination by the shared subunit Ubc7. In addition to targeting misfolded proteins, both of these complexes also target certain functional proteins in response to specific conditions.

In the first set of experiments, Foresti and coworkers looked at the proteomes of strains deleted individually for Doa10, Hrd1, or Ubc7.  To their surprise, they found a set of proteins, including Erg11 and Nsg1, that are unaffected by the deletion of either Doa10 or Hrd1, but whose levels are increased in strains deleted for Ubc7.  This suggested there is a branch of the ERAD pathway that involves Ubc7 but is independent of Doa10 and Hrd1.  The authors set out to find this undiscovered third branch lurking somewhere within the yeast.

Some possible candidates for being part of the ERAD pathway were two paralog proteins Asi1 and Asi3,  and their associated protein Asi2.  Based on their sequences, Asi1 and Asi3 are putative ubiquitin-protein ligases like Doa10 and Hrd1.  Interestingly, all three Asi proteins localize to the inner nuclear membrane, which connects to the ER at nuclear pores.

When Foresti and coworkers deleted any one of the three Asi proteins, degradation of Erg 11 and Nsg1, both involved in sterol synthesis, was blocked. However deletion of Asi1, Asi2, or Asi3 didn’t affect all proteins involved in sterol biosynthesis, since Erg1 was unaffected.  Biochemical experiments confirmed that Erg11 binds to a complex composed of these three Asi proteins.

Since the ERAD pathway is important for degradation of misfolded proteins, the authors set out next to determine whether the Asi complex plays a role in this process as well.  That would be a somewhat surprising finding, since misfolded proteins aren’t generally found near the INM. But through a complicated set of experiments summarized below, Foresti and coworkers confirmed that the Asi complex does also have a role in this process.

They first tested several proteins that are known ERAD substrates, but mutations in the ASI genes had no measurable effect on them. Because some misfolded proteins are targeted by more than one ERAD complex, the authors next looked to see whether the Asi pathway contributed to either the Hrd1 or the Doa10 pathways. Testing the accumulation of several substrates in strains with different combinations of asi, hrd1, and doa10 mutations, they found that one mutant protein that misfolds, Sec61-2, had high steady state levels in a hrd1 knockout, but even higher steady state levels in a double knockout of hrd1 and asi1 or hrd1 and asi3.  So both the Asi and Hrd1 pathways appeared to work on this misfolded protein.

The researchers hypothesized that the Asi branch may target misfolded proteins for degradation as they travel through the inner nuclear membrane on the way to the ER.  To test this idea, they compared the steady state levels and localizations of two differently mutated versions of the Sec61 protein – one that localized to the inner nuclear membrane and one that did not, in both wild-type cells and a variety of deletion strains.

The bottom line from these experiments was that the mutant protein that was located at the inner nuclear membrane was more dependent on the Asi complex than the mutant that wasn’t.  Not only that, but the mutant Sec61 protein that was directed to the inner nuclear membrane changed its localization to the nuclear envelope in an asi1 deletion strain.  Both of these results are consistent with a role for the Asi complex in targeting proteins for degradation while they are in the inner nuclear membrane.

The final set of experiments confirmed the importance of the Asi complex in ER protein quality control.  Yeast responds to the presence of too many misfolded proteins in the ER with a signaling pathway called the unfolded protein response (UPR).  Strains in which this pathway is compromised, for instance by deleting IRE1, need a functional ERAD to thrive.  The authors found that deleting HRD1, IRE1, and ASI1 had a much more severe effect on viability than did just deleting HRD1 and IRE1.  This supports the idea that the Asi complex is important in ER protein quality control.

Foresti and coworkers have thus uncovered a previously undiscovered branch of the ERAD pathway in yeast by doing a broad, unbiased proteomics study.  The key proteins they identified, Asi1, Asi2, and Asi3, were originally discovered for their genetic effects on the transcriptional repression of amino acid permeases (hence their name, Amino acid Signaling Independent).  Their detailed biochemical functions were unknown until now.

A lesson here is that just because a process looks like it is pretty well locked down, this doesn’t mean that there aren’t hidden parts yet to be discovered. And just because a gene is implicated in one process, don’t assume it isn’t also involved in other processes as well. Looking from a different angle can allow you to see things you had missed before.

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

Categories: Research Spotlight

Tags: ERAD, inner nuclear membrane, Saccharomyces cerevisiae, ubiquitin-mediated degradation

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