New & Noteworthy

When One Spark Isn’t Enough

August 07, 2013

As anyone who has ever tried to start a fire with flint knows, a single spark is rarely enough.  You need to get a bunch of sparks all working at once to end up with that roaring campfire.  And with the wrong kindling or wood, even lots of powerful sparks just can’t get it done.

A new study in the yeast S. cerevisiae by Lang and coworkers suggests that evolution may be similar.  A single helpful DNA change may not be enough to give an individual yeast that leg up it needs to spread through the population.  Turns out that more often than not it needs something like 5-7 mutations.   And again, even that may not be enough if the rest of its DNA isn’t up to par.  A set of powerful sparks on soggy wood still won’t light a fire.

Lang and coworkers followed 40 different yeast populations for a thousand generations as the yeast adapted to a new environment (rich medium).  They sequenced each population to 100-fold depth at 12 different time points.  Not only did this allow the researchers to watch mutations rise and fall over time, it also let them screen out sequencing errors.  Real mutations will correlate over time, sequencing errors won’t.

The key finding of their research was that mutations that increased in the population over time almost always came in bunches (or cohorts) and that not all the mutations were beneficial.  Neutral mutations invariably hitchhiked along with strongly beneficial ones.

A great example of this involves the ELO1 and GAS1 genes.  These two mutations arose together in a yeast population but when the researchers looked at each individually, only GAS1 was beneficial.  ELO1 appeared to go along for the ride.

Another key point of this study is that mutations do not happen in a vacuum…beneficial mutations only “catch” in the context of a good background.  This is clearly shown in one of the populations they followed. 

In this population, yeast with a mutation in the SPC3 gene began to spread through the population.  After about 300 generations, though, a second yeast with mutations in the WHI2 and ROT2 genes began to outcompete the SPC3 mutant.  If things stayed like this, the SPC3 mutation would disappear from the population even though it was obviously helpful.

What happened instead was that a yeast with the SPC3 mutation developed a useful mutation in the YUR1 gene.  This combination was strong enough for this yeast to stay in the game until one of them developed a third mutation in the WHI2 gene.  This triple threat proved too much for the yeast with the mutations in the WHI2 and ROT2 genes – they were driven to extinction.

No wonder people refer to evolution as a dynamic process!  This example shows just how tumultuous it actually is.  Even helpful mutations like the ones in ROT2 and WHI2 can disappear over time if they happen in a weaker background.  And presumably even potentially harmful mutations can spread if they hitchhike along with a cohort of strongly beneficial mutations.

These results not only shed light on how evolution works, but could also spark other discoveries on how cancer progresses, how bacteria become resistant to antibiotics, and how viruses deal with our immune system, just to name three.  And that could help kindle a brighter future.  

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

Categories: Research Spotlight

Tags: evolution, Saccharomyces cerevisiae

The Baby Bear of Proteins

July 31, 2013

In the story of Goldilocks and the Three Bears, Goldilocks always likes Baby Bear’s stuff best. Baby Bear has the most comfortable bed, the best porridge, and so on.

The reason Goldilocks likes Baby Bear’s things are that they are just right. They are neither too hard nor too soft, too hot nor too cold, too big nor too little.

Turns out that when it comes to certain proteins, the yeast S. cerevisiae is sort of like Goldilocks…it likes to have them at just the right levels. Too much or too little protein can throw things out of whack.

This idea is supported in a new study in GENETICS, where Sasanuma and coworkers find that a key helicase in yeast, Srs2p, needs to be present in just the right amounts for meiotic recombination to go off without a hitch. In particular, they show that this protein affects meiotic recombination by interfering with the assembly of filaments containing another protein, Rad51p.

Meiotic recombination starts off with Spo11p making a double stranded (DS) break in the DNA. This DS DNA is then trimmed back so that there is a 3′ overhang of single stranded DNA which is then coated with replication protein A (RPA), Rad51p, and Dmc1p. The coated single stranded DNA then invades a stretch of homologous DNA and recombination can begin. 

One of the first things Sasanuma and coworkers did was to show that toying with Srs2p levels has a negative effect on meiosis in general. Too little Srs2p brings spore viability down to 36.8% of wild type, and overexpressing it brings spore viability down to 22.4%.  Clearly Srs2p is a bit like Goldilocks…the amount has to be just right.

The authors next set out to determine how overexpressing Srs2p affects meiosis so profoundly. They showed that too much Srs2p delays the start of meiosis, causes chromosomes to end up in the wrong places, and stunts the repair of DS DNA breaks. Basically, extra Srs2p inhibits meiotic recombination.

They next looked at areas on the DNA where both Rad51p AND Dcm1p were bound, and found that too much Srs2p keeps Rad51p but not Dcm1p off the DNA. When either of these proteins binds to DNA, it forms foci that are visible as dots when the proteins are detected with fluorescent antibodies. While a wild type strain had roughly equal numbers of Dcm1p and Rad51p foci, there were four fold fewer Rad51p foci when Srs2p was overexpressed. Clearly Srs2p was keeping Rad51p-DNA complexes from forming. 

Srs2p can act as a translocase, and it can also bind Rad51p. Sasanuma and coworkers asked which of these functions is essential to its ability to disassemble Rad51p filaments on DNA. Using srs2 mutants that were blocked in just one of these functions, they showed that the translocase mutant was completely unable to remove Rad51p from DNA during meiosis. The Rad51p-binding mutant could still cause Rad51p to dissociate from chromosomes, although at a reduced rate compared to wild type. So the translocase activity is essential, while Rad51p binding is not.

Although it was known that in vitro Srs2p can cause Rad51p-DNA filaments to disassemble, this study is the first to establish that it actually happens in vivo during meiosis. The requirement for the translocase activity suggests that Srs2p may actually move along the filaments as it disassembles them. And this work also shows that just like Goldilocks with her bowl of porridge, the cell needs an amount of Srs2p that is not too big, not too little, but just right.

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

Categories: Research Spotlight

Tags: helicase, meiotic recombination, Saccharomyces cerevisiae

Size Isn’t All That Matters

July 23, 2013

Sometimes the pressures and stresses of everyday life can make some people go a little crazy…they lose a few of their marbles.  The same thing can happen to a cell too.  The only difference is that instead of losing their marbles, cells can lose their chromosomes!

There are all sorts of mechanisms in place to make sure that a cell has just the right number of chromosomes.  Still, sometimes all these systems fail and a chromosome is lost.  This can be catastrophic for a cell and, as an important part of cancer, catastrophic for the whole body too.

Given how important having the right number of chromosomes is, it is surprising how little we know about what makes a particular chromosome more likely to fly the coop.  Kumaran and coworkers set out to change this in their new study in PLOS ONE.

First off, they showed that some chromosomes in S. cerevisiae are indeed more likely to be lost than other ones and that there was a surprisingly wide range of stabilities.  For example, chromosomes XIII and XIV were thousands of times more stable than chromosome III.

One key factor in stability was chromosome size—the smaller the chromosome the more likely it was to be lost.  But chromosome III showed that size was not the whole story.  It was five times more likely to be lost than the smallest chromosome.

The authors next set out to determine what about chromosome III made it so flighty.  By creating a hybrid of chromosomes III and IX, they were able to show that there was no single site that made chromosome III so unstable.  They were also able to rule out the idea that HML, HMR, and the MAT locus made chromosome III more likely to be lost.

They next focused on the centromere because it is such an important player in chromosome segregation.  They created a series of plasmids using the centromeres from chromosomes III, IX, XII, XIV, and XV and found that the ones from chromosome III and chromosome XV were around 5-fold less stable than the other chromosomes. While they do not have a good explanation for why chromosome III and XV fared the same in their assay, the result did suggest that at least part of the instability of chromosome III could be explained by its centromere.

As a final experiment, they determined the frequency of chromosome loss in mad2 deletion mutants.  They did this because MAD2 is involved in the spindle checkpoint and so is a key mediator of chromosome stability.  They found that deleting this gene significantly increased the loss of other chromosomes, but chromosome III was 3-6 fold less affected by the loss of MAD2.  It was almost as if the centromere of chromosome III was already somewhat compromised for its interaction with the spindle.

The authors aren’t sure yet how chromosome III got to be so unstable. It could be that random mutations just made its centromere less effective. But another interesting possibility is that it might be under selective pressure. Carrying the mating type loci, chromosome III could be considered to be equivalent to a sex chromosome in larger eukaryotes, and we know that those chromosomes are under different evolutionary constraints from other chromosomes. Maybe S. cerevisiae just can’t take the pressure!

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

Categories: Research Spotlight

Tags: chromosome stability, Saccharomyces cerevisiae

Oxidation: Maybe Not SO Bad After All

July 11, 2013

You don’t have to be a scientist to get the message that oxidation is bad and antioxidants are good. Just go to the vitamin aisle of your local supermarket, or listen to the ads on late-night TV.  You’ll quickly find out that oxidation caused by free radicals is the reason for aging, and antioxidants are the fountain of youth.  Of course you shouldn’t believe everything you hear…

Things just aren’t that clear when you take a good hard look at aging. Yes, oxidation happens, but there actually isn’t solid experimental proof that it causes aging. In mice, this connection has not panned out at all: lowering the ability to sop up oxidants, by knocking out an antioxidant enzyme, does not shorten the mouse’s life.

In a recent eLife paper, joint first authors Brandes and Tienson and their coworkers used our favorite experimental subject, Saccharomyces cerevisiae, to see if oxidation is a cause or just a consequence of aging. They generated a ton of data about oxidation during aging and did not find any evidence for causation.  Instead they came to the surprising conclusion that the trigger for aging may actually be a sudden drop in the levels of the coenzyme NADPH.

The first step, published previously by this group, was to come up with a very sensitive assay for protein oxidation. The amino acid cysteine can act as a sensor for levels of oxidation, as its sulfur-containing thiol group can be oxidized and reduced. Their technique, known as OxICAT, detects the ratio of reduced to oxidized thiol groups on cysteine residues for individual proteins. They can do this for hundreds of proteins at the same time.

In the current study, they looked at the oxidation state of cysteine residues in about 300 different proteins and also measured the levels of several different metabolites related to the redox state of the cell. All of these data were collected over time in aging yeast cells, both under normal conditions and under conditions simulating caloric restriction or starvation.  These last conditions were included because a lower-calorie diet has been shown to slow down aging, in yeast as well as in animals.

Oxidation of proteins definitely did increase over time. But if oxidation were the cause of cell death, you would expect that it would increase steadily and at some maximum point, the cells would die. Surprisingly, that didn’t happen.

Instead, different groups of proteins were oxidized with different kinetics. The most sensitive proteins (about 10% of the set that they studied) were oxidized 48 hours before the cells started to lose viability. This set included some conserved proteins that are important in maintaining oxidation-reduction balance in the cell, such as the thioredoxin reductase Trr1p. 

But it wasn’t only those especially sensitive proteins that were oxidized. In a second wave of oxidation, almost all the remaining proteins (80%) were oxidized at 24 hours before death. And even with so many proteins oxidized the cells were still metabolically active, with ATP levels near normal. So massive oxidation did not equal instant death for these cells.

As predicted, a low-calorie diet slowed down the whole process. The pattern looked a lot like it did in cells on a normal diet, but there was more time between the waves of oxidation and before the end of viability.

The authors also looked at what happened to different metabolites during aging. One key metabolite is the coenzyme NADPH: it donates electrons to the thioredoxin system that helps balance oxidation and reduction. They found that even before any changes in oxidation are detectable, levels of NADPH decrease very suddenly. The authors speculate that this decrease starts the collapse in redox potential that ends in the death of the cell. The oxidation of protein thiols is an effect rather than a cause, and could actually be a way for the cell to sense its redox state and possibly regulate it. NADPH levels have been seen to decrease in aging rats as well, suggesting that this could be a universal part of the aging process.

The results of this study are too voluminous to describe fully here, but they raise a lot of intriguing questions. Some proteins never got oxidized – what protected them? Are NADPH levels really the trigger for aging, and if so, what causes the sudden decrease? Is oxidation of cysteines actually part of a sensory mechanism? And if that’s true, would preventing oxidation really be such a good thing? This may be another good reason to turn off late-night TV.

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

Categories: Research Spotlight

Tags: aging, oxidation, redox, Saccharomyces cerevisiae

Hanging on by a Thread

July 02, 2013

When Nik Wallenda recently made his incredible tightrope walk over a 1500 foot-deep gorge, the attachment of the cable he walked on was critical. If that had failed, it would have been a very unhappy ending for Nik.

Something equally dramatic can happen in a cell.  If  the attachment of spindle microtubules to chromosomes during cell division fails, then the chromosomes don’t end up in the right place. When this happens, the cell can end up dead, or even worse, cancerous.  This is as bad as falling off a tightrope without a net!

In a cell, the chromosome is attached to the spindle with something called the kinetochore. It is like the spike driven into the side of the gorge the tightrope walker is going over. One end is attached to the chromosome (the side of the gorge) and the other is attached to the spindle (the rope that is tied to the spike).

This is where the analogy ends though…a kinetochore is way more complicated than a metal spike. It is a huge, multi-protein complex with lots of specialized parts. The way in which the whole complex assembles still isn’t completely understood.

In a new paper in GENETICS, Akiyoshi and coworkers unraveled a bit of the mystery behind it.  They found that phosphorylation by a highly conserved protein kinase known as Aurora B (Ipl1p in S. cerevisiae) of one kinetochore subunit, Dsn1p, provides some of the glue that holds the structure together.  More specifically, they found that phosphorylated Dsn1p does a better job at keeping inner kinetochore proteins attached to the complex.  It drives the spike deeper into the gorge.

The researchers mutated two residues in Dsn1p that are sites for Ipl1p phosphorylation.  They mutated one or both to alanine, which prevents phosphorylation, or to aspartic acid, which mimics the phosphorylated state.  They found that preventing phosphorylation of these sites loosened the complex and keeping them “phosphorylated” tightened it. 

First, to try to look at what happens when Dsn1p isn’t phosphorylated by Ipl1p, they mutated the two sites to alanine. Either site could be mutated with no apparent effects, but mutating both was lethal. Clearly these sites are doing something!

The researchers got around this lethality issue by mutating a third site in Dsn1p. This site is a target for phosphorylation by a different kinase, Cdk kinase (Cdc28p). The idea is that preventing phosphorylation by Ipl1p makes Dsn1p unstable, but then preventing phosphorylation by Cdc28p can stabilize the mutant protein.

Now that they had a living yeast strain in which Dsn1p wasn’t phosphorylated by Ipl1p, they could look to see what was different about the kinetochore in this mutant. When they pulled down the mutant Dsn1p using antibody and a Flag-tag, it brought down normal levels of outer kinetochore proteins but reduced levels of inner kinetochore proteins. So this suggested that Ipl1p phosphorylation promotes interactions between Dsn1p and inner kinetochore proteins.

Supporting this idea, an ipl1 mutant that phosphorylated Dsn1p to a lower extent showed lower-than-wild-type levels of inner kinetochore proteins associated with Dsn1p.  And, when they looked at a mutant where those Dsn1p residues were changed to aspartic acid, mimicking constant phosphorylation, higher levels of inner kinetochore proteins were pulled down. All of this evidence, and more, points to Ipl1p phosphorylation of Dsn1p as critical for attachment of inner kinetochore proteins to the kinetochore complex.

In yeast there is just one Aurora kinase, and Dsn1p is just one of its substrates. In human cells there are multiple versions of Aurora, and they are implicated in cancer development. Clearly, yeast will be a helpful model in understanding all the details of how Aurora influences kinetochore structure and chromosome segregation. And that will be a much more impressive and useful feat than a tightrope walk!

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: Aurora kinase, kinetochore, Saccharomyces cerevisiae

Saccharomyces cerevisiae, the Party Animal

June 26, 2013

Like a barfly before his liver gives out, the yeast S. cerevisiae can tolerate incredibly high levels of ethanol.  But unlike the town drunk, S. cerevisiae uses this skill to its own advantage.

In the wild, this yeast ferments sugars to flood the local environment with alcohol.  The end result is that it does just fine but any nearby microorganisms are killed.

Humans also take advantage of this unique property of S. cerevisiae.  Not only does it allow us to brew beer and ferment grapes into wine without worrying too much about bacterial contamination, but it also helps us generate ethanol as a biofuel.  What a cool and useful little beast!

Given how important this property is for both yeast and us, it is perhaps surprising how little we know about how S. cerevisiae pulls this off.  A new study out in PLOS Genetics by Pais and coworkers sets out to rectify this situation.

The study yielded a number of interesting findings.  It might seem obvious that an organism that can make a lot of ethanol should able to grow in the high-ethanol environment that it created. However, by looking at these characteristics in 68 different S. cerevisiae strains, the authors found that the ability to produce ethanol was at least partially separate at the genetic level from the ability to thrive in it. Pais and coworkers called the first process “high ethanol accumulation capacity” and the second “tolerance of cell proliferation to high ethanol levels.”

Second, they identified DNA differences in three different genes – ADE1, URA3, and KIN3 – that all work together to give certain strains of yeast their high ethanol accumulation capacity.  The most interesting of these three is KIN3.

Kin3p is a protein kinase that has a role in DNA repair.  Since ethanol is a known mutagen, it may be that the DNA differences in the KIN3 gene make its protein better at DNA repair, rescuing the cell from the DNA damage from high ethanol levels.

The authors found these genes as part of a larger study to identify at the genetic level why some strains of S. cerevisiae did better than others in ethanol.  They focused on two strains, CBS1585 and BY710.  CBS1585 is a sake yeast strain that can tolerate and grow in high levels of ethanol while BY710 is a laboratory strain that doesn’t do well with either (although still better than most any other beast out there in nature!).

They created diploids using these two strains, sporulated them into haploids, and then screened these haploids for their ability to deal with high levels of alcohol.  As would be predicted from their survey of the 68 strains, the haploids could be grouped into three distinct but overlapping pools: 

1)   High ethanol accumulation capacity in the absence of cell proliferation

2)   Cell proliferation at high ethanol levels

3)   Poor tolerance and growth in high ethanol

The authors then used pooled-segregant whole-genome sequence analysis to identify the DNA regions critical to the first two functions.  Basically this is just what it sounds like.  They isolated DNA from the three pools and looked for differences in pools 1 and 2 that weren’t in 3.  (OK that is dangerously simplified, but that is the gist of it.)

This is how they identified ADE1, URA3, and KIN3 as important in high ethanol accumulation capacity.  We will have to wait for them to pinpoint the important genes in the regions they identified for pool 2 to begin to understand why some strains can proliferate in the presence of high alcohol concentrations.

Once they have identified all of the genes that make certain yeast strains so good at dealing with alcohol, we may be able to engineer a yeast that can make more ethanol for less money.  We can make a great little alcohol producer even better!  

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

Categories: Research Spotlight

Tags: biofuel, brewing, ethanol tolerance, Saccharomyces cerevisiae

Flip-Flopping Yeasts

June 20, 2013

We all have certain things we can’t live without. But what’s essential to one person may be completely trivial to another. For example, a teenager who can’t live without his video games is fine without that antique tea set,  while the opposite may be true for grandma. 

In a new article in Genome Biology and Evolution, Ryan and coworkers find that the same holds true for different yeast species.  One gene that is essential in one yeast is dispensable in another.  And furthermore, they tend to be essential or nonessential in sets. Just like grandma’s tea set and the teenager’s games.

It’s been seen in S. cerevisiae that the genes that encode the proteins in a complex tend to be either mostly essential or mostly nonessential.  It is like the teapot and the cups and saucers all being essential to grandma or all being nonessential to the teenager.  This is called modular essentiality.

Ryan and coworkers found that if some protein complex is essential in one yeast, most or all of those genes will be essential in that species.  On the other hand, if that protein complex is dispensable in a different yeast, then most or all of those genes will be nonessential.  The genes encoding the entire complex flip together from essential to nonessential – again, just like the tea set.  It isn’t as if one of the tea cups happens to stay essential when the teenager gets ahold of the set! 

To do this work, Ryan and coworkers started out by looking at S. cerevisiae.  As all of us here at SGD know, this was an excellent choice!  But not just because we work on it…

Because the S. cerevisiae genome sequence has been available for quite a while, we know which genes are essential to life, which genes interact genetically, and which proteins interact physically with each other. We also have a very good list of the protein complexes that exist in yeast and what their subunits are.

Ryan and coworkers used updated data to confirm that modular essentiality exists in S. cerevisiae.  Most of the proteins in an essential complex tend to come from essential genes and most of the proteins in nonessential complexes come from nonessential genes.  There is very little overlap…the tea set does not often contain a video game!

Next the authors asked whether this modular essentiality is found in other species too. At the moment, Schizosaccharomyces pombe, or fission yeast, is the only other eukaryote with complete data on the essentiality of genes. Although it’s also a single-celled yeast, S. pombe is about as far away from S. cerevisiae as you can get and still be a yeast. The two are thought to have diverged as much as 400 million years ago.

Even though it is so different, S. pombe also shows modular essentiality. And using an incomplete set of data from knockout mice, the authors see a similar pattern! So it looks like modular essentiality is at least conserved across fungi, and may be universal.

Next they asked if complexes that have flipped from essential to nonessential over time still maintain their modular essentiality.  Do all the tea cups become nonessential, or just some of them? 

Most (83%) of the genes that are present as one-to-one orthologs in both yeasts are either essential in both or nonessential in both. Ryan and coworkers focused on the other 17%, where a gene was essential in one species but not in the other.

In the cases where essentiality is “flipped” between the species, whole protein complexes tend to flip as a unit. The subunits of a complex that is nonessential in budding yeast are mostly nonessential, while the subunits of the analogous complex in fission yeast are mostly essential.

An example of this is the large subunit of the mitochondrial ribosome. Mitochondrial translation is optional for S. cerevisiae, but obligatory for S. pombe. In keeping with this, almost all the proteins that make up the S. cerevisiae large mitochondrial ribosomal subunit are nonessential. In S. pombe, the situation is flipped.

So the essentiality of a complex mirrors the lifestyle of its owner, just like the teenager and his grandmother.  The two yeasts, with their different lifestyles, place different importance on the mitochondrial ribosome. This wasn’t a big surprise, since this lifestyle difference was already known. But other complexes that are flipped between the species may point to things that we don’t yet know about their physiology.

These results support the idea that modular essentiality is universal, which would mean that in various organisms we can expect that mutants in subunits of a complex will share the same phenotype, disease association, and drug sensitivity. Obviously there are important implications here for antifungal drug design or for disease treatment: if you want to stop a complex from working, any of its subunits (or perhaps several at the same time) might prove to be good targets.

But another bigger point is how much we can learn from a deep understanding of an organism’s genome.  By teasing apart what is essential and what isn’t we can learn a lot about the beast we’re studying.  And someday, maybe a lot about ourselves.

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

Categories: Research Spotlight

Tags: essentiality, evolution, Saccharomyces cerevisiae

Alternative Ways to Increase a Cell’s Shelf Life

June 05, 2013

Like milk or eggs, most cells with linear chromosomes have a shelf life. Each time these cells divide, they lose a little off the end of their chromosomes. Eventually, too much is lost and the cells crap out. Or, to use a more scientific term, they become senescent.

But this is not the fate of every cell. Some cells, like those that go on to become sperm or eggs, use a reverse transcriptase called telomerase to extend their telomeres as part of their normal life cycle. And they aren’t the only ones. Around 85% of cancers hijack the telomerase and use it for their own nefarious ends.

The other 15% of cancers use a variety of different mechanisms to keep their telomeres from getting too short (Cesare and Reddel, 2010). All these different ways are lumped together in a single category called alternative lengthening of telomeres or ALT. The telomeres are lengthened in these cells by recombination with other telomeres, either those on other chromosomes or those that exist as shed, extrachromasomal bits. 

While telomere extension may keep cells alive, it can sometimes be a double-edged sword. A double stranded DNA break is usually recognized as DNA damage. However, if the break happens near a telomere seed (a sequence that looks like a telomere), then the DNA damage response can be suppressed and the end can be extended into a new telomere, in a process called chromosome healing. But now the cell could be in trouble, with new, partial chromosomes being created and getting pulled this way and that.

In a new study out in GENETICS, Lai and Heierhorst decided to investigate whether chromosome healing happens in yeast cells that have stayed alive because of ALT.  What they found was that chromosome healing at telomere seeds was suppressed in these post-senescence survivors.

They created these ALT dependent, post-senescence survivors from an est2 mutant strain that lacked the catalytic subunit of telomerase.  Without telomerase, the only way for these cells to survive is by using ALT. 

In the first experiment, they looked at whether the post-senescence survivors could create a new telomere by chromosome healing.  The authors used a galactose inducible HO endonuclease to create a double stranded break near an 81 base pair sequence known to be a telomere seed sequence in wild type. 

Broken DNA usually signals cells to pause the cell cycle until the damage is repaired. This is known as the DNA damage checkpoint. During chromosome healing in wild type, this checkpoint is suppressed so the chromosome break isn’t recognized as DNA damage.

In the post-senescence survivors, even after 21 hours there was no evidence of a telomere forming.  They didn’t suppress the DNA damage checkpoint either.

Lai and Heierhorst determined that these ALT-dependent cells could still repair a different break that was not near a telomere seed sequence. They just couldn’t repair the break at the telomere seed. And this wasn’t because the DNA damage checkpoint was active. When they prevented the checkpoint by using a rad53 mutant, the telomere still wasn’t repaired.

Instead, the post-senescence survivors eventually repaired the break by some other mechanism, generating lots of differing products in the process. When they repaired breaks at sites that were not telomere seeds, they were able to use homologous recombination. But homologous recombination was suppressed at the telomere seed site.

Since ALT is used in cancer cells, and happens most often in some of the least-curable types of cancer, whatever we can learn about the process in yeast is valuable. It may give us clues on how to change the expiration date of those cancer cells to “ASAP”.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: cancer, DNA damage checkpoint, Saccharomyces cerevisiae, telomere

Proteins Hate Crowds Too

May 29, 2013

A goldfish will swim faster through water than it will through gravy or Jell-O. And according to the results of a new study by Miermont and coworkers, it looks like the same may be true for proteins in a yeast cell.

Now obviously the authors didn’t replace the insides of a yeast cell with gravy or Jell-O. Instead, they used severe osmotic stress to remove water from the cell, making the interior more viscous and the proteins more crowded.

The movement of a wide range of proteins within the cell slowed to a crawl in these shrunken cells. In fact, if the cell was subjected to enough stress, the proteins in the cell essentially stopped moving at all. This lack of movement wasn’t because the high osmotic stress had killed the yeast…they recovered just fine when put back into a more osmotically friendly environment.

The first case the authors looked at was the pathway that helps yeast respond to osmotic stress, the HOG pathway. Once a cell is subjected to osmotic stress, Hog1p is phosphorylated and translocated into the nucleus where it can then turn on the genes needed to respond to this environmental insult.

Miermont and coworkers traced the movement of Hog1p using a Hog1p-GFP fusion protein. At 1 M sorbitol, which subjects the cell to mild osmotic stress, this fusion protein was phosphorylated within two minutes and had reached the nucleus within five.  There were already signs of cell shrinkage even under these mild conditions.

As sorbitol concentrations increased, the phosphorylation and nuclear localization took longer and longer to happen. At 1.8 M sorbitol, Hog1p-GFP didn’t reach maximum fluorescence in the nucleus until 55 minutes. At 2 M sorbitol and above, the cells were shrunken down to their minimal volume, which was 40% of their original size. Under those conditions, only 25% of cells had any nuclear fluorescence even after several hours.

The authors used fluorescence recovery after photobleaching (FRAP) to confirm that these effects were due to slowed protein movement. Basically they zapped a small portion of a cell to burn out the fluorescence of the Hog1p-GFP in that region and then timed how long it took fluorescing Hog1p-GFP from other parts of the cytoplasm to diffuse into that area. To prevent the complication of nuclear translocation in these experiments, they used a pbs2 mutant strain, in which the HOG pathway is blocked and Hog1p stays in the cytoplasm.

They found that fluorescence recovery took less than one second in normal media, about five seconds in 1 M sorbitol, and never happened at 2 M sorbitol. Clearly the protein’s movement was slowed with increasing osmotic pressure. But the effects weren’t permanent: once the cells were put back into normal media, the protein returned to its original kinetics.

The authors expanded their studies to look at proteins that shuttle between the nucleus and cytoplasm in other pathways, including Msn2p, Yap1p, Crz1p, and Mig1p, and obtained similar results. All four proteins took longer to reach the nucleus after being exposed to high osmotic stress. They also looked at other cellular processes where protein movement is critical, such as endocytosis, and again found that increased osmotic stress led to slower moving proteins.

So it looks like molecular crowding can definitely slow down protein movement and affect how a cell functions. For example, the time it takes to respond to environmental stimuli could be significantly slowed, affecting the cell’s chances for survival.  Since yeast cells in the wild encounter high-sugar environments (like rotting grapes), their protein density must be regulated so that they can get through stresses like this. They are ultimately much better adapted than that goldfish in the bowl of Jell-O.

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

Categories: Research Spotlight

Tags: osmotic stress, protein crowding, Saccharomyces cerevisiae

A Radical Discovery About a Well-Known Enzyme

May 22, 2013

Living your life puts a lot of wear and tear on you. A big reason is that as your cells go about their business, they churn out lots of damaging chemicals. 

One of the worst offenders is the free radical superoxide, O₂^–. Cells can’t help producing this powerful oxidant during normal metabolism, but it’s so toxic that it can destroy proteins and damage DNA.

Cells have come up with a two-step process to deal with this toxic waste. In the first step, they use the enzyme superoxide dismutase (Sod1p is the cytosolic form in yeast) to convert superoxide into the less harmful hydrogen peroxide (H₂O₂) and water. The cells then use catalases to take care of the H₂O₂, converting it to water and molecular oxygen. 

We’ve known about the first enzyme, superoxide dismutase, for decades. It has always been thought to have a simple role, sitting in the cytoplasm and detoxifying O₂^–. But new research shows that its job is considerably more interesting than that: it also has a role in a regulatory process known as the Crabtree effect.

The Crabtree effect is named after the scientist who first described it way back in 1929. Some types of cells are able to produce energy by either fermentation or respiration in the presence of oxygen. Since these two processes have different metabolic costs and consequences, which one to use is a critically important choice.

If lots of glucose is around, yeast cells choose fermentation. They prevent respiration by repressing production of the necessary enzymes, and this glucose-dependent repression is the Crabtree effect. It happens not only in yeast, but also in some types of proliferating cancer cells.

A new study by Reddi and Culotta shows that Sod1p is actually a key player in the Crabtree effect. In response to oxygen, glucose, and superoxide levels, it stabilizes two key kinases that are involved in glucose repression.

It was recently found that the sod1 null mutant can’t repress respiration when glucose is around.  This is different from the wild type, which is subject to the Crabtree effect. 

Reddi and Culotta started by investigating this observation and found that SOD1 is part of the glucose repression pathway that also involves the two homologous protein kinases Yck1p and Yck2p. They found that Sod1p binds to Yck1p, which wasn’t totally unexpected since this interaction had been seen before in a large-scale screen. The unexpected part was that Sod1p binding actually stabilizes Yck1p and Yck2p.  These stabilized kinases can now phosphorylate targets that propagate the glucose signal down the pathway and ultimately repress respiration.

Now the question is why does Sod1p binding stabilize the kinases? It turns out that its enzymatic activity is crucial for stabilization. One idea is that the hydrogen peroxide that Sod1p makes in the neighborhood of the kinases could inactivate ubiquitin ligases that would target them for degradation. Ubiquitin ligases are rich in cysteine residues, and so could be especially sensitive to oxidation by H₂O₂.

This regulation might also feed into other pathways: these kinases are also involved in response to amino acid levels, and the sod1 null mutant was seen to affect the amino acid sensing pathway in this study.

Most excitingly, this mechanism is not just a peculiarity of yeast Sod1p. The authors mixed and matched yeast, worm, and mammalian superoxide dismutases and casein kinase gamma (the mammalian equivalent of Yck1p/Yck2p), and found that binding and stabilization works in the same way across all these species.

Superoxide dismutases may have been drafted into this regulatory role during evolution because they are the only molecules that sense superoxide, whose levels reflect both glucose and oxygen conditions. A radical idea indeed!

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

Categories: Research Spotlight

Tags: fermentation, regulation, respiration, Saccharomyces cerevisiae

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