New & Noteworthy

Another possibility has to do with the fact that ψ’s make an RNA more stable. Making certain mRNAs more stable could increase the number of protein molecules they can produce: yet another way to affect gene expression post-transcriptionally. A stability study of mRNA and/or more proteomics might help determine whether this is the function of the unusual modifications.

Whatever the reason, it definitely looks like another bit of biological dogma has been overturned with the help of our faithful and reliable friend yeast. Yes Virginia, mRNA almost certainly has the modified nucleotide ψ. And, as usual, thanks to yeast for teaching us the fundamentals of our own basic biology.

* CMC stands for N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulphonate

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

Categories: Research Spotlight

Tags: pseudouridine, RNA modification, Saccharomyces cerevisiae

Yeast as a Painkiller Factory

September 04, 2014

Imagine you were designing a factory to make a very special product.  You’d study the process carefully, buy the right equipment, and bring in the right people. 

So if one step made a lot of dust, while another step had to be dust-free – you’d be sure to separate them into different rooms of your factory.  And you’d make sure that the instructions were written in a language that your experts could understand! 

In a new paper in Nature Chemical Biology, Thodey and coworkers designed a factory in just this way to make some very important molecules: the opiate drugs that millions of people rely on every day to control pain. Because of this new factory, opium poppies won’t be needed for making these drugs (although they’ll still be very pretty!).  The factory’s location: inside cells of our favorite yeast, S. cerevisiae.

The researchers first tried to coax the yeast to produce the natural opiates morphine and codeine. They recruited experts in the field (or, from the field), taking three opium poppy genes for enzymes in the opiate synthesis pathway: thebaine 6-O-demethylase (T6ODM), codeine O-demethylase (CODM), and codeinone reductase (COR).

Of course, simply transforming yeast with a plant gene doesn’t do much good.  Yeast and poppies don’t speak the same language at the transcription level (and even their translation dialects are hard to understand).  So the researchers put the poppy genes under the control of efficient yeast transcriptional regulatory sequences such as promoters and terminators, and optimized their codons for yeast.

Thodey and colleagues tweaked the system to try to steer it in the direction of the products they wanted. They fed the yeast additional monosodium glutamate and glutamine to increase intracellular levels of 2-oxoglutarate, which is required during catalysis by the T6ODM and CODM enzymes. They also varied the relative expression levels of the three poppy enzymes by varying the copy numbers of their genes in yeast.

Although these tweaks improved things, almost half the product was still the undesirable neomorphine. To address this, the researchers looked even more closely at the details of the pathway.

When morphine synthesis is going right, the neopinone made by T6ODM spontaneously rearranges to the codeinone that COR uses to continue along the pathway.  But if COR grabs the neopinone before there is time for the rearrangement, the end result of the pathway is neomorphine, which does no one any good.

When you design a factory, it’s important that your assembly line doesn’t move too fast! In the yeast factory, when neopinone gets to the COR enzyme too quickly, the end result is not what you want – although maybe not this messy.

Going back to their blueprint, Thodey and colleagues decided to separate T6ODM and COR into different parts of the factory, to allow more time for this rearrangement. They added a tag to COR that would direct it to the endoplasmic reticulum membrane, while T6ODM stayed in the cytoplasm. Now it would take longer for neopinone to reach COR, giving it plenty of time to rearrange into codeinone. Sure enough, morphine production went way up.

This was great, but the researchers decided to take it a step further. Semisynthetic opioids such as hydrocodone, oxycodone, and hydromorphone are medically useful because they work better in some cases than the natural opiates. Currently, these are produced by chemical modification of the opiates produced by poppies. Could yeast do this job too?  Of course!

Turning to different expert workers, Thodey and colleagues tried expressing the enzymes NADP+-dependent morphine dehydrogenase (morA) and NADH-dependent morphinone reductase (morB) from the bacterium Pseudomonas putida* along with the poppy enzymes. Again, the process needed a lot of tweaking, more than we can describe here. But the end result was a strain that produced both hydrocodone and oxycodone.

Putting together all their results, the researchers were able to construct three yeast strains, each like an assembly line tailored for different products. One assembly line is optimized for codeine and morphine, another for hydromorphone, and one for hydrocodone and oxycodone.

The next steps will be to scale up this process to industrial levels, and also to construct yeast strains that carry out the entire process starting from simple sugars, rather than needing to be fed the precursor thebaine. Substituting yeast cultures for opium poppy fields will have a huge global impact that goes far beyond pharmaceutical production.

It’s important to note that this factory could never have been constructed without knowing how to make its fundamental building blocks. Basic research in yeast molecular biology and genetics, which may seem arcane to some, was essential to provide the knowledge necessary to express and manipulate these foreign genes in yeast. Just another reason that we’re “high” on yeast research!

* Read more about Pseudomonas putida, a bacterial workhorse with an appetite for all kinds of weird substances.

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

Categories: Research Spotlight

Tags: opiate biosynthesis, pathway engineering, Saccharomyces cerevisiae

RNase P, Unmasked

August 28, 2014

Masks for a masquerade party come in a dazzling array of shapes and sizes.  And yet they all pretty much serve the same purpose — they hide the identity of the wearer.

Biology sometimes has its own dazzling array of cellular machines all doing the same thing.  One of the best examples of this is RNase P.   This enzyme trims tRNA precursors into mature tRNAs and has pretty much been around in one form or another since there were cells.  And yet, despite this common heritage and its one apparent job, it seems that no two are exactly alike.

In bacteria, RNase P is a piece of RNA that serves as the enzymatic component, complexed with a single protein.  Most Archaea and eukaryotes kept the RNA and added a varying number of protein subunits to make some wildly complex enzymes.  But in a few eukaryotes, the RNA has been dropped completely and a single protein substituted to provide the enzymatic activity. 

A new study out in PLOS Biology by Weber and coworkers shows that, despite this structural diversity, all the different forms of RNase P pretty much do the same thing.  Just like someone can hide who they are with any old mask, a cell can trim its tRNA precursors with any old RNase P.  Well, at least the simple RNase P of Arabidopsis thaliana, comprised of a single enzymatic protein subunit, can replace the enzymatic RNA and at least one protein subunit from the much more complex RNase P of our friend Saccharomyces cerevisiae!

This suggests that evolution has done something weird here.  It took what most likely started out as an RNA enzyme and made various changes to it over time.  Despite these changes, the enzyme kept doing the same thing: trimming tRNA precursors.  It is as if the enzyme went through a bewildering set of evolutionary changes and ended up at nearly the same place doing the same thing.   

How did Weber and coworkers arrive at this startling finding? Yeast RNase P consists of nine protein subunits and an RNA component that comes from the RPR1 gene.  The first thing Weber and coworkers did was to show that the lethal phenotype of a rpr1 knockout could be rescued by the single-subunit RNase P from either the plant Arabidopsis thaliana or the trypanosome Trypanosoma brucei.  The RNase P in these beasts consists of only a single polypeptide.

The authors next integrated the RNase P gene of A. thaliana into the genome of a yeast cell lacking both RPR1 and one of the protein subunits of RNase P, Rpr2p, and put it through a set of rigorous tests.  To their surprise, they found that this strain does a perfectly fine job of processing tRNA precursors.  There was no buildup of intermediates and, if anything, the A. thaliana RNase P proved to be a bit more efficient at trimming these tRNA precursors.

Of course just because the simpler RNase P can substitute for the RNA subunit of the more complex RNase P, that does not mean the two do the exact same thing.  It could be that the more complex form of RNase P has a broader set of functions, but that the only function absolutely required for life is the trimming of tRNA precursors.  But this does not appear to be the case.

Previous research showing that unprocessed forms of other RNAs accumulate at the restrictive temperature in an rpr1-ts mutant had suggested that yeast RNase P also processes a number of other RNAs besides tRNAs.  Since Weber and coworkers didn’t see these unprocessed forms accumulating in their strain, either the simple A. thaliana RNase P was able to process those other RNAs, or they’re actually not RNase P substrates. 

By analyzing the phenotypes of several different RNase P mutants, they showed that the other RNAs aren’t RNase P substrates; apparently their accumulation in the rpr1-ts mutant is an indirect effect.  All in all, these results show that the added complexity of yeast RNase P did not arise so that the enzyme could also process these other RNAs.

The authors next set out to see if there was any subtle difference between the two strains.  In other words, does replacing the RNA component of yeast RNase P with the catalytic protein subunit from A. thaliana have any effect on the yeast whatsoever? 

Weber and coworkers tested this by comparing the growth of the two strains under a wide range of conditions.  They saw no significant effects in any of the over 30 conditions tested.  If the yeast RNase P has any added features over the A. thaliana one, they are very, very subtle.

Pushing to see if they could find any differences, they even set the two up in direct competition to see which was the best suited for survival.  They did this by adding GFP to one or the other strain so that they could follow it, putting the two strains together, and growing them for many generations to see if one routinely outcompeted the other.  Neither did…it was a draw.  There appears to be no advantage to having the yeast RNase P despite its complexity!

This is weird.  It is almost like round trip evolution.  RNase P starts out as a single RNA that processes tRNA precursors.  Then as it moves around the tree of life, it picks up various bells and whistles and occasionally is even replaced by a protein.  And yet in the end, all RNase P’s are strangely equivalent.  As if all of that evolving was for naught!

Obviously there are still plenty of unanswered questions.  Why did yeast build up this complexity if there is seemingly no advantage?  And is the protein subunit superior to the RNA subunit?  If so, this last question would at least explain why a few beasts evolved away from the RNA catalytic subunit to the protein one – but still wouldn’t answer why all those proteins are glomming onto the perfectly adequate RNA that probably predates proteins.  More studies in yeast may help us “unmask” the answer to this fundamental question.

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

Categories: Research Spotlight

Tags: evolution, RNase P, Saccharomyces cerevisiae, tRNA processing

Special Delivery for Cytotoxic Proteins

August 21, 2014

Say you want to send a letter to your friend on the other side of the country. First off you’ll need to put the right address and postage on the envelope. Then you’ll need the U.S. Postal Service (USPS) to take your letter and deliver it to the right person. The stamp tells the USPS to deliver the letter, and the address indicates where it should be delivered (unimpeded by snow nor rain nor heat nor gloom of night, of course!).

It turns out something similar happens in human cells with aggregated proteins. Aggregated proteins are “stamped” by attachment of the small protein ubiquitin and “addressed” to the Atg8 protein. Atg8p triggers the aggregated proteins’ incorporation into autophagosomes for eventual degradation in the lysosome.

And just as it can be devastating if your mail doesn’t get to where it needs to go, so too can it be devastating for these aggregates to accumulate instead of being properly delivered. A buildup of these aggregates is a big factor in Alzheimer’s and Huntington’s diseases.

Enter the cellular USPS. Just as is the case for a prepared letter, the human cell has a service that delivers the ubiquinated proteins to the autophagosome, in the form of the protein adaptor p62 (SQSTM1) and its relative, NBR1.

These adaptor proteins can act as a postal service because they recognize both the aggregated proteins’ stamp (ubiquitin) and their addressee (Atg8p). Specifically, they each possess an ubiquitin-conjugate binding domain (UBA) and an Atg8-interacting motif (AIM). The protein p62 in particular has been shown to associate with protein aggregates linked to neurodegenerative diseases like Huntington’s disease.

In a new paper published in Cell, Lu et al. asked whether there is a link between the ubiquitin and autophagy systems in yeast. If so, yeast might provide some clues about diseases like Huntington’s. Proteins stamped with ubiquitin are known to be addressed to the proteasome for degradation in yeast, but no link between ubiquitination and autophagy had previously been seen, even though many central components of autophagy were actually first described in yeast.

Indeed, the authors showed that cells specifically deficient in the autophagy pathway (atg8∆, atg1∆, or atg7∆), accumulated ubiquitin conjugates under autophagy-inducing conditions. This suggests that the ubiquitin and autophagy pathways are connected in yeast, as is the case for humans.

Next, the researchers looked to see if there is an adaptor in yeast analogous to p62 in humans. When they pulled down proteins that bind yeast Atg8p under starvation conditions, they found ubiquitin conjugates and, using mass spectrometry, further identified peptides from a few other proteins – one of which was Cue5p.

Could Cue5p, like p62 in humans, be the postal service that recognizes both stamped ubiquitin conjugates and the addressee Atg8p in yeast? Strikingly, Cue5p had both a CUE domain that binds ubiquitin and an Atg8p-interacting motif (AIM). The authors confirmed in vivo that Cue5p binds ubiquitin conjugates and Atg8p using these domains, particularly under starvation conditions. They also showed that it acts specifically at the stage of ubiquitin-conjugate recognition and on aggregated proteins, without affecting the process of autophagy itself.

Given that Cue5p functions similarly to p62 and p62 is known to associate with protein aggregates involved in neurodegenerative disease, Lu et al. were quick to look for Cue5p substrates. Analyzing ubiquitin-conjugated proteins that accumulated in cue5 mutant cells, they identified 24 different proteins. Although these 24 Cue5p substrates had diverse functions, the common thread was that many had a tendency to aggregate under certain conditions such as high temperature.

Could Cue5p then actually facilitate removal of cytotoxic protein aggregates in neurodegenerative diseases? Indeed, the authors showed that CUE5 helped clear cytotoxic variants of the human huntingtin protein (Htt-96Q) when it was expressed in yeast, and that Htt-96Q is ubiquitinated in yeast.

These experiments started with an observation in human cells that prompted discovery of an analogous system and adaptor protein in yeast. Now the authors turned the tables and used yeast to look for new adaptor proteins in human cells. Using bioinformatics, they identified a human CUE-domain protein, Tollip, which, although different in its domain organization from Cue5p, contains 2 AIM motifs.

To make a long story (and a lot of work!) short, they showed that Tollip binds both human Atg8p and ubiquitin conjugates and clears cytotoxic variants of huntingtin in human cells. Expressed in yeast, it similarly binds ubiquitin conjugates and Atg8p and suppresses the hypersensitivity of cue5∆ cells to the variant huntingtin protein Htt-96Q. So Tollip is a newly defined adaptor protein and functional homolog of Cue5p!

Letter carriers of one sort or another have been around for as long as human civilization has existed, from homing pigeons to FedEx. Now we know that for even longer, cells from yeast to human have been using similar ways to recognize stamped proteins and deliver them to the right address. And once again, yeast has helped us understand the inner secrets of human cells.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: autophagy, cytotoxic proteins, Saccharomyces cerevisiae, ubiquitin-mediated degradation, yeast model for human disease

Pinpointing Peroxisomes

August 14, 2014

One way to think about the cell is that organelles float around in it like those globs in a lava lamp.  This is obviously a simplification, but it’s also true that organelles aren’t locked into place.  As usual, the real picture lies somewhere in between these two extremes.

What we know about the architecture of the cell has mostly been discovered using classical cell biology and genetic techniques. But in a paper published in Molecular BioSystems, Cohen et al. uncovered some very interesting small details using a very large-scale approach.

The authors were interested in peroxisomes, where a lot of critical metabolic reactions happen (or fail to happen, in several human diseases). The researchers were able to see that peroxisomes not only interact with other organelles, but they contact the endoplasmic reticulum (ER) and mitochondria in a way that could be extremely important for cellular metabolism. And surprisingly, it was by combining a variety of different high-throughput techniques that Cohen and colleagues could uncover this fine structure.

The first step was to set up two reporter constructs to look for genes involved in two different peroxisomal processes.

One reporter was a red fluorescent protein, mCherry, modified to carry a peroxisomal targeting signal and show whether import into peroxisomes was normal. Another reporter, a peroxisomal membrane protein (Ant1p) tagged with green fluorescent protein (GFP), would show whether peroxisomal membranes were normal.

The reporters were crossed into mutant collections, creating one strain for each gene in the genome that had either a complete deletion (for nonessential genes) or a knock-down allele (for essential genes), plus both reporters. Now the researchers could systematically test for genes that, when mutated, affected one or both of these aspects of peroxisomal biogenesis.

To visualize the mutant phenotypes, they used a sophisticated technique termed “high-content screening.” This is an automated way to analyze micrographs that both pinpoints the intracellular location of a fluorescent reporter and measures its quantity. Screening the mutant collection in this way showed that 56 strains had altered distribution of the two different reporter proteins.  Some had a reduction in peroxisomal protein import (mCherry fluorescence), while some had fewer or no peroxisomes and some had peroxisomes that were smaller than normal (GFP fluorescence).

One result that caught the researchers’ eyes was that one of the strains with smaller peroxisomes had a mutation in the MDM10 gene. Mdm10p is part of the ERMES (ER-Mitochondria Encounter Structure) complex that tethers mitochondria to the ER, and this wasn’t previously known to have any connection with peroxisomes. Strains that were mutant in other ERMES subunits had the same phenotype, confirming that the complex has something to do with peroxisome structure.  Other results from the screens added weight to the idea of a three-way connection between peroxisomes, the ER, and mitochondria, and the authors went on to show that peroxisomes often sit at the ERMES complex where mitochondria contact the ER.

Next, to test whether mitochondria might have specific subdomains where peroxisomes interact, the authors used yet another large-scale screen. In the C-terminal GFP fusion library, where each yeast open reading frame is C-terminally tagged with GFP, 96 strains showed a punctate pattern of the fluorescent signal – meaning that the protein was concentrated in spots, rather than evenly distributed.  They labeled the mitochondria with a red fluorescent marker protein in these strains and, again using the high-content screening system, identified protein spots that co-localized with mitochondria. The most intense hit was for Pda1p, a subunit of the mitochondrial enzyme pyruvate dehydrogenase (PDH), and a similar result was obtained for another PDH subunit. So PDH isn’t distributed uniformly in the mitochondrion, but is instead concentrated in clusters.

Looking more closely using the various reporter constructs in their collections, the authors found that peroxisomes and the ERMES complex most often co-localized with those mitochondrial globs of PDH. It would make metabolic sense for peroxisomes to hang out near PDH on mitochondria because this could increase the local concentration of metabolites that they both use.

Intriguingly, Cohen et al. also found that mitochondria and peroxisomes co-localized in mammalian cells. Given that many diseases are linked to peroxisomal metabolism, this is an important avenue to investigate.

So while organelles don’t float around in the cell quite as fluidly as the globs in a lava lamp, the data generated from large-scale approaches boiled down to learning some very fine-grained detail about cellular architecture. We think that’s, like, groovy.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: endoplasmic reticulum, mitochondria, peroxisomes, Saccharomyces cerevisiae

Shmoos Lost in Translation

August 07, 2014

To mate, the yeast Saccharomyces cerevisiae needs to shmoo — to generate a projection that reaches out to a nearby yeast of the opposite sex, until the yeast cell is shaped like the Al Capp cartoon character.  And to shmoo yeast needs, among other things, polyamines like spermidine.

Spermidine is important for one of the most interesting proteins in the world, the translation initiation and elongation factor eIF5A.  Not only is this protein pretty much conserved in just about every living thing, but it is also the only protein to have the unique amino acid hypusine.  And to make things even more fascinating, there are two other conserved proteins whose only job is to convert a single lysine residue of eIF5A into hypusine, using polyamines like spermidine.  Simply mind boggling.

In a new study in GENETICS, Li and coworkers provide compelling evidence that spermidine is important in yeast shmooing because of its involvement in the hypusinylation of eIF5A.   They also found that one reason eIF5A is so important in this process is that it is necessary for translating Bni1p, a formin needed to organize the actin cables of the shmoo.  Without these actin cables, the shmoo can’t form.

It looks like yeast needs eIF5A to translate Bni1p because of the long stretches of prolines found in this protein.  This suggests that like its bacterial ortholog EF-P, a key job for eIF5A is to help the cell deal with polyproline stretches in proteins.

To show this the researchers made a set of targeted mutations to check whether hypusinylation of eIF5A is necessary for shmooing.  When they knocked out LIA1, one of the enzymes that uses spermidine to convert lysine to hypusine, the resulting yeast failed to shmoo.  Since the only known target of the Lia1 protein is eIF5A, this suggests that hypusinylation of eIF5A is critical to its function in shmooing.

They also used temperature sensitive mutants of eIF5A to show that this gene (HYP2, also known as TIF51A) is involved in shmooing.  At the nonpermissive temperature, only 7.7% of yeast with the less severe mutant allele, tif51A-1, shmooed, while none of the yeast with the more severe mutation, tif51A-3, were able to shmoo.  These two results taken together establish the importance of eIF5A in shmooing.

Because eIF5A was known to be important for translating polyproline regions, the researchers looked for yeast proteins with such stretches, with the idea that their failure to be translated may be behind the need for eIF5A in shmooing.  They found 549 such proteins, and a comparison of their Gene Ontology (GO) annotations showed four overrepresented categories including “mating projection” (shmoo).  They focused on a protein from this group, Bni1p, because it was known to be involved in shmoo formation and it was one of only two proteins with ten or more prolines in a row. 

Bni1p is important for organizing the actin cables that are needed to make a shmoo.  Li and coworkers showed that the temperature sensitive mutants of eIF5A and bni1 mutants had similar phenotypes in terms of actin organization in the shmoo.

So the idea here is that yeast need eIF5A to shmoo because they need eIF5A to translate Bni1p, and Bni1p is needed to set up the actin framework of the shmoo.  In this hypothesis, it is the indirect action of eIF5A that prevents the shmooing. To test this hypothesis, the authors generated a bni1 mutant that lacked the polyproline regions. 

They compared the transcript levels of wild type BNI1 and the mutant lacking the polyproline stretches using RT-qPCR and found that the presence of eIF5A didn’t matter much.  The transcript levels of the mutant and wild type BNI1 were pretty much the same.

It was a different story for the protein levels.  Using Western blots Li and coworkers saw very little wild type Bni1p, but lots of the mutant protein.  The yeast cells struggled to translate wild type Bni1p but had no trouble with the mutant.  The easiest explanation is that eIF5A is needed to help the yeast translate polyproline regions of proteins, including Bni1p. 

Finally, to confirm the eIF5A and Bni1p connection, they showed that additional Bni1p could partially overcome the shmoo defect of the temperature sensitive mutants of eIF5A.  Since this suppression was only partial, and since the mutant phenotype of the eIF5A mutant is more severe than that of the bni1 mutant, there are probably other proteins involved in shmooing that require eIF5A for translation. Some likely candidates are those proteins containing polyproline stretches that are annotated to the GO term “mating projection”.

Although a connection between oddly-shaped yeast cells and human fertility and/or disease may not seem obvious, there might indeed be one. It turns out that eIF5A is so highly conserved  that human eIF5A works just fine when expressed in yeast, and mammalian formins, like Bni1p, are also proline-rich. Formins are necessary for polarized growth, which is a feature of both reproductive cell and cancer cell growth, and spermidine is required for fertilization.

Hard to believe that yeast channeling a cartoon character can teach us so much about the most fascinating of proteins, eIF5A.  And maybe even shed light on our own fertility. 

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

Categories: Research Spotlight

Tags: eIF5A, hypusine, Saccharomyces cerevisiae, translation

Time Flies Like an Arrow, Fruit Flies Like a Grande Yeast

July 31, 2014

Here at SGD we tend to have a totally positive opinion of yeast.  As we have said before, they give us bread, booze, a great model organism, and even our livelihoods.  But in truth, Saccharomyces cerevisiae has a few minor faults.

For example, you can thank yeast for all those irritating fruit flies buzzing around your brown bananas.  Fruit flies aren’t attracted to the rotting fruit itself.  They are instead attracted to chemicals the yeast cells are pumping out as they nosh on that old banana.

In a new study, Schiabor and coworkers set out to identify the genetic differences that make some yeast strains more attractive to fruit flies as compared to other strains.  They found that the flies can actually tell the difference between “petite” yeast, with defective mitochondria, and “grande” yeast whose mitochondria are normal.  The mitochondria play a huge role in determining which volatile chemicals a yeast will release, and so determine which yeast are the most attractive to a fruit fly.  But the mitochondria are probably not involved in the way that you might be thinking…

In the first experiment, the authors tested a bunch of different yeast strains to find the ones that fruit flies prefer. As expected, they found a wide range of yeast attractiveness.  They decided to focus on BY4741 as the more appealing strain and BY4742 as the less appealing one.

Schiabor and coworkers chose these two strains both because they are isogenic and because they are the strains from which the systematic yeast deletion collection was made.  These two attributes mean that it should be relatively easy to track down the genetic difference in each strain’s attractiveness to fruit flies.

The first obvious candidate was the different auxotrophies in each strain. Although the strains are isogenic overall, they have a few small differences: BY4741 is a met17 mutant and is mating type a, while BY4742 is a leu2 mutant and is mating type α. Since amino acids are very important in creating various volatile chemicals, the mutations in the amino acid biosynthetic genes seemed a likely cause of the difference in the way the two strains smelled to fruit flies. However, the authors found that none of the auxotrophic mutations mattered.  When they mated the two strains and did tetrad analysis to obtain every possible genetic combination, they found that each of the eight new strains was preferred over BY4742.

Given the non-autosomal inheritance of attractiveness, an obvious candidate was the mitochondria. This hunch was confirmed in a couple of ways.  First, Schiabor and coworkers showed that every strain except BY4742 grew well on glycerol, and second, they found that an isolate of BY4742 with functional mitochondria, BY4742g, was as attractive to fruit flies as BY4741.  Apparently their stock of BY4742 had lost mitochondrial function (which can happen fairly easily for some strains), and clearly the mitochondria matter here!

Through a series of experiments we don’t have the space to describe here, the authors found that the lack of attractiveness was not due to an inability to respire.  Instead, by growing each strain on different nitrogen sources, they were able to provide evidence that mitochondrial functions like proline catabolism and/or branched amino acid anabolism were more likely to be involved.  It can sometimes be hard to remember that the mitochondrion is more than the powerhouse of the cell we all learned about in high school: a lot of very important metabolic reactions other than respiration happen within the mitochondrial compartment.

The authors think that yeast with good working mitochondria are the most useful to fruit flies, which is why fruit flies have evolved to be attracted to those yeast.  This all makes sense, as yeast and fruit flies have a mutually beneficial relationship.  Yeast serve as food for fruit fly larvae, and the ethanol they produce also protects those same fruit fly larvae from predators.  Fruit flies can open up parts of the fruit the yeast can’t get to and help move the yeast to different places.   

The bottom line is that you can blame yeast mitochondria for that swarm of fruit flies hovering over your fruit bowl.  One day maybe we can come up with a way that our fruit will only allow petite yeast to grow.  Then we’ll have a bit of time to enjoy fruit that isn’t attractive to fruit flies.  Until, of course, the flies evolve to prefer petite yeast…

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

Categories: Research Spotlight

Tags: Drosophila, mitochondria, Saccharomyces cerevisiae

Esa1p, the Balancing Artist

July 15, 2014

In the art of rock balancing, the artist positions large rocks with exquisite precision. If he or she succeeds, the rocks counterbalance each other and stay in seemingly impossible positions to make a surprising and beautiful sculpture. But a little uneven pressure is enough to make the whole thing collapse.

It turns out that the cellular acetylation state is just as precisely balanced. In a new GENETICS paper, Torres-Machorro and Pillus identify Esa1p, an acetyltransferase, as the balancing artist in Saccharomyces cerevisiae cells.

Acetylation is an important type of protein modification. Histones, the proteins that interact with DNA to provide structure to chromosomes, are acetylated by histone acetyltransferases (HATs) and deacetylated by histone deacetylases (HDACs). Some HATs and HDACs also act on non-histone proteins.

The acetylation state in a cell is a dynamic process.  All those HATs are adding acetyl groups at the same time that HDACs are removing them.  The final level of acetylation depends on the activities of each of these classes of proteins.

Acetylation of histones has been associated with increases in gene expression and deacetylation with decreases.  So to keep gene expression levels in balance, it is very important to keep acetylation balanced as well.  Throwing acetylation patterns just a bit out of whack can have profound consequences on global gene expression that can ultimately lead to cell death. 

The authors focused on one particular HAT, Esa1p, that acetylates histones H4 and H2A and also has non-histone targets. They were intrigued by the fact that yeast cells cannot survive without Esa1p, since no other HAT or HDAC subunit is essential in yeast.

An obvious explanation for lethality is that losing this protein leads to too low a level of acetylation.  They reasoned that if they also knocked out an HDAC, then the overall acetylation levels might increase and so rescue the esa1 null mutant.  And they were right.

Using a plasmid-shuffling method, they created various double mutant strains of esa1 and HDAC genes, and found that a strain that was mutant in esa1 and also in either the SDS3 or DEP1 genes was viable. SDS3 and DEP1 both encode subunits of the Rpd3L HDAC complex.

Torres-Machorro and Pillus next characterized the esa1 sds3 double mutant further.  They found that although the sds3 mutation suppressed the inviability of the esa1 mutant, it did not suppress other phenotypes such as sensitivity to high temperature and DNA damaging agents.

The authors found that the sds3 mutation subtly increased histone H4 acetylation, which was low in the absence of Esa1p.  However, acetylation levels of a different histone, H3, remained high even in the absence of Esa1p. This suggested that the fundamental problem in the esa1 null mutant was an imbalance in the global state of histone acetylation.

To test this hypothesis, the researchers used a variety of different genetic methods to tweak the balance of cellular acetylation in the esa1 sds3 mutant. They created mutations in histones H3 and H4 that made it seem as if acetylation was low or high, and they also mutated other genes for HDAC subunits. It is as if they were passers-by who decided to poke at a balanced rock sculpture to see what it took to bring the whole thing down.

Although the details are too numerous to report here, the results showed that by using these genetic methods to tweak the overall acetylation state of the cell, the fitness of the esa1 sds3 strain could be improved: phenotypes such as slow growth, sensitivity to high temperature or DNA damaging agents, or cell cycle defects were suppressed to some extent by the various manipulations.  This lends support to the hypothesis that Esa1p is the master balancer of acetylation levels in the cell and that this is its essential function.

This balancing act may happen in human cells too. Esa1p has a human ortholog, TIP60, that has been implicated in cancer and other diseases. Like Esa1p, TIP60 is essential and is involved in the DNA damage response.

So yeast teaches us that the acetylation of proteins is balanced on a knife’s edge.  Even the slightest changes can lead to a collapse in global gene regulation, which can have catastrophic effects like cancer. All that we learn about Esa1p, the acetylation balancing artist, may have much broader implications for human health.

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

Categories: Research Spotlight, Yeast and Human Disease

Tags: histone acetylation, Saccharomyces cerevisiae, yeast model for human disease

Adding Introns to Synthetic Biology’s Toolbox

July 03, 2014

As any good handyman knows, the more tools you have in your tool chest, the better the chance that you can find what you need to solve a problem.  The same goes for synthetic biologists.  The more parts they can mix and match, the more likely they are to engineer the exact level of gene expression they need.

In the last few years synthetic biologists have amassed a wide variety of transcription and translation elements that can be combined in different ways to exquisitely tune the level of expression of their gene of interest.  And now, in a new study out in PLOS Genetics, Yofe and coworkers have added introns to the list of parts available for our favorite yeast Saccharomyces cerevisiae.

Yeast isn’t loaded with introns, but it does have a reasonable number that can be co-opted for synthetic biology.  The authors inserted 240 of these introns individually into the same position near the 5’ end of the yellow fluorescent protein (YFP) gene and monitored the level of fluorescence of each individual strain over a 24 hour period.  They chose the 5’ end of the gene because yeast has a bias for introns being located there.

The authors found that these reporters spanned a 100-fold range of gene expression, that every intron caused a decrease in the level of gene expression, and that even though many of these introns respond to environmental stimuli in their natural context, their effect on gene expression here was immune to the environmental changes the authors tested.  Taken together, these results suggest that introns could be used in yeast systems for dampening over-exuberant gene expression in ways that are independent of growth conditions.  If all of this holds up, introns will prove to be very useful tools indeed.

Yofe and coworkers next wanted to use this library to figure out some of the rules for why some introns cause lowered activity compared to others.  The simplest possibility, that longer introns cause a larger decrease in gene expression, turned out not to be true.  There was no correlation between the size of the intron and its effect on the level of fluorescence. 

Next they scanned the sequences of their constructs to look for elements that might increase or decrease splicing efficiency.  These splicing regulatory elements (SREs) are better understood in larger eukaryotes, but there is evidence that they are important in yeast as well.  The authors identified a number of intron splicing enhancers (ISEs) and intron splicing silencers (ISSs) that were highly enriched near the splice sites. 

To confirm that these sequences did in fact affect splicing efficiency (and hence gene expression), they showed that mutating the enhancer motif TTTATGCT to the silencer motif TTTGTGTA in two reporters resulted in a 22% and a 13% decrease in gene expression.  This proof of principle experiment suggests that future synthetic biologists may be able to further tweak the expression of their genes by manipulating these SREs.

In a final set of experiments the authors used the library to identify rules that can be used to predict how inserting various introns into different positions will affect a gene’s activity.  They found that the most important features were the presence of SREs and the RNA structures at the intron-exon junction.  Synthetic biologists should be able to use these rules to intelligently design their reporter systems.

These experiments are the first step towards adding introns to the ever growing set of tools available to synthetic biologists for modulating gene expression.  We are getting closer to figuring out how genes are controlled and being able to use that knowledge to our advantage.  Or to put it another way, we have taken another baby step towards being able to control a gene as well as a yeast cell does.

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

Categories: Research Spotlight

Tags: Saccharomyces cerevisiae, splicing, synthetic biology

Next