New & Noteworthy

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.

Like the X-men, mutated yeast might one day be the superheroes that save us from the worst effects of global warming. Image by Gage Skidmore via Wikimedia Commons

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: evolution, Saccharomyces cerevisiae, biofuel, thermotolerance

Help SGD by Taking a Brief Survey

October 21, 2014

If you could wave a magic wand and change something about SGD, what would it be? We want to know!

We need your feedback to make SGD even more useful to the biomedical research community. Which features are most important to you and how could they be improved? Which new data, tools, or resources will you need from SGD over the next few years as your research evolves?

We would greatly appreciate your thoughts on how we can serve you better. It will take just a few minutes to take our survey. Click the button below, or access the survey from the button in our website header. Thank you in advance for your time.

Categories: News and Views

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.

Working brakes are important for both large and small machines, including RNA polymerase. Image from Wikimedia Commons

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

SGD Fall 2014 Newsletter

October 15, 2014

SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Fall 2014 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.

Categories: Newsletter

You Can Thank Fruit Flies for Those Yummy Beers

October 14, 2014

A wonderful side effect of the close relationship between yeast and fruit flies is great tasting beer. Image from Wikimedia Commons

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, Saccharomyces cerevisiae, Drosophila

New and improved Locus Summary pages

October 13, 2014

We are pleased to announce that the redesign of our gene-specific pages, which has been ongoing over the past year, is now complete with the release of the reworked Locus Summary page. The page contains all of the information on the previous Locus Summary page, and has a more modern look and feel. Note that the order and organization of the sections has changed, and the order of the tabs across the top of the page has changed as well. New elements on the page include a navigation bar on the left to take you to the different sections of the page, a redesigned map showing genomic context in the sequence section, and a new interactive histogram summarizing expression data. Biochemical pathway information now appears in its own section (see an example), and we have added a History section to replace the previous Locus History tab. If there are no data of a particular type (for example, Pathways), then that section is absent from the page.

Please explore this new page and send us your feedback.

Categories: New Data, Website changes

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.

Yeast and bacteria may not be this cute, but they too can enjoy a mutually beneficial friendship.

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: glucose repression, Saccharomyces cerevisiae, bacteria, prions

Redesigned Expression pages

October 06, 2014

The Expression pages have been redesigned and now include a clickable histogram depicting conditions and datasets in which the gene of interest is up- or down-regulated. Expression data are derived from records contained in the Gene Expression Omnibus, and datasets are assigned one or more categories to facilitate grouping, filtering and browsing. Short descriptions of the focus of each experiment are also provided. The PCL files generated for each dataset are used to populate the expression analysis tool SPELL. Also included on the pages are network diagrams which display genes that share expression profiles. The Expression pages provide seamless access to the SPELL tool at SGD, as well as external resources such as Cyclebase, GermOnline, YMGV and FuncBase.

Please explore these new pages, accessible via the Expression tab on your favorite Locus Summary page, and send us your feedback.

Categories: New Data, Website changes

Not too long, not too short

October 02, 2014

Just like Justin Bieber’s hair, cells make sure telomeres are always the exact right length. Image from Wikimedia Commons

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, telomere, telomerase

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.

Looking at a problem from a different angle can reveal something you couldn’t see before. Note either faces or a vase, a rabbit or a duck! Images from Wikimedia Commons

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, ubiquitin-mediated degradation, Saccharomyces cerevisiae

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