New & Noteworthy

Pathway Between Copper and Disease Revealed in Yeast

July 08, 2022

Several lethal genetic disorders in humans are caused by mutations that cause symptoms of copper (Cu) deficiency, even in the presence of copper. These copper-deficiency disorders are fatal and include Menkes disease, Friedreich’s ataxia, and neurological and cardiac defects in infants due to lack of copper supply to cytochrome c oxidase in mitochondria. Given that no treatments are currently available for these terrible disorders, researchers have been interested in drugs that might improve copper bioavailability. The copper-binding oncological drug elesclomol (ES) has been identified as a candidate.

In a recent report by Garza et al. in the Journal of Biological Chemistry, the authors use the facility of yeast genetics to ask detailed questions about how ES affects metal homeostasis in yeast cells. It was previously established that perturbation in the levels of one metal tends to cause perturbations in supply of other metals, and thus they asked directed questions about both copper and iron (Fe). Deficiency in copper can cause linked deficiencies in bioavailable iron, and both are critically important for metal-dependent enzymes in mitochondria.

The crux of the team’s findings was that supplementing copper-deficient yeast cells with Cu-bound ES (ES-Cu) not only increased Cu levels, but nearly doubled Fe levels in mitochondria. They were able to show that ES transports copper by an alternate route that bypasses the major yeast copper importer (Ctr1p). While this is an intriguing and perhaps encouraging result, perturbations in metals have so much potential to be toxic that it remains critically important to understand the relationships between the components.

The authors found that application of preformed ES-Cu complex is more efficient than ES at transporting Cu, and that transport of Cu across the plasma membrane by the drug occurs by passive transfusion, not active transport. Further, they made the critical discovery that copper delivered by ES goes first to the Golgi lumen, not directly to mitochondria as the authors had expected based on studies in other models. At the Golgi, the Cu is made available to the copper-transporting ATPase Ccc2p, which in turn assists in metalating Fet3p, a multicopper oxidase that oxidizes ferrous (Fe2+) to ferric iron (Fe3+). Once activated with copper in the Golgi, Fet3p-Cu is transported to the plasma membrane, where it oxidizes iron to Fe³⁺, the form that can be taken up by the iron transporter Ftr1p. This increased transport of iron leads to increased bioavailability in mitochondria. Thus, the link between copper and iron by means of an alternative copper transporter becomes more clear.

Interestingly, copper and iron metabolism are linked in both humans and yeast. The yeast protein Fet3p has two homologs in humans (ceruloplasmin and hephaestin), both of which are multicopper oxidase proteins critical for normal iron metabolism. From these powerful studies in yeast, it appears that disorders of Cu metabolism cause defects in Fe metabolism due to disrupted metalation of these cuproenzymes within the Golgi. Indeed, this is an intriguing finding that opens avenues for possible therapies, and it would be hard to imagine making this connection without the use of the yeast model.

Categories: Research Spotlight

Tags: copper, copper transport, cytochrome c oxidase, iron, iron transport, mitochondria, Saccharomyces cerevisiae, yeast model for human disease

Polarity Linked to Lifespan in Mother Yeast Cells

April 01, 2022

In last week’s post, we discussed asymmetry in pH between mother and daughter cells. This week we’re continuing the theme of mother/daughter inheritance patterns with a study of asymmetrical mitochondrial inheritance. The underlying hope is to garner clues about aging and lifespan, since mother cells have reduced lifespan relative to their daughters.

Yeast cells are normally symmetrical, i.e. round. One of the first steps during cell growth is the breaking of symmetry to create poles, where one pole will become the bud tip and the other pole will become the mother cell tip. In a recent paper in iScience, Yang et al. describe how the mother cell tip is normally distal to the bud tip and that higher-functioning mitochondria localize to both poles. The mitochondrial F box protein Mfb1p was shown to be key for tethering mitochondria at the mother cell tip, which prevents every higher-functioning mitochondria from going to the bud. Mfb1p remains associated with the mother cell tip throughout the entire cycle, and is the only protein known to do this.

Interestingly, Mfb1p is itself asymmetrically localized, where it associates with mitochondria in the mother cell but remains excluded from the daughter cell until just before cytokinesis.

To assess the link between aging and polarity, the authors labeled bud scars with dyes that distinguished between new and old scars. In highly polarized cells, new scars will form directly next to one another. They found polarized bud site selection in >97% of young cells, which was quickly reduced to 89% after 6–10 divisions and plateaued at ~70% for the oldest cells. Thus, aging as a factor of polarity was detectable early in lifespan and continued to decline to a fixed level. Likewise, the polarized localization of Mfb1p to mother cell tips also declined with age, where young cells showed Mfb1p tethered to mitochondria almost exclusively at the mother cell tips, but over time the localization dispersed throughout the mother cell. Interestingly, deletion of RSR1/BUD1 (required for polarized bud site selection) disrupted this polarized localization of Mfb1p within the mother, yet didn’t cause Mfb1p to go to the bud (i.e. cause loss of asymmetry). Thus, polarization and asymmetry could be separated in the bud1Δ mutant cells—and these cells also showed reduced lifespan. The same was seen in bud2Δ and bud5Δ cells.

Given that both bud1Δ and mfb1Δ cells had reduced lifespan, the authors asked whether the two mutations affected lifespan in the same way by examining the double mutant for additive effects. Seeing no additive effects, they concluded that BUD1 and MFB1 operate in the same pathway for controlling mitochondrial distribution and the associated effects on aging.

Whereas the study we highlighted last week did not find a link between aging and pH in yeast, Yang et al. show that mitochondrial localization and inheritance patterns have a clear relationship with replicative lifespan. The secrets of aging continue to invite study, and future results should be equally intriguing.  

Categories: Research Spotlight

Tags: mitochondria, mitochondrial inheritance, polarity in yeast, Saccharomyces cerevisiae, yeast inheritance, yeast model for aging

Mixing Mitochondria Makes Magic

May 31, 2018

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In the Harry Potter universe, there are two materials that make up a wand: the wood, which comes from trees like cedar and holly, and the core, which is a magical substance such as the feather of a phoenix or a unicorn hair.

Every wand has unique properties that depend mainly on the combination of its wood and core. Different wood-core pairings give different characteristics that can either antagonize or synergize with the wizard using it. When a wand meets up with its ideal owner, it will begin to learn from and teach its human partner. Such auspicious pairings can continuously improve the wizard’s spell-casting, helping the wizard perform better and better under ever more varied circumstances. However, a poor pairing between a wand and a wizard can be devastating, enfeebling the wizard’s magic or even causing it to backfire.

And just like wizards and wands, it turns out that mitochondrial DNA and nuclear DNA in a cell need to be properly paired to perform the “magic” of running a cell in the most efficient way.

Mitochondria are dynamic structures inside eukaryotic cells that provide much of the energy to keep a cell humming along.

Mitochondria contain their own DNA, encoding genes necessary for the organelle to do its work. Although mitochondrial DNA is physically separate from nuclear DNA, it turns out that the two need to work together if the cell is to make functioning mitochondria.

Like the wand-wizard pairing in Harry Potter’s world, the combination of a specific mitochondrial genome (the wand) with a particular nuclear genome (the wizard) is important for making a healthy mitochondrion. Some mitochondrial-nuclear combinations work well and others not so much, but not a lot is known about where different mitochondrial DNAs come from and how they end up paired with their favored nuclear genomes.

Knowing more about this may help us understand how mitochondrial genomes evolve during interspecific hybridizations, such as in lager beer yeast and certain other fermentation yeasts.

A new study in GENETICS from Wolters et al. shows that when S. cerevisiae yeast cells go through the mating process, there is often mixing of mitochondrial genomes to give new combinations of mitochondrial genes — almost as if lots of new wood-core combinations of wands were being created.

How do these new mitochondrial combinations arise? When two haploid yeast cells mate, they merge to form a single diploid cell that contains mitochondria from both of its parents. Sometimes, these mitochondria exchange pieces of DNA, mixing-and-matching genes in a process known as mitochondrial recombination.

The authors found that a surprising proportion of mated yeast cells (~40%) had recombinant mitochondrial DNA. And in many cases, the recombined mitochondrial genomes work even better with the nuclear genome to make a super healthy cell. Often these optimal pairings allowed the cells to develop new powers, tolerating higher temperatures and more oxygen-stressed conditions than the original parent cells — in other words, the cell has found its optimal “wand”!

But other pairing combinations were inauspicious, giving sickly or dead mitochondria that can harm the cell, especially when it is growing under stressful conditions. For instance, when the authors swapped the mitochondrial-nuclear pairing for two different but very fit cell types, these new pairings gave unhealthy cells, meaning that the original fit cells had already found their perfect “wand”.

So just like when Harry Potter was in Ollivander’s wand shop and finally found his holly-phoenix feather wand and felt unified with its amazing magic, yeast cells can acquire new super powers when their nuclear and mitochondrial genomes are perfectly paired!

by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.

…and we wish a fond farewell to Barry Starr, Ph.D. who has left the Stanford Department of Genetics for new horizons. We miss you Barry and wish you well!

Categories: Research Spotlight

Tags: environmental stress, mitochondria, recombination

The Latest Buzz on Stressed-Out Mitochondria

September 30, 2015

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Stinging wasps get our attention, and with good reason—getting stung hurts a lot! If you see wasps going into a nest within the walls of your house, you’ll likely try to block their access.

But this could backfire: instead of being able to peacefully go into their nest, a swarm of angry wasps could be buzzing around looking for trouble. It might get so bad that you’ll need to call in an exterminator to take care of the problem.

Mitochondrial proteins might seem a lot less scary than wasps, but it turns out that they can also cause trouble if they can’t get into mitochondria. In two new letters to Nature, Wrobel and colleagues and Wang and Chen used complementary approaches to ask what happens when dysfunctional mitochondria aren’t able to import all of the proteins that are waiting to get in. 

What they both found was that these piled up proteins cause real problems for the cells. In desperation, the cells slow down protein synthesis to reduce the excess, and also turn to their own exterminator, the proteasome, to keep these proteins under control.

This is a paradigm shift in thinking about how poorly functioning mitochondria cause disease. In the past, almost everyone focused on how damaged mitochondria couldn’t make enough energy for a cell. Now it looks like there are other ways for a nonworking mitochondria to do a cell in. And new targets for scientists to go after in treating mitochondrial disease.

As we all know, mitochondria are the powerhouses of the cell, where energy is generated, and they’re also the site of many other essential biochemical reactions. They’re composed of about 1,000 proteins, and nearly all of those are synthesized in the cytoplasm and then imported into mitochondria by an intricate system of transporters.

In order to find out what happens when these 1,000 proteins don’t get into mitochondria as efficiently as they should, both groups created strains whose mitochondrial import was impaired.

Wrobel and colleagues used the temperature-sensitive mia40-4int mutation, affecting an essential component of the mitochondrial import system. Wang and Chen started with the aac2-A128P mutation in PET9 (which is also known as AAC2), an ADP/ATP carrier of the mitochondrial inner membrane. Overexpression of the aac2-A128P allele causes mitochondrial dysfunction and eventual cell death.

Wrobel and colleagues decided to get a comprehensive look at what happens in the mia40-4int mutant by assaying its transcriptome and proteome, using RNA-seq and stable isotope labelling by amino acids in cell culture (SILAC), respectively. Surprisingly, one of the biggest differences from wild type that they saw in the import-defective mutant was a decrease in cytoplasmic translation. Whether they looked at the mRNAs encoding ribosomal proteins, the proteins themselves, or the polysome content and translational activity of the cells, everything pointed to down-regulation of translation. And at the same time, the proteasome—the molecular machine that breaks down unwanted proteins—was activated.

To verify that what they were seeing wasn’t peculiar to the mia40-4int mutant, Wrobel and colleagues slowed down mitochondrial import in several other ways: using different mia40 mutant alleles, other import mutants, or treatment with a chemical that destroys the mitochondrial membrane potential required for import. Under these different conditions causing the accumulation of mitochondrial precursor proteins in the cytoplasm, they still saw decreased cytoplasmic translation and increased proteasome activity.

Wang and Chen took a different approach, looking to see whether over-expression of any other genes could compensate for the lethality of overexpressing the aac2-A128P allele. The researchers transformed the mutant with a library of yeast genes on a multicopy plasmid, and found 40 genes whose expression could keep it alive.

The suppressor genes found by Wang and Chen were all involved in some aspect of synthesis or degradation of cytoplasmic proteins, just like the genes found by Wrobel and colleagues whose expression was altered in the mia40 mutant. And Wang and Chen also verified that these suppressors weren’t specific to the aac2-A128P mutation: they suppressed a variety of other mutations that decreased import.

Both groups observed precursors of mitochondrial proteins accumulating in the cytosol of the mutant strains they studied. Wang and Chen saw a couple other very interesting proteins increase in abundance: Gis2 and Nog2. These proteins are involved in regulating ribosome function, and the researchers speculate that their stabilization during this stress response contributes to the translational down-regulation. Intriguingly, their human orthologs are implicated in neuromuscular degenerative disease.

So, using orthogonal approaches, the two groups converged on the same model: a newly discovered cellular pathway that regulates cytosolic translation and protein degradation in order to deal with the stress of inefficient mitochondrial import. Wrobel and colleagues have named it UPRam, for Unfolded Protein Response activated by mistargeting of proteins, while Wang and Chen call it mPOS, mitochondrial Precursor Over-accumulation Stress.

Before this work, it was unknown whether cytosolic pathways were even affected by mitochondrial dysfunction. Now we know that the cell has a specific response when mitochondrial precursor proteins begin swarming in the cytosol, unable to get into their home: it slows down the production of those proteins and calls in the proteasome exterminator to take care of them.

We usually think of mitochondrial disease symptoms as being caused by the reduced energy generation of sick mitochondria, or by the lack of other key events that happen in mitochondria—for example, the synthesis of the iron-sulfur clusters that some vitally important enzymes need. Now, these findings raise the possibility that proteostatic stress on the cell caused by the accumulation of mitochondrial precursors could also lead to impaired cell function and disease.

Perhaps drugs that inhibited cytoplasmic translation, or activated the proteasome exterminator, would be helpful in reducing the buzzing swarm of mitochondrial precursor proteins. Wouldn’t it be wonderful if this knowledge suggested new avenues of treatment to take some of the sting out of human mitochondrial disease?

by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD

Categories: Research Spotlight

Tags: mitochondria, proteasome, proteostatic stress, Saccharomyces cerevisiae

How to Make A Safe and Fun Mitochondrion

January 13, 2015

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Influencing mitochondrial import to treat disease:

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Categories: Research Spotlight, Yeast and Human Disease

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

Mother Yeast Keeps Daughters Tidy

December 18, 2014

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

Categories: Research Spotlight

Tags: mitochondria, protein aggregation, Saccharomyces cerevisiae

Ribosomes Caught in the Act

November 24, 2014

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

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

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

Pinpointing Translation Locations

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

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

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

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

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

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

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

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

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

Osm1p Goes Both Ways

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

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

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

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

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

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

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

Categories: Research Spotlight

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

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

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

Thinking Inside the Box (or Mitochondrion)

March 28, 2013

The mantra in real estate is that location is everything. The same may be true for some cases of bioengineering. You may not get the best yield unless a whole pathway is in the right cellular compartment.

This is what Avalos and coworkers found for synthesizing branched chain alcohols in the yeast Saccharomyces cerevisiae. These scientists were able to increase yield by 260% by putting the whole pathway of enzymes into the mitochondrion. This is way better than anything anyone else has been able to achieve.

This matters because branched alcohols like isobutanol may prove to be better biofuels than ethanol. We can get more energy out of isobutanol than we can out of ethanol…it has more bang for the buck. Good idea in theory, but producing large quantities of isobutanol has not worked too well in practice.

Yeast is just not very good at making these alcohols, and efforts to improve yields have been anything but inspiring so far. Overexpressing the enzymes in the metabolic pathways that generate isobutanol increased yield by only about 10%. Unfortunately, 10% of almost nothing is still pretty close to nothing.

One of the key metabolic pathways involved in generating isobutanol and other branched chain alcohols is split between the mitochondria and the cytoplasm. Normally, the valine biosynthesis pathway converts pyruvate to valine and alpha-ketoisovalerate in the mitochondria; then those two intermediates, after transport to the cytoplasm, are further converted to isobutanol by the Erlich pathway for valine degradation. Avalos and coworkers reasoned that the failure to increase yield might be because of some rate limiting step in getting the intermediates from the mitochondria to the cytoplasm. And it looks like they may have been right.

They compared the effects of overexpressing the pathway enzymes in the cytoplasm and mitochondria and found the mitochondrial approach won hands down. Overexpression in the cytoplasm bumped yield up 10% while overexpression bumped it up 260%. And this increase wasn’t just for isobutanol. Yields of two other energy rich alcohols, isopentanol and 2-methyl-1-butanol, also went up significantly.

Part of the explanation almost certainly has to do with transport of intermediates between the mitochondrion and the cytoplasm, but that may not be the whole story. The mitochondrion might be a useful environment for other reasons too. For example, its smaller volume means an increase in the concentration of reactants, and its higher pH, lower oxygen content, and more reducing redox potential may be better for certain reactions. It also contains many key intermediates like heme, steroids, biotin and so on.

On the way to improving isobutanol yield, these scientists made it easier for others to test whether moving their pathway to the mitochondria can help increase the yield of their favorite metabolite. Avalos and coworkers created a system of plasmids that easily allows researchers to attach the N-terminal mitochondrial localization signal from Cox4p, subunit IV of the yeast cytochrome c oxidase, to genes of their choice. This will make it much simpler to test whether a pathway’s yield is enhanced by moving it into the mitochondrion.

These results show there is more to increasing yield than overexpression or codon optimization. Sometimes scientists need to take a good hard look at their particular pathway and think outside of the box for new ways to optimize yield. Or sometimes they just need to think within the box that is the mitochondrion.

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

Categories: Research Spotlight

Tags: biofuel, mitochondria, Saccharomyces cerevisiae

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