March 23, 2016
Diagnosing why something has gone wrong in a complicated system can be difficult. There are so many bells and whistles that you can easily get lost.
That’s why it can sometimes help to turn to a simple system and then apply what you have learned to the more complicated one. This will, of course, sound familiar to any scientists studying that marvel of a model organism, Saccharomyces cerevisiae.
For example, it is amazing what you can glean from this yeast about human brain and blood diseases. Even though, of course, baker’s yeast has neither blood nor a brain!
This becomes very clear in a study out in PLOS Genetics by Fernandez-Murray and coworkers. In this study they use yeast to help figure out why mutations in the SLC25A38 gene in people leads to something called congenital sideroblastic anemia. And even better, their work hints at a possible treatment.
People with sideroblastic anemia make too little hemoglobin in their red blood cells and have too much iron in the mitochondria close to the nucleus (perinuclear mitochondria). The current treatment for this condition is not ideal, involving lots of transfusions and iron chelation.
Sometimes people are born with this anemia and sometimes people get it later in life. One subset of the inherited version happens because a gene with an unknown function, SLC25A38, isn’t working correctly. This group of patients is the focus of this study.
Fernandez-Murray and coworkers started out by using yeast to figure out what the yeast homolog, HEM25, does in a yeast cell. When the gene was deleted, the cells made about 50% less heme than wild type yeast and adding back the human gene, SLC25A38, to this deletion strain restored heme levels. Looks like they had made a yeast model of this inherited anemia.
Previous work had suggested that SLC25A38 might be a glycine or serine transporter and the next set of experiments confirmed it as a glycine transporter in a couple of ways. In both, they took advantage of cases in which yeast can use glycine as their sole nitrogen source if the glycine can make it into the mitochondria.
In the first case, they showed that yeast cells deleted for HEM25 grew poorly on plates where glycine was available as the only nitrogen source. In the second case, they showed that cells deleted for both SER1 and HEM25 grew poorly on plates where again glycine was the only nitrogen source. This last result confirms HEM25 as a glycine transporter since yeast deleted for the SER1 phosphoserine aminotransferase can only grow in the absence of serine if they can get glycine into their mitochondria. (There isn’t space to go into it here, but they also showed that HEM25 was not a serine transporter.)
OK so now they had created a yeast cell that mimicked the effect of sideroblastic anemia and figured out why people with a mutated SLC25A38 gene had the condition. Now it was time to find a treatment.
The researchers came up with three possibilities. The first treatment was just to give the yeast extra glycine, the second was to drive glycine synthesis within the cell by adding a lot of serine, and the third was to add a downstream precursor of heme synthesis, 5-aminolevulinic acid (5-Ala).
They tested each scenario on yeast cells deleted for HEM25 and found that both glycine and 5-Ala worked to restore heme synthesis, but that added serine had no effect. Both glycine and 5-Ala returned heme levels to that seen in wild type.
Of course we aren’t yeast, so they next tested their treatment on something a bit more complicated — zebrafish. By using morpholino technology to knock down both copies of the zebrafish SLC25A38 homolog, SLC25A38a and SLC25A38b, the researchers managed to lower a zebrafish’s heme levels to about 50% of normal.
When they gave these zebrafish extra glycine or 5-Ala, their heme levels did not improve. They were still anemic!
After a bit of thought, the researchers realized that folate might be what the zebrafish were missing. In work that we didn’t have time to go over before, the researchers had shown that a folate dependent pathway was critical for getting heme levels up to normal.
Yeast could get away without added folate in these experiments because they make their own. However, zebrafish, like people, do not.
So the final step was to try to add both glycine and folate to these fish. Now the zebrafish’s heme levels returned to about 80% of normal.
These results suggest a better treatment for some people with sideroblastic anemia — added folate and glycine. And it all came from studying the problem in the simpler, bloodless S. cerevisiae. Nice work again yeast.
by Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
March 06, 2014
Imagine the heater at your house is run by a homemade copper-zinc battery. You are counting on a delivery of a copper solution that will keep the thing going. Unfortunately it fails to come, which means the battery doesn’t work and you are left out in the cold.
Turns out that something similar can happen in cells too. The respiratory chain that makes most of our energy needs copper to work. In a recent study, Ghosh and coworkers showed that if Coa6p doesn’t do its job delivering copper to the respiratory chain, the cell can’t make enough energy.
This isn’t just interesting biology. In this same study, the researchers showed that mutations in the COA6 gene cause devastating disease in humans and zebrafish. And their discovery that added copper can cure the “disease” in yeast just might have therapeutic applications for humans.
The respiratory chain is a group of large enzyme complexes that sit in the mitochondrial inner membrane and pass electrons from one to another during cellular respiration. This process generates most of the energy that a cell needs. Hundreds of genes, in both the nuclear and mitochondrial genomes, are involved in keeping this respiratory chain working.
Yeast has been the ideal experimental organism for studying these genes, because it can survive just fine without respiration. If it can’t respire for any reason, yeast simply switches over to fermentation, generating the alcohol and CO2 byproducts that we know and love.
Human cells aren’t as versatile though. Genes involved in respiration can cause mitochondrial respiratory chain disease (MRCD) when mutated. This is one of the most common kinds of genetic defect, with over 100 different genes known so far that can cause this phenotype.
Ghosh and colleagues wondered whether there were as-yet-unidentified human genes involved in maintaining the respiratory chain. They reasoned that any such genes would be highly conserved across species, because they are so important to life, and that the proteins they encoded would localize to mitochondria.
One of the candidates, C1orf31, caught their eye for a couple of reasons. First, some variations in this gene had been found in the DNA of a MRCD patient. And second, the yeast homolog, COA6, encoded a mitochondrial protein that had been implicated in assembly of one of the respiratory complexes, Complex IV or cytochrome c oxidase.
They first did some more detailed characterization of COA6 in yeast. They were able to verify that the coa6 null mutant had reduced respiratory growth because it had lower levels of fully assembled Complex IV.
They also looked to see what happens in human cell culture. When they knocked down expression of the human homolog, they also saw less assembly of Complex IV. This suggested that the function of this protein is conserved across species.
Next they turned to a sequencing study of an MRCD patient who had, sadly, died of a heart defect (hypertrophic cardiomyopathy) before reaching his first birthday. The sequence showed a mutation in a conserved cysteine-containing motif of COA6. To see whether this might be the cause of the defect, they created the analogous mutation in yeast COA6. The mutant protein was completely nonfunctional in yeast.
To nail down the physiological role of COA6 in a multicellular organism, they turned to zebrafish. The embryos of these fish are transparent, so it’s easy to follow organ development. Given the phenotype, the fact that they can live without a functional cardiovascular system for a few days after fertilization was important too.
When the researchers knocked down expression of COA6 in zebrafish, they found that the embryos’ hearts failed to develop normally and they eventually died. The abnormal development of the fish hearts paralleled that seen in the human MRCD patient carrying the C1orf31/COA6 mutation. And reduced levels of Complex IV were present in the fish embryos.
Going back to yeast for one more experiment, Ghosh and colleagues decided to see whether Coa6p might be involved in delivering copper to Complex IV. They knew that Complex IV uses copper ions as a cofactor, and furthermore Coa6p had similarities to several other yeast proteins that are known to be involved in the copper delivery.
They tested this by supplying the coa6 null mutant with large amounts of copper. Sure enough, its respiratory growth defect and Complex IV assembly problems were reversed. The delivery of copper kept the energy flowing in these cells. And this result showed that Coa6p is involved in getting copper to Complex IV.
These experiments showcase the need for model organism research even in the face of ever more sophisticated techniques applied to human cells. The mutation in human C1orf31/COA6 was discovered in a next-generation sequencing study, but yeast genetics established the relationship between the mutation and its phenotype. The zebrafish system allowed the researchers to follow the effects of the mutation in an embryo from the earliest moments after fertilization. And the rescue of the yeast mutant by copper supplementation offers an intriguing therapeutic possibility for some types of MRCD. Just another testament to the awesome power of model organism research!
YeastMine now lets you explore human homologs and disease phenotypes. Enter “COA6” into the template Yeast Gene -> OMIM Human Homolog(s) -> OMIM Disease Phenotype(s) to link to the Gene page for human COA6 (the connection between COA6 and disease is too new to be represented in OMIM). To browse some diseases related to mitochondrial function, enter “mitochondrial” into the template OMIM Disease Phenotype(s) -> Human Gene(s) -> Yeast Homolog(s).
by Maria Costanzo, Ph.D., Senior Biocurator, SGD