New & Noteworthy
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.
March 25, 2013
It is with great sadness that we note the passing of Carl Singer on February 8, 2013. Carl was a staunch supporter of the international yeast community. His good humor, generosity and infectious enthusiasm have been bright highlights of every yeast meeting for many decades. His creativity and engineering prowess have exerted a similarly powerful impact in labs world-wide, contributing to the quality of yeast research and the ease of performing it. He will be sorely missed.
Please send memories celebrating his life to his family at email@example.com. A celebration of Carl’s life will be held at 2 PM on Tuesday, March 26th, at St. Michael’s Church, Minehead, UK.
Thanks to Terry Cooper for drafting this obituary.
March 21, 2013
At first our favorite small eukaryote, S. cerevisiae, might not seem like a great model for cancer studies. After all, budding yeast can’t tell us anything about some of the pathways that go wrong in cancer, like growth factor signaling. And it clearly can’t help explain what happens in specific tissues of the human body. But in other ways, it actually turns out to be a great model.
For example, all the details of cell cycle control were originally worked out in yeast. And now a whole new batch of genes has been found that influence a phenomenon, chromosome instability (CIN), that is important in both yeast and cancer cells.
As the name implies, chromosomes are unstable in cells suffering from CIN. Big chunks of DNA are lost, or break off and fuse to different chromosomes, turning the genome into an aneuploid mess. And this mess has consequences.
CIN can cause new mutations or make old ones have a stronger effect. Eventually these mutations can affect genes that are important for keeping a cell’s growth in line. Once these are compromised, a tumor cell is born.
Since CIN is pretty common in yeast, we might be able to better understand it in cancer cells by studying it in yeast. The Hieter lab at the University of British Columbia has come up with a powerful screen to get yeast to confess why it CINs.
A previous study from the group set the stage by finding a large group of mutants that have CIN phenotypes, implying that those genes are involved in keeping chromosome structure stable. In a new paper in G3: Genes, Genomes, Genetics, van Pel et al. uncovered the network of interactions among the genes in this set, using synthetic genetic array (SGA) technology. And they confirmed that the human homologs of some of these genes interact in the same way as in yeast, making them potential targets for cancer therapies.
The idea behind SGA studies is that if two proteins are involved in the same process, then a strain carrying mutations in both of their genes will be much worse off than a strain carrying either single mutation. In the worst case, the double mutant will be dead. This is known as a synthetic lethal interaction.
Yeast is a great model for doing these sorts of studies on a very large scale. We can construct networks showing how lots of different genes interact, and most importantly, find the genes that are central to many interactions. These “hubs” are likely to be the key players in those processes.
The researchers looked specifically for interactions between genes that are involved with CIN in yeast and are also similar to human cancer-related genes. They came up with various interaction hubs that will be interesting research subjects for a long time to come. In this study, they focused on one of these: genes involved with the DNA replication fork.
One of these in particular, CTF4, is a hub for both physical and genetic interactions. Unfortunately, Ctf4p doesn’t look like a good target for chemotherapy. It’s thought to act as a scaffold, and lacks any known activity that could potentially be inhibited by a drug. However, the interaction network around CTF4 that van Pel et al. uncovered suggests another way to target this hub. If a gene that interacts with CTF4 itself has a synthetic lethal interaction with another gene, and we could re-create the synthetic lethal phenotype in a cancer cell, we might be able to knock out the whole process. And that is just what they found in human cells.
First the authors identified a couple of human genes that were predicted from the yeast screen to be close to human CTF4 in the interaction network and to have a synthetic lethal interaction with each other. They then lowered the expression of one using small interfering RNA (siRNA), and reduced the activity of the other with a known inhibitor. Neither treatment alone had much effect, but combining them significantly reduced cell viability.
Since cancer cells frequently carry mutations in CIN genes, it should be possible to create a synthetic lethal interaction, guided by the yeast interaction network, where one partner is mutated in cancer cells (equivalent to using siRNA in this study) and the other partner is inhibited with a drug. Since it relies on a cancer-specific mutation, this approach has the potential to selectively target cancer cells while not disturbing normal cells, the ultimate goal for chemotherapy.
March 20, 2013
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March 14, 2013
You can’t teach an old dog new tricks, or so the saying goes. But imagine you found that your old dog knew a complicated trick and had been doing it all her life, right under your nose, without your ever noticing it! You’d be surprised – about as surprised as the Hinnebusch group at NIH when they discovered that some long-studied S. cerevisiae genes had an unexpected trick of their own.
They were working on the VPS* (vacuolar protein sorting) genes. While known for a very long time to be important in protein trafficking within the cell, Gaur and coworkers found that two of these genes, VPS15 and VPS34, play an important role in RNA polymerase II (pol II) transcription elongation too. Now there is an unexpected new trick…like your dog learning to use a litter box!
There had been a few hints in recent years that the VPS genes, especially VPS15 and VPS34, might have something to do with transcription. Following up on these, the researchers tested whether vps15 and vps34 null mutants were sensitive to the drugs 6-azauracil and mycophenolic acid. Sensitivity to these drugs is a hallmark of known transcription elongation factors. Sure enough, they were as sensitive as a mutant in SPT4, encoding a known transcription elongation factor. Further experiments with reporter genes and pol II occupancy studies showed that pol II had trouble getting all the way to the end of its transcripts in the vps mutant strains.
There was a bit of genetic interaction evidence that had suggested that there might be a connection between VPS15, VPS34, and the NuA4 histone acetyltransferase complex. This is important, since NuA4 is known to modify chromatin to help transcription elongation. Looking more closely, the researchers found that Vps34p and Vps15p were needed for recruitment of NuA4 to an actively transcribing reporter gene.
Other lines of investigation all pointed to the conclusion that these VPS proteins have a role in transcription. They were required for positioning of several transcribing genes at the nuclear pore, could be cross-linked to the coding sequences of transcribing genes, and could be seen localizing at nucleus-vacuole junctions near nuclear pores.
One appealing hypothesis to explain this has to do with what both genes actually do. Vps34p synthesizes phosphatidylinositol 3-phosphate (PI(3)P) in membranes, while Vps15p is a protein kinase required for Vps34p function. The idea is that when Vps15p and Vps34p produce PI(3)P at the nuclear pore near transcribing genes, this recruits the NuA4 complex and other transcription cofactors that can bind phosphoinositides like PI(3)P. There are hints that this mechanism may also be at work in mammalian and plant cells.
There’s a lot more work to be done to nail down the exact role of these proteins in transcription. But this story is a good reminder to researchers that new and interesting discoveries may always be hiding in plain sight.
* These genes were also called VPL for Vacuolar Protein Localization and VPT for Vacuolar Protein Targeting