New & Noteworthy
SGD Help: Interaction Overview and Network
August 24, 2015
SGD includes data on many thousands of genetic and physical interactions between the genes and proteins of Saccharomyces cerevisiae, as curated by our friends at the BioGRID database. We provide two different graphical displays that help you get a very quick and intuitive overview of known interactions for a particular gene or protein.
All interactions for a gene and its product are listed on its interactions page (see an example). At the top of the page, the Interactions Overview shows at a glance how many interactions have been curated and whether they are physical or genetic. This video explains the details of the Interactions Overview diagram:
Farther down on the Interactions page, the Interaction Network is a visual representation of genetic interactions for a particular gene and the protein-protein interactions for its gene product. The network is interactive, allowing you to choose to view either genetic or physical interactions or both. Using the slider, you can set a minimum number of experiments supporting the interactions displayed. Learn how to use the interactive features of the Interaction Network by watching this brief video:
Categories: Tutorial
Tags: BioGRID, genetic interactions, protein-protein interactions
Ancient Hybridization Causes Revision of Yeast’s Calendar
August 19, 2015
Just as this monk had trouble pinpointing when Jesus was born, so too did yeast biologists have trouble figuring out when the genome of S. cerevisiae doubled in size. But they had trouble for very different reasons… Image via Wikimedia Commons
Most people assume that Jesus Christ was born around 1 A.D. or 1 B.C. or something like that. After all, that dating system is based on when he was born. 1 A.D. is by definition “the first year of the Lord.”
Now of course no one was actually marking years this way from the get go. In fact, this dating system wasn’t really invented until 525 A.D. by the monk Dionysius Exiguus.
And as can sometimes happen when you look that far back in time, he didn’t quite get the date of Jesus’ birth right. In point of fact, Jesus was most likely born between 4 and 6 B.C.
While not nearly as momentous, yeast biologists may have done something similar with the genome of our friend Saccharomyces cerevisiae. For many years scientists have believed that this yeast underwent a whole genome duplication (WGD) event around 100 million years ago.
But if the conclusions reached by Marcet-Houben and Gabaldón in a new PLOS Biology article are correct, then what looks like the WGD event actually happened before there was a S. cerevisiae around to duplicate its genome.
Which would mean that there couldn’t have been a WGD. There is no way for a species to double its genome if it doesn’t yet exist! Instead the authors propose that what looks like a WGD was actually a hybridization of two related yeasts from longer ago than 100 million years.
Once you get past the vocabulary, the idea behind this study is actually pretty easy. Basically, they took pairs of genes that most likely came from a duplication event (ohnologs) and figured out when they diverged away from one another. This is the same idea behind figuring out when chimps and humans shared a common ancestor by comparing homologous genes.
When Marcet-Houben and Gabaldón compared every potential ohnolog in S. cerevisiae, they found that a WGD event could explain the origin of only 15% of these genes. These 15% could be traced back to a time after S. cerevisiae was already around.
The other 85% all looked to have been duplicated before S. cerevisiae yet existed. Which of course means these could not have come from a WGD. A genome that does not yet exist cannot be duplicated. This set of genes must have arisen in a different way.
An origin story that makes more sense than a WGD for these genes is one in which two related species hybridize to form one new species. This kind of thing definitely happens, especially with yeast (and if you like lagers, you can be glad it does!).
Here the idea is that the related species share a subset of their genes from when they had a common ancestor. When these species fuse, the new beast has both sets of genes. A cursory look might suggest that these genes were from a duplication event, especially if there are many large tracts of them. After all, many genes share a lot of homology between species.
It is only with a closer look that you might trace these genes back to a common ancestor that came before the species you are studying even existed. This is, in essence, what Marcet-Houben and Gabaldón found.
From the phylogeny of reference species that they created, the authors were able to get a general idea about which clades these prehybridization species may have come from. The largest peak of duplication from their analysis came from before Saccharomyces split from a clade containing Kluyveromyces, Lachancea, and Eremothecium (KLE). The other major peak came before Saccharomyces separated from a clade that contains Zygosaccharomyces rouxii and Torulaspora delbrueckii (ZT). So the simplest interpretation is that at some point long ago, an ancestor of modern S. cerevisiae was formed from the hybridization of a pre-KLE and a pre-ZT species.
Showing that something like this happened so long ago is fraught with peril. All sorts of things can happen to a genome in more than a hundred million years. And duplicated genes are even trickier because they can mutate at different rates when one copy is gaining a new function. (After all, that is one way that new genes are born.)
So Marcet-Houben and Gabaldón threw everything but the kitchen sink at the genome sequences. They tried at least three different methods for comparing the various sequences, using alternative reference phylogenies and a variety of techniques to show what might happen to their results if genes were mutating at different rates.
With each method they got similar results. Many or even most of the “duplication” events happened before S. cerevisiae was even a species. And on top of all of this they were able to show that they got a similar result when they used a known hybrid, S. pastorianus, and its two founding species, S. cerevisiae and S. eubayanus.
All of this taken together argues that S. cerevisiae did not undergo a WGD in the deep, dark past. Instead, it is the result of two closely related species getting together and creating a new species.
Now this does not necessarily mean there was no WGD in our favorite yeast’s past. There are at least two ways that a hybrid species might have formed, and as you can see in this image from Marcet-Houben and Gabaldón’s article, one of them involves a duplicated genome:
Fig 6. Hybridization scenarios. From Marcet-Houben and Gabaldón (2015), PLOS Biol. 13(8):e1002220.
In the first scenario, shown on the top left, two diploids of different species fuse together to create a yeast with two sets of chromosomes. Eventually, through mutation, translocation, gene loss and whatever other genome sculpting mechanisms are handy, the yeast ends up with double the number of chromosomes of its predecessors.
In the second possibility, shown on the bottom left, two haploids fuse. This fused yeast then undergoes a whole genome duplication and then goes through similar processes as the first model to get to the current genome.
So, although there may have been a WGD, it looks unlikely that it happened to S. cerevisiae. Just as the placement of historic events in our calendar changes when more information is available, the generation of genome sequences for more and more yeast species and new methods for analyzing them are giving us deeper insight into the history of our friend S. cerevisiae.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: evolution, hybridization, Saccharomyces cerevisiae, whole-genome duplication
Redesigning Life, Ethically
August 13, 2015
Yeast is an essential ingredient in the recipes for our most delicious food and drink. But now, researchers are working on a recipe for yeast itself! Image by Maria Costanzo
For thousands of years, humans have used yeast as an essential part of recipes for bread, wine, and beer. But now we’re turning the tables on yeast. Instead of creating recipes with yeast, researchers are creating recipes for yeast.
Now of course we have been making minor tweaks here and there for years. But what we’re talking about now is changing out the whole recipe book: creating a whole new genome for S. cerevisiae.
The Synthetic Yeast 2.0 Project (Sc2.0) has the ambitious goal of re-designing and synthesizing the entire yeast genome, some 12 million base pairs. Along with the scientific challenges, the researchers face some tricky ethical issues as well. After all, they’re creating the blueprint for an entire living eukaryotic cell!
Fortunately, the Sc2.0 researchers have thought long and hard about these issues. They’ve issued a statement of ethics and governance in a new article in GENETICS that also reviews the current regulatory and ethical landscape for synthetic biology. The statement by Sliva and colleagues sets the course for Sc2.0 and serves as a model for oversight of other synthetic biology projects.
We wrote about the science in this space before, when the project published its first major milestone, the synthesis of chromosome III. It’s fascinating stuff: the scientists are not only re-synthesizing the genome, but are re-designing it to be leaner and more useful in the lab. They’re adding features like loxP sites that can be used to alter the structure of the genome for evolution experiments, and engineering the tRNA genes so that one codon can be repurposed to code for a novel amino acid.
But even though these are seemingly benign changes to a relatively harmless beast, there are ethical issues inherent in modifying a living organism in such a big way. While the authors focus on Sc2.0, the issues they discuss are relevant to other synthetic biology projects that combine genes from several organisms in novel pathways, such as the efforts to create an opiate biosynthetic pathway in yeast.
While we can only touch on the highlights of their statement here, one of the principles most strongly emphasized by Sliva and colleagues is that all Sc2.0 work will be done for peaceful purposes that benefit society. To promote transparency, they are making outreach a priority, engaging with and educating the public about the project. Sc2.0 will be a public resource, with no intellectual property rights or restrictions on data or materials.
The researchers are also committed to safety. They have engineered multiple auxotrophies into all working strains so that they need special media to survive, even though it seems unlikely that an Sc2.0 strain on the loose would be harmful or would have a competitive advantage over wild strains. And although it’s not required for working with organisms like yeast that are classified “Generally Regarded as Safe” by the FDA, all participants in the project receive biosafety training.
Currently, there is relatively little official policy in place for the field of synthetic biology. Two safety measures currently recommended for DNA synthesis companies by the U.S. Department of Health and Human Services are that the companies check that the sequences they synthesize don’t correspond to toxins or harmful organisms, and that they also verify the identities and institutions of their customers. While compliance with these guidelines is voluntary, the Sc2.0 project has decided to support only companies that follow these safety measures.
To ensure that the policies outlined in their Statement of Ethics and Governance are followed, the Sc2.0 project will maintain an Executive Committee comprised of people both internal and external to the project who have broad expertise in policy, ethics, and science. All of the participants in the project are accountable to this committee, which will actively monitor the work to ensure that the guidelines are followed.
It’s obvious that this is no half-baked scheme, but rather an impressively well-planned recipe for cooking up a yeast cell from scratch. But, we expect nothing less from our friend S. cerevisiae and the talented researchers in the yeast community than to be at the forefront of modern science!
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
Tags: Saccharomyces cerevisiae, synthetic biology
SGD Help Video: Genome Snapshot
August 10, 2015
Have you ever wondered just how many genes are found in the genome of the S. cerevisiae reference strain S288C, or how well characterized they are? SGD’s Genome Snapshot gives you a graphical overview of the annotation state of the genome, updated daily. This brief video gives you a tour of the page and explains the information shown in each section.
Categories: Tutorial
Tags: Genome Snapshot, video
Private Nurses Help Birth Ribosomal Proteins
August 06, 2015
Some ribosomal proteins need to be closely chaperoned even as they’re being born. Image courtesy of the National Library of Medicine via Wikimedia Commons
Some kids are born troublemakers, wreaking havoc and destruction everywhere they go. They can’t help themselves; it’s in their nature to be that way. But if they have concerned and protective adults in their lives, children can overcome this tendency and grow up to become productive members of society.
Within the cell, ribosomal proteins are problem children. Although they grow up to have essential and productive roles, as newborns they can cause big trouble.
Many of them have highly charged, unstructured regions that give them a tendency to aggregate with other proteins. And they have a complicated journey to adulthood, since ribosome assembly happens in multiple cellular compartments. With an estimated 160,000 ribosomal proteins synthesized every minute in rapidly growing S. cerevisiae cells, these troublemakers could cause major problems if left to their own devices.
To help control this unruly mob, certain proteins in the cell act as designated chaperones for ribosomal proteins. In a new paper in Nature Communications, Pausch and colleagues found that, surprisingly, these specialized nurses catch their client proteins even as they’re being born. They swaddle them from the first moment they start to emerge as nascent proteins, and keep them from causing any harm until they can be delivered safely to their final destination.
The researchers first looked at the proteins Rrb1 and Sqt1. Previous work had suggested they might act as specific chaperones for the ribosomal proteins Rpl3 and Rpl10, respectively. And Pausch and colleagues confirmed these results, showing that TAP-tagged Rrb1 pulled down only Rpl3 and Sqt1 only pulled down Rpl10. Each of these troublesome tots had its own personal chaperone!
But surprisingly, very little of the protein was needed for these chaperones to keep ahold of their respective charges. When the authors trimmed down the ribosomal proteins to shorter and shorter lengths, they saw that just the N-terminal 15-20 amino acids of each ribosomal protein were necessary and sufficient for interaction with its chaperone.
They decided to use X-ray crystallography to look in detail at the Sqt1-Rpl10 interaction. First they determined the crystal structure of Sqt1 on its own, and found that it forms an eight-bladed WD-repeat beta-propeller, looking much like a round electric fan. The amino acids positioned on the surface of the blades are negatively charged.
Next, the authors co-crystallized Sqt1 with a peptide corresponding to amino acids 2-15 of Rpl10. The structure showed that the positively charged peptide was cradled in the negatively charged surface.
To test whether these charged residues were important for the interaction, they mutated the charged residues of Sqt1 and of the peptide and combined them in various ways. Sure enough, changing the charged residues of either partner disrupted or diminished the interaction.
The Sqt1 chaperone resembles an electric fan, with eight blades rather than four. Image via Wikimedia Commons
Pausch and colleagues went on to test whether those same charged residues are important in vivo. An sqt1 mutation changing glutamate residue 315 to lysine (E315K), that abolished the Sqt1-Rpl10 interaction in vitro, was lethal for yeast cells, confirming the importance of the interaction.
The researchers also detected many allele-specific genetic interactions between the charged residues of the two proteins, and even found that switching the charges in an interacting pair of amino acids (changing an Sqt1 residue to a positive charge and its Rpl10 binding partner to a negative charge) would improve growth compared to either single mutant.
The lethality of that sqt1-E315K mutation, and even the lethality of an sqt1 null mutation, were weakly suppressed by overproduction of Rpl10. So yeast cells can get by (just barely) with an un-chaperoned Rpl10, as long as there’s enough of it around. This result also confirmed that Rpl10 is the only client of Sqt1.
As yet another verification that Sqt1 acts as a chaperone, the authors looked to see what happens to the Rpl10 protein in sqt1 mutants. If cells carrying wild-type SQT1 are lysed and separated into a pellet and supernatant, most Rpl10 spins down in the pellet but a significant amount is soluble in the supernatant. However, if the cells carry any of several sqt1 mutant alleles that alter the charged residues and diminish the interaction with Rpl10, all of the Rpl10 is found glommed together in the pellet.
The two chaperone-ribosomal protein interactions that Pausch and colleagues investigated, Sqt1-Rpl10 and Rrb1-Rpl3, both involved the extreme N termini of the ribosomal proteins. Previous studies had also shown that two other chaperones for ribosomal proteins, Yar1 and Syo1, also interact with the N termini of their clients. So the authors wondered whether interactions between ribosomal proteins and their chaperones might even start during translation of the ribosomal proteins.
In a final experiment, the researchers treated yeast with cycloheximide to freeze translation and then pulled down each of the four chaperones via affinity tags. Each chaperone specifically pulled down the mRNA encoding its client protein, showing that it was binding to the nascent protein as it first started to emerge from the translating ribosome.
So this study has defined a new step in ribosomal biogenesis. Certain specific ribosomal proteins are such troublemakers that it’s too dangerous for the cell to just release them into the cytoplasm after they’re translated.
Instead, these bouncing baby proteins are caught by their individual nurses before they’re even fully born, and wrapped up to protect both the ribosomal proteins themselves and the rest of the cell. Since ribosomal biogenesis is highly conserved across species and since defects in it are associated with many different diseases, further study of these cellular midwives could have important implications for human health. Perhaps some gentle guidance could help put wayward ribosomes on the right track.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
Tags: chaperones, ribosomal proteins, Saccharomyces cerevisiae
Another Small Victory for Lamarck
July 29, 2015
Yeast gives us an example of an adaptation that is positively Lamarckian! Image of Jean Baptiste de Lamarck via Wikimedia Commons
Examples of various ways that the environment affects gene expression have become so commonplace that new examples don’t make much of a splash anymore. It is as if Lamarck and Darwin had never argued about how natural selection works.
The main reason we aren’t surprised anymore is that we have a pretty good handle on how most of these changes are happening. Something in our environment causes chemical groups to be added or removed from our DNA and/or its associated proteins, causing a change in gene expression. These kinds of epigenetic changes happen a lot and are now seen as the norm.
What doesn’t happen much at all is that the underlying DNA gets changed in a predictable way to change gene expression in response to something in the environment. Which is why a recent study in PNAS by Jack and coworkers makes you stand up and take notice.
In this study the researchers provide evidence that suggests that yeast will expand the number of copies of its rDNA locus to match the level of nutrients in the environment (presumably to make more ribosomes to take advantage of all those nutrients). The yeast is responding to the environment by changing the content of its genome rather than just changing how it is used.
This is big. It is almost as if Lamarck was right about some part of natural selection. Oh wait, that’s exactly what it is!
What makes this so cool is that it suggests that natural selection doesn’t necessarily just happen when a random genetic variant wins out in a population. Sometimes the environment itself can induce the winning genome change–and these aren’t just epigenetic changes. (Go Lamarck!)
Jack and coworkers focused on the rDNA locus of the yeast Saccharomyces cerevisiae. This is a fairly fluid part of the yeast genome that consists of multiple copies of the 35S and 5S pre-rRNA genes. The average yeast cell has around 180 copies of this locus and there is a normal range of 150-200 per cell. If a yeast somehow ends up with 80 or fewer copies, it quickly increases the number back to that golden 150-200 range via a Fob1 dependent mechanism.
The authors created a strain of yeast that lacked Fob1 and had only 35 tandem repeats in its rDNA region. This strain, rDNA35, could not expand its rDNA unless Fob1 was added back. They now had a strain in which they could test what affected rDNA expansion, by transforming the strain with a plasmid expressing Fob1 and growing the transformants under different conditions.
The most surprising experiment was the final one of the paper. The authors grew the rDNA35 strain in either 2% or 0.5% glucose and found that rDNA amplification was slowed significantly in low glucose. The authors interpret this to mean that the genome change, the expansion of rDNA, is dependent on nutrient availability. A signaling pathway is able to adjust the rate of rDNA expansion.
Yeast will, of course, grow more slowly at low levels of glucose than they will at higher levels. But the authors were able to show that slow growth was not the reason for the slowed expansion of rDNA at low glucose. They were able to separate the two effects by overexpressing Pnc1, a nicotinamidase.
Overexpression of Pnc1 led to a decreased rate of copy number increase even at the higher glucose levels without affecting growth rate. So rDNA expansion can be separated from slow growth under the right conditions. And as you’ll see below, Pnc1 makes perfect sense given how at least part of nutrient level-dependent rDNA amplification works.
In looking for factors that might affect the rate of rDNA expansion, Jack and coworkers focused on the TOR signaling pathway, since previous work had suggested that it might be important in this process and it is known to respond to nutrient availability. The authors confirmed it was a key player by showing that rapamycin, a TOR inhibitor, kept the rDNA35 strain from expanding its rDNA in the presence of FOB1.
Again they ran into the problem of disentangling cell growth and the rDNA expansion, as rapamycin slows cell growth. The next set of experiments showed that the lack of expansion was almost certainly not due to the slower growth rate.
It is known that rapamycin affects histone deacetylases (HDACs) including Sir2. Jack and coworkers found that nicotinamide, a Sir2 inhibitor, increased the rate of expansion of rDNA without affecting growth rate. So rDNA amplification was not dependent on growth rate.
Which brings us back to Pnc1, that enzyme that cleaves nicotinamide! Presumably endogenous levels of nicotinamide are able to inhibit Sir2 and so encourage the rDNA expansion. Overexpressing Pnc1 releases Sir2 which can then impede the expansion of rDNA.
While that was a bit complicated, the idea is simple and potentially profound. Yeast can sense the level of nutrients in their environment at least partially through the TOR signaling pathway and adjust the actual content of their genome accordingly.
The involvement of nicotinamide in this regulatory process makes this result even cooler, as it has important roles in aging and cellular metabolism from yeast to man. For example, it plays a key role in the life extending properties of caloric restriction in yeast and possibly in more complicated eukaryotes as well. (Click here for a fascinating look at NAD, a compound that contains nicotinamide.)
So, in the presence of low nutrient levels, yeast expand their rDNA much more slowly than they would at higher nutrient levels. Yeast can tailor its genome in response to its environment so it can better utilize that environment.
This work raises the fascinating possibility that this process might even happen at genomic regions other than the rDNA locus. Yeast still has plenty of surprises in store—including giving a Lamarck a little boost.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight
Tags: environmental response, nicotinamide, ribosomal DNA amplification, Saccharomyces cerevisiae
SGD Help Videos: Working with Lists in YeastMine
July 28, 2015
Understanding lists and knowing how to work with them is crucial to getting the most out of YeastMine. This set of short videos explains everything you need to know.
YeastMine allows you to save objects in lists. Typically, these objects are genes, but you can also make lists of other objects such as Gene Ontology terms or PubMed IDs. One way to create a list in YeastMine is to run a query and save the results in a list. Another way is to type in or upload your own list.
Whenever you create a list in YeastMine, you’re immediately presented with information about the genes in the list, such as Gene Ontology and pathway enrichment, interactions, orthologs, and more. This can help you decide what kind of further analysis you’d like to do.
And what if you create a list but then realize that you forgot to include a gene? No worries. It’s easy to edit your saved lists.
Once you have a list of genes, you can feed it into any template query whose name begins with “Gene” to get results for all of the genes in the list. This powerful feature lets you run successive queries to narrow down your results. For example, you could make a list of all the proteins in a given size range, then query that list to see which ones are located in the nucleus, and finally ask how many of these nuclear proteins have human homologs.
And finally, once you’ve created and saved lists you can do a lot of different things with them: combine them, find their intersection, find genes that are not shared between two lists, or find genes that are in one list but not another. This provides a powerful way to combine or compare results from different YeastMine queries.
As always, please contact us if you have any questions about YeastMine. We’re happy to help!
Saving Search Results as a List
Creating and Using Gene Lists
Adding Objects to a Saved List
Feeding Lists into Templates
List Operations
Categories: Tutorial
Two Tales of Two Tails
July 22, 2015
Tails let animals sense and interact with their environment at a distance from their bodies. It turns out that some proteins use their “tails” in a similar way. Except they take it one step further.
Some mythical creatures have tails that coil around each other. Septin proteins are not all that different! Image via Wikimedia Commons
In addition to sensing the environment, they can also use their tails as sort of fishing poles to catch proteins they need to interact with. And they do this with great specificity…it is like having that perfect lure that always catches one type of fish.
A great example of this is the septin family of proteins which includes many members that are “tailed” proteins. Septins are highly conserved proteins that typically have a globular GTP-binding domain adjacent to an elongated C-terminal extension.
Septins form structures that act as the boundaries between different cellular parts. In budding yeast cells, they form the septum that separates the mother and bud, and recruit the cytokinesis machinery that allows the daughter cell to separate from the mother cell. In larger animals, they can be found in places such as the dendritic spines of neurons, sperm flagella, or cilia. And in humans, septin mutations have been linked to cancer and neurological diseases.
Until now, the details of how septins recruit other proteins to boundary sites have been elusive. But in two new papers in GENETICS, Finnigan and coworkers in the Thorner lab at Berkeley dove into this question and gained real insight into the lures these proteins use.
In their first paper they reported an extremely comprehensive genetic analysis to dissect the functions of two of the least characterized septins, Shs1 and Cdc11. In the second paper they used both genetic and physical methods to show how these septins recruit myosin to the septum to form the contractile ring that pinches off the bud from the mother.
The bottom line: their C-terminal tails are extremely important. They intertwine with other proteins’ tails like love-struck seahorses. And their specificity comes from these same tails—certain tails only coil around other tails.
The S. cerevisiae genome encodes a family of septins that assemble with each other to form octameric rods that consist of four different septins. The rods have both end-to-end and side-to-side interactions with each other, forming a ladder-like superstructure.
The septins Cdc11 and Shs1 are the most closely related members of the septin family, and the most recently evolved. They cap the ends of the septin rods. In otherwise wild-type cells, Cdc11 is essential for life while Shs1 is not.
Because SHS1 can be deleted without causing a major phenotype, the first step in investigating its function was to find genetic conditions under which its function becomes more obvious. The authors created four different genetic backgrounds in which the function of other septins was compromised by different mutations. Cells that had mutations in both SHS1 and in other septin genes had obvious problems, such as elongated buds or the inability to grow at high temperatures.
Now Finnigan and colleagues were set to do a detailed genetic analysis to figure out what different parts of Shs1 do by testing mutant versions in these different backgrounds. We can’t possibly recapitulate all the results here, but we’ll do our best to cover the highlights.
Almost all septins, whether in yeast or mammals, end with a tail: a long stretch called the C-terminal extension (CTE) that contains sequence patterns characteristic of a coiled-coil structure. The researchers found that the coiled coil regions of Shs1 and Cdc11were essential to their functions. (And no, they didn’t create any mutations by writing their names in the coiled coil sequence!)
Finnigan and colleagues tried swapping CTEs between different septins. When Cdc11 carried the Shs1 CTE and vice versa, the cells grew just fine. However, this swappability didn’t extend to other septins that are positioned internally in the septin rods. The CTEs of the end subunits Cdc11 and Shs1 could be exchanged for each other, but these CTEs only worked when they were on the ends of the rods.
Since coiled coils are often involved in interactions between proteins, Finnigan and colleagues wondered whether the essential function of the Cdc11 and Shs1 CTEs might be to recruit other proteins to the bud neck.
To test this, they searched published data to identify proteins that are well-known to be localized to the bud neck at the time in the cell cycle when septins are present. They found 30 such proteins, and overexpressed GFP-tagged versions of each in a strain where both Cdc11 and Shs1 lacked their CTEs.
Of the 30 proteins, only overexpressed Bni5 suppressed the growth defect of this strain. To test directly whether binding to Bni5 is a critical function of Shs1, Finnigan and colleagues fused the two genes to each other, so that Bni5 replaced the CTE of Shs1. This fusion protein could compensate for the lack of both Cdc11 and Shs1 CTEs.
To confirm that the important function of the CTEs is to hold Bni5 in the right place, they came up with an alternative test using a “nanobody”, which is a very small, very high-affinity single-chain antibody. They replaced the CTE of either Cdc11 or Shs1 with a nanobody that recognized GFP, and expressed a GFP-Bni5 fusion in these strains. In both cases, tethering Bni5 to the septin via the nanobody obviated the need for the CTE.
Finally, the authors asked why it is important for Bni5 to be located on the septin rods. Previous work had suggested that Bni5 recruits Myo1 (myosin), an important component of the contractile ring at the bud neck. They used the same nanobody constructs to test this, simply expressing GFP-Myo1 in the strains where the nanobody replaced the CTEs of Cdc11 or Shs1. Sure enough, tethering Myo1 to the terminal septins eliminated the need for Bni5.
So we now know that tails are absolutely essential for the functions of the alternative terminal septins Shs1 and Cdc11. These fishing poles let them hold on to the Bni5 “bait,” which in turn catches Myo1 to provide the muscle for cytokinesis to occur. Since septins are so highly conserved, it’s probable that these results will be directly applicable to higher organisms: there are mammalian septins that also occupy the end positions of septin rods, analogous to Cdc11 and Shs1. And that’s no fish story!
Not only septins use their tails to fish.
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
Tags: cytokinesis, Saccharomyces cerevisiae, septins
SGD Help Video: Gene Name Reservation
July 13, 2015
The eminent Drosophila geneticist Michael Ashburner famously said: “Biologists would rather share their toothbrush than share a gene name.” It’s true that assigning names to genes is often a sticky subject.
In the Saccharomyces cerevisiae community we’re very lucky to have well-defined guidelines for genetic nomenclature, an established system for reserving gene names, and criteria for making them “standard,” or official, names. This system was agreed upon by yeast researchers nearly two decades ago and has served the community well.
Take a look at this video to get an overview of how the gene naming system works. And as always, please contact us with any questions or suggestions.
Categories: Tutorial
Tags: gene nomenclature, Saccharomyces cerevisiae, video
The Gift for the Man Who Has Everything
July 08, 2015
Gifts can be hard to buy for some people. They have everything they need and not many outside interests. What to do?
Having trouble finding that personal gift for that impossible to buy for person? How about a vanity protein with their name written right into the amino acid sequence? Image by D. Barry Starr
You could name a star after them or get them some knick knack they don’t need. Or you could design a personalized protein that has their name in it, solve the structure and present them with the picture.
This is what Deiss and coworkers did to celebrate the 50th birthday of their colleague Andrei N. Lupas, a key figure in studying coiled-coil proteins. They created a personalized protein based on Gcn4 from Saccharomyces cerevisiae. And of course Gcn4 is a coiled-coil protein!
Coiled-coil proteins are the perfect clay for biosculpting a personalized protein. They follow a relatively simple set of rules which makes it easy to predict how they will fold. There isn’t much of the “protein folding problem” with these user-friendly proteins.
Basically these proteins consist of repeated 7 amino acid motifs that each form an alpha helix. They have hydrophobic residues down one face of the helix so that they will tend to oligomerize with each other to keep the hydrophobic residues away from the water. These helices spontaneously coil up like a rope (hence their name).
The 7 amino acids of a repeat are usually represented as a–b–c–d–e–f–g and are arranged in the pattern hxxhcxc, with h being hydrophobic residues, c being charged residues and x being most any other amino acid. So a and d must be hydrophobic, and e and g charged. That’s pretty much it!
Deiss and coworkers used the name Andrei N. Lupas to create a personalized coiled coil. They replaced 12 amino acids in Gcn4 with the amino acids represented by the letters in his name. Well, they were able to do that for most of the letters.
First off, they had to Roman things up a bit and turn the U into a V (there is no amino acid with the single amino acid code U). So here is the amino acid sequence they used and how they lined it up with the 7-amino acid repeats:
In this arrangement, the hydrophobic residues are asparagine, isoleucine, and valine, and the charged residues are aspartic acid, glutamic acid, proline, and serine. Obviously the last two are not optimal, especially the proline. Proline has an especially rigid conformation and is known to wreak havoc with alpha helices.
When the authors analyzed the protein, they found that as predicted, the proline disrupted the part of the alpha helix with which it was associated. But not enough to completely destroy the coiled coil structure. X-ray diffraction showed that this protein was still able to trimerize properly. They had created a distorted but functional personalized protein. What other kind would anyone want!
And it isn’t as if proline is completely absent from the heptad repeats of coiled-coil proteins. A quick search by the authors found two viral fusion proteins, 1ZTM and 3RRT, that could form a trimer even though they too had prolines. In both of these proteins the proline is in the f position.
They also found 4 dimers with a proline in a heptad repeat. In these cases the proline is at b or c. So no known natural coiled-coil proteins have a proline at the e position. Talk about personalized!
How cool is all of this, and who wouldn’t want a protein of their very own? Unfortunately, not everyone can easily have one.
For example, President Barack Obama would have real trouble since there are no amino acids designated with a B or an O and there is no obvious way to transform these letters into ones that are present in the single letter code. Jeb Bush is out too, but maybe we can do something with Hillary Clinton. Let’s see if we can line up the amino acids of her first name to create a personalized Gcn4 just for her.
“HILLARY” isn’t too bad by itself. All the letters are amino acids (yay) and a and d are hydrophobic (isoleucine and alanine). Aspartic acid works very well for e and while probably not perfect, histidine isn’t too bad for g. The tyrosine at position f is not ideal either but is way better than a proline. This thing might replace one heptad repeat in Gcn4 without causing too many problems.
So what about your name? Can you turn yours into a heptad repeat to create your own personalized Gcn4?
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight