December 22, 2018
To promote the use of yeast as a catalyst for biomedical research, SGD utilizes the Disease Ontology (DO) to describe human diseases that are associated with yeast homologs. Disease Ontology annotations to yeast genes are now available through SGD’s new Disease pages. Each page corresponds to a Disease Ontology term, such as amyotrophic lateral sclerosis, and lists out all yeast genes annotated to the term by SGD.
Yeast genes with one or more human disease associations will also have a new Disease Summary tab (example: MIP1), accessible from the genes’ respective locus pages. The Disease summary tab shows all manually curated, high-throughput, and computational disease annotations for the yeast gene. Additionally, these pages feature a network diagram that depicts shared disease annotations for other yeast genes and their human homologs.
Categories: New Data
December 14, 2018
Macromolecular complexes, already retrievable from SGD’s YeastMine data warehouse, are now available on new pages on the SGD website. These new Complex pages (example: GAL3-GAL80 complex) provide manually curated information about the complex as well as helpful links and diagrams. Key features of Complex pages include:
Complex pages can be accessed by running a search for the complex, or by visiting the gene summary pages of its subunits. For example, to find the GAL3-GAL80 complex page, simply run a search for “GAL3-GAL80” and click on the Complexes category (symbolized by the gold dot). Or, go to the GAL3 or GAL80 gene page and locate the Complex section.
SGD curated these macromolecular complex data in collaboration with curators at EMBL-EBI’s Complex Portal. Be sure to check out the page for your favorite complex, and let us know if you have any feedback or questions.
Categories: New Data
November 20, 2018
The alternative reference strain SK1 is a rapid and synchronously sporulating diploid constructed by Kane and Roth in the early 1970’s to study carbohydrate metabolism under sporulation conditions. Whereas the reference strain S288C is notorious for being poor at sporulation, SK1 undergoes sporulation readily, and as such has been widely used to study the genetics of sporulation and meiosis.
The genome of SK1, which was temporarily removed from SGD, is now available once again with an updated sequence provided by Scott Keeney from the Sloan Kettering Institute. You can access the updated SK1 sequence for your favorite genes from the Sequence tab, in the Alternative Reference Strains section (example: ECM22 Sequence page). To access the entire SK1 genome sequence, visit the SK1 Strain page.
In addition, we have updated following Sequence and Analysis tools to utilize the latest SK1 sequence:
November 14, 2018
YeastRGB is a new resource for exploring protein abundance and localization. Utilizing data from the classical C-terminally tagged GFP yeast library along with new-generation collections derived from SWAp Tag (SWAT) technology, YeastRGB enables simultaneous visualization of dozens of yeast strains imaged with multiple fluorescent tags.
From SGD, you can access YeastRGB through any Protein page (example: Atp12p). The YeastRGB link is located in the Resources section, under Localization. Alternatively, you can visit the YeastRGB website and search for your favorite genes or keywords.
November 07, 2018
The 30th Fungal Genetics Conference takes place next year March 12-17, 2019 at Asilomar Conference Grounds in Pacific Grove, CA. The biennial Fungal Genetics Conference is a place where scientists working on any aspect of fungal genetics–such as gene regulation, evolutionary biology, cell development, fungal-host interactions and more–can come together in a common platform to share ideas and collaborate.
The schedule of events is now available. The conference features multiple workshops, Plenary Sessions with central themes on various aspects of fungal biology, and dozens of diverse Concurrent Sessions where you can attend talks on topics most relevant to your research. The 2019 Perkins/Metzenberg Lecture, which provides perspectives given by a leader in the field of fungal genetics, will be presented by John Taylor from the University of California, Berkeley.
October 30, 2018
Fungal Pathogen Genomics is an exciting week-long course that provides experimental biologists working on fungal organisms with hands-on experience in genomic-scale data analysis. Through a collaborative teaching effort between the web-based fungal data mining resources FungiDB, EnsemblFungi, PomBase, SGD/CGD, MycoCosm, and JGI, students will learn how to utilize the unique tools provided by each database, develop testable hypotheses, and analyze various ‘omics’ datasets across multiple databases.
Daily activities in Fungal Pathogen Genomics will include both individual and group exercises, lectures on the bioinformatics resources provided by various databases, and presentations by distinguished guest speakers. Examples of what you will learn at Fungal Pathogen Genomics include:
Don’t miss out – apply now!
October 05, 2018
SGD periodically sends out its newsletter to colleagues designated as contacts in SGD. This Fall 2018 newsletter is also available on the community wiki. If you would like to receive the SGD newsletter in the future please use the Colleague Submission/Update form to let us know.
September 24, 2018
Resistance to poisoning is a good thing in the cartoon world, where both good and bad guys and gals often have superpowers that render them resistant to all sorts of toxins and poisons. But does that happen in the “real” world that we live in?
Well, some people would say that Keith Richards and cockroaches are the ultimate examples of earthly organisms resistant to every known toxin! But there are other more mundane examples of humans that are resistant or tolerant to certain chemical or organic compounds.
For example, almost everyone knows someone (or themselves) who can’t drink milk, because if they do, they feel really sick, with uncomfortable or even severe gastrointestinal symptoms. These people are said to be “lactose intolerant” (which is NOT an allergy to milk), because their gut can’t digest a sugar called lactose found only in milk and milk-based products.
Interestingly, all humans have the “lactase” gene that allows their body to digest lactose, but lactose intolerant folks have a particular “switch” sequence in their DNA that causes the gene to get totally turned off once they’re not babies anymore.
However, people who CAN drink milk as adults (“lactose tolerant” people) have a change in their DNA sequence that inactivates the switch. So these people have the lactose-digesting gene turned on and functioning for their whole lives and can consume milk and dairy products even as adults.
This is an example of genomic diversity among human beings, where people’s individual genome sequences have differences that can make them resistant to the negative effects of some foods or chemicals, while other people have little or no tolerance at all.
This type of DNA sequence variation across all the members of a species is called “standing genetic variation” and it’s a very good thing. Why? Because when a changing or novel environment challenges a population, standing genetic variation increases the chance that some individuals can adapt to new types of foods (like what happened with the lactase gene) or have other characteristics that make them better suited to survive in the new environment…or even be resistant to new diseases!
But what does all of this have to do with yeast? Well, just like lactose intolerant humans, yeast can get sick when they try to “eat” certain things too, and will fail to grow well in the presence of such compounds. But could there be some yeast individuals somewhere in the world that have tolerance to certain normally-toxic compounds?
This is exactly the question that Higgins and co-workers asked, and in the September issue of Genetics they describe how they found naturally occurring yeast that can tolerate high levels of compounds called “ionic liquids.” And they also discovered the underlying naturally-occurring genetic variations in two genes that make the yeasts more tolerant to these compounds!
So what are ionic liquids and why were these investigators interested in them? Ionic liquids are a type of salt that can exist in a liquid state at temperatures under the boiling point of water or even at room temperature.
One type in particular, “imidizolium ionic liquids” (IILs) are often used in production of biofuels because they efficiently solubilize plant biomass cellulose and help turn it into glucose. The glucose can then be fermented (often by our yeast friend Saccharomyces cerevisiae) into bioethanol or other biofuels.
However, most commonly used S. cerevisiae strains, when grown in the presence of IILs, get very ill. But if IIL-tolerant yeasts could be found, they could help improve the production of cellulosic ethanol and other bioproducts.
Higgins and co-workers decided to make use of the standing genetic variation within the S. cerevisiae species by taking hundreds of different strains of S. cerevisiae, isolated from around the world, and seeing if any of them could grow better in the presence of IILs.
And indeed they found some strains that were extremely IIL-tolerant compared to other S. cerevisiae strains such as beer, wine or lab strains, or even those commonly used in biofuel production! The yeast strain that was the most IIL-tolerant was, surprisingly, a clinical isolate from Newcastle, England.
The researchers then took genomic DNA from this IIL-tolerant strain and chopped it up into large pieces and put the pieces (carried on special plasmids called fosmids) into the common S288C lab strain (which is intolerant to high IILs) to see which regions could allow the lab strain to now grow in the presence of high levels of IILs.
They found two genes that conferred tolerance to the IILs: the SGE1 gene, plus a previously unnamed gene, YDR090C, which had not been studied very much. The proteins made from both genes appear to be located in the plasma membrane of the cell. The authors propose that the tolerant version of the SGE1 protein, already known to be a multidrug efflux pump that exports toxic cationic dyes out of the cytoplasm, is directly involved in the pumping the IILs out of the cells to help yeast tolerate these toxic compounds.
The researchers were not able to exactly figure out what YDR090C is doing in the membrane to help cells be resistant to the bad effects of IILs, but they did find out that cells with a deletion of the gene were less tolerant to IILs. They thus named this gene “ILT1” for “Ionic Liquid Tolerant”.
The authors also found that the SGE1 gene from the IIL-resistant “wild” strain from England had a change in its protein sequence relative to the lab strain, but rather than making the protein more efficient at pumping, it looks like the changed protein is more abundant in the cell and thus can just pump out more of the obnoxious IILs.
Whenever they put this resistant gene version into an IIL-intolerant yeast, it did the trick of allowing the strain to grow better in high IILs concentration. This discovery might allow greater use of IIL-treated biomass for the production of biofuels, as it shows one powerful method of increasing the tolerance of biofuel-producing yeast to toxic IILs.
So this is indeed a case where looking at the “standing genetic variation” of a species has helped discover new and useful biotechnological functions in yeast, and also shown us that resistance (to IILs at least) is NOT futile, and may indeed help us make yeast more efficient at making more biofuels!
Resistance might be futile if you’re up against the Borg, but at least yeast can resist IILs!
by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.
Categories: Research Spotlight
August 22, 2018
In Star Wars: The Force Awakens, the evil First Order rises up and threatens to eliminate the New Republic. The protagonists, who join forces of the Resistance, must find the reclusive Jedi Master Luke Skywalker so that he can take up arms against the First Order and save the galaxy.
(Warning: Star Wars spoilers below!)
One of the protagonists, Rey, keeps precious cargo with her as she searches for Luke: the lightsaber of Anakin Skywalker, Luke’s father. She must deliver the lightsaber to Luke in order to galvanize his joining the Resistance and to help motivate him to resume his role as a Jedi Master.
Just like how the galaxy would be doomed if Rey could not reach Luke, budding yeast cells would be doomed if a Hsp70 protein chaperone could not interact with another chaperone, Hsp90. But Hsp70 isn’t trying to deliver a lightsaber to Hsp90—instead, Hsp70 is trying to deliver “client” protein substrates, so that Hsp90 can help fold and mature these proteins.
In Star Wars, the trusty droid R2-D2 was ultimately there to help Rey find Luke with a holographic map to Luke’s location. But is our friend Saccharomyces cerevisiae just as fortunate? In fact, it just so happens that S. cerevisiae also has an “R2-D2” of its own: the co-chaperone Sti1p. Just like how R2-D2 helps Rey find Luke, Sti1p helps bring Hsp70 and Hsp90 together so that Hsp90 can receive its protein substrates and save the galaxy (or at least help fold proteins and save the yeast cell).
To give some background, Hsp90 (encoded by HSP82 and HSC82 in yeast) is a molecular chaperone that assists in the folding and maturation of specific protein substrates, or “clients”. It functions as a homodimer that undergoes ATP-regulated cycles of “opening” up to receive clients, closing, and then opening again. Many clients of Hsp90 first bind to a chaperone of the Hsp70 family, such as Ssa2p, which assists in the early stages of protein folding before interacting with Hsp90 and passing on the client.
The co-chaperone Sti1p comes into the picture by bridging Hsp70 and Hsp90 and helping them interact, so that the Hsp70-bound client can be delivered to Hsp90. Although this function of Sti1p has long been known, the exact mechanistic details have been obscure, and mutational studies have suggested that Sti1p does more than just bridge the two chaperones together.
Thanks to a recent GENETICS study by Reidy and coworkers, we now know more about how Sti1p helps save the galaxy: it not only helps Hsp90 interact with Hsp70, but also prepares Hsp90 to receive its client protein and advance in its reaction cycle.
To uncover the role of Sti1p in the Hsp90 cycle, the authors examined Hsp90 mutants that were dependent on Sti1p for viability. They mapped both previously-known and newly found Sti1-dependent mutants and found that all of the mutations clustered in just two sites. They designated the sites Sti1-dependent N and C-terminal domain proximal, or “SdN” and “SdC”, respectively.
Because previous studies showed that some Hsp90 SdN mutants don’t interact well with Hsp70, and that analogous SdC mutations in E. coli weaken interactions with Hsp90 client proteins, the authors hypothesized that Sti1p assists Hsp90 with these functions in particular.
To clarify how Sti1p and Hsp90 cooperate, the authors utilized a combination of mutational studies, pull downs with purified proteins, mass spectrometry, and more. They observed that SdN mutations in Hsp90 reduce interactions with Hsp70, while SdC mutations do not. Further, they found that the Sti1p dependency of SdN mutants could be cured through a novel suppressor mutation (E402R), which increases the interaction of Hsp90 with Hsp70. These results suggest that Hsp90 interacts with Hsp70 through the SdN region, and that Sti1p is needed to bring the two chaperones together if they aren’t able to do so well enough on their own.
Importantly, although the E402R suppressor mutation was able to “cure” SdN mutants of their Sti1p dependency, it was unable to do so in SdC mutants. This indicated that SdC mutants are defective in a function that Sti1p assists with, but one that’s separate from having Hsp70 interact with Hsp90.
To uncover why SdC mutants depend upon Sti1p for viability, the authors investigated suppressor mutations. The authors isolated multiple SdC suppressors and also found that a previously characterized mutation, A107N, is able to ameliorate the effects of SdC mutations. Previous studies on A107N show that this mutation promotes closure of the open-state Hsp90 heterodimer, which is an important step of the Hsp90 reaction cycle. The authors found that other SdC suppressor mutations were consistent with A107N and could relieve the Sti1p dependence of SdC but not SdN mutants. These results indicate that Sti1p not only promotes Hsp90-Hsp70 interaction, but also has an additional function in promoting Hsp90 heterodimer closure and progression of the Hsp90 cycle.
So it turns out that Sti1p is like R2-D2 in more ways than one. In its first role, Sti1p helps Hsp70 and Hsp90 interact, much like how R2-D2 helps Rey find Luke in The Force Awakens. In its second role, Sti1p helps Hsp90 accept its substrates, progress through its reaction cycle, and perform its function. This is similar to what R2-D2 does in Star Wars: The Last Jedi. In the movie, Luke initially shows reluctance to resume his role as a Jedi Master and help save the galaxy, despite being found by Rey. But thankfully, R2-D2 was there to motivate Luke to return to his heroic duties, much like how Sti1p is there to “motivate” Hsp90 to capture client proteins and do its job.
Thanks to the efforts of Reidy and coworkers, how Sti1p helps save the
galaxy yeast cell is that much clearer. Not only does Sti1p help Hsp70 interact with Hsp90 and deliver lightsabers client proteins, but it also helps Hsp90 do its job as a Jedi Master chaperone by promoting progression of the Hsp90 reaction cycle!
by Kevin MacPherson, M.S.
Categories: Research Spotlight
July 31, 2018
If there was a World Cup soccer championship for cellular proteins, it’s a pretty sure bet that calcineurin wouldn’t make the team. That’s because this protein is one of those players that just can’t help but use their hands! And as pretty much everyone knows, that’s a big no-no for soccer players (except goalies, of course).
Conserved across virtually all eukaryotic organisms — from plants and protozoa to fungi and humans — calcineurin is a very abundant calcium-binding protein. In fact, it’s so abundant that it makes up 1% of the total protein content in a cow’s brain!
But what does this ubiquitous protein do? Well, it’s “merely” responsible for regulating many diverse, fundamental life processes… things such as fertilization, development, behavior, life span, responses to environmental cues, immune responses, cell death, and so on.
For eukaryotes, certain environmental and developmental cues, such as hormones or nutrient availability, can initially signal their presence by causing a change in the cell’s internal calcium levels. Calcineurin helps detect these calcium level changes and then passes the signal on in a chain of events.
Calcineurin is a calcium-regulated protein phosphatase, meaning that when it is activated by calcium level changes, it removes a phosphate group(s) from other proteins, particularly transcription factors. When these transcription factors have their phosphate group(s) chopped off by calcineurin, they travel into the cell’s nucleus and turn on a specific set of genes needed to make the cell respond appropriately to the original environmental or developmental cue.
Calcineurin is actually made up of two different proteins that bind together. One is a catalytic protein subunit (called CNA) and the other is a regulatory subunit (CNB). In our yeast friend Saccharomyces cerevisiae, the regulatory CNB protein subunit of the calcineurin complex is encoded by the CNB1 gene.
The Cnb1 protein contains a set of four almost-identical short amino acid domains that are conserved in the CNB proteins of all other organisms. These motifs are called “EF hand” domains because each of them looks like a spread thumb and forefinger of a human hand. And crucially, each “EF hand” can grab and hold onto calcium ions (Ca2+). The four EF hands of the Cnb1 protein are called EF1, EF2, EF3 and EF4, respectively.
But besides holding onto calcium ions, what else do these hands do? Why are there 4 of them? Are each of the hands doing the same thing? Does the right hand know what the left (or the middle, or the other middle) hand is doing?
The authors made a series of 4 different mutant CNB1 genes, each one having a disabling mutation in one of the 4 EF hands so that the hand can’t grab calcium ions any more. They put these mutant-handed CNB1 genes into yeast cells that had their normal CNB1 gene completely removed. The yeast cell thus ends up depending on a Cnb1 protein with one mutant hand and three functional hands. In a way, they are making Cnb1p have one of its 4 hands tied behind its back, and then seeing how well it can do its job!
How did they test how well each of the mutant-handed proteins works? Remember that the Cnb1 protein is the regulatory subunit of calcineurin, and calcineurin activates a transcription factor by dephosphorylating it. In yeast, the transcription factor regulated by calcineurin is encoded by the CRZ1 gene. The Crz1 protein recognizes and binds a certain DNA sequence (called CDRE) located just upstream of each one of its target genes and turns these genes on, ultimately changing the yeast cell’s behavior during calcium signaling. The authors put a special reporter gene into their yeast strains; this reporter gene has the special CDRE DNA sequence fused to an often-used bacterial gene called lacZ. The amount of lacZ protein produced by the yeast cells (which positively correlates with calcineurin function) can be sensitively monitored by an easy test tube assay.
Using this test tube assay, Connolly and co-workers tested how well each of the EF hand mutants worked. First they tested how well each could turn on the CRZ1-regulated genes, and also how well the mutant proteins detected calcium. Then they also tested how well each of the mutant-handed Cnb1 proteins worked in high salt environments, during the mating response, under oxidative stress conditions, and even in the presence of immunosuppressive drugs! Why the latter? Calcineurin is a target of immunosuppressive drugs, which are used when people get organ transplants to stop their own bodies from attacking the “foreign” organ. Yeast calcineurin is so similar to human calcineurin that it too is affected by these drugs!
The results were clear (well, actually yellow in the assay)—in all cases, the Cnb1 protein was able to have its EF4 hand disabled and still function perfectly or almost as well as the intact Cnb1 protein! But whenever one of the other EF hands was disabled, the function of calcineurin suffered, and this was true for each of the many ways it was tested, from salt to mating to immunosuppressive drugs.
It appears that when any of the useful hands (EF1, 2 or 3) were mutated, it causes Cnb1p to improperly change its shape in response to calcium, and this misshapen protein can’t do its job of activating Crz1p and ultimately getting the cell to respond to calcium-mediated signals properly.
And (#APOYG alert!) these yeast genetic results for the 4 EF hands match very closely to what’s been seen for mammalian calcineurin EF hand mutants in test tube (“in vitro“) experiments, giving an even stronger confirmation to these mammalian results. Maybe yeast will help develop new strategies for calcineurin-related diseases!
So now back to soccer… As we’ve found out, calcineurin HAS to use its hands, so it’s not a good pick for a regular soccer position player. But maybe it could be a super-awesome goalie since it has 4 hands! It even seems that you can tie its EF4 hand behind its back and Cnb1p can still guard the goal just fine with its remaining 3 hands – the EF4 hand seems to be a totally useless appendage! But if you disable any of the other hands, then it causes the Cnb1 protein to bend awkwardly and not do its job anymore.
Thanks to the efforts of Connolly and coworkers, we now know that it’s not quite “all hands on deck” for Cnb1p, but rather “EF 1, 2, and 3 hands on deck” in order to carry out its “goal” of regulating cellular responses to calcium!
by Barbara Dunn, Ph.D. and Kevin MacPherson, M.S.
Categories: Research Spotlight