New & Noteworthy

Proteins Hate Crowds Too

May 29, 2013

A goldfish will swim faster through water than it will through gravy or Jell-O. And according to the results of a new study by Miermont and coworkers, it looks like the same may be true for proteins in a yeast cell.

Now obviously the authors didn’t replace the insides of a yeast cell with gravy or Jell-O. Instead, they used severe osmotic stress to remove water from the cell, making the interior more viscous and the proteins more crowded.

The movement of a wide range of proteins within the cell slowed to a crawl in these shrunken cells. In fact, if the cell was subjected to enough stress, the proteins in the cell essentially stopped moving at all. This lack of movement wasn’t because the high osmotic stress had killed the yeast…they recovered just fine when put back into a more osmotically friendly environment.

The first case the authors looked at was the pathway that helps yeast respond to osmotic stress, the HOG pathway. Once a cell is subjected to osmotic stress, Hog1p is phosphorylated and translocated into the nucleus where it can then turn on the genes needed to respond to this environmental insult.

Miermont and coworkers traced the movement of Hog1p using a Hog1p-GFP fusion protein. At 1 M sorbitol, which subjects the cell to mild osmotic stress, this fusion protein was phosphorylated within two minutes and had reached the nucleus within five.  There were already signs of cell shrinkage even under these mild conditions.

As sorbitol concentrations increased, the phosphorylation and nuclear localization took longer and longer to happen. At 1.8 M sorbitol, Hog1p-GFP didn’t reach maximum fluorescence in the nucleus until 55 minutes. At 2 M sorbitol and above, the cells were shrunken down to their minimal volume, which was 40% of their original size. Under those conditions, only 25% of cells had any nuclear fluorescence even after several hours.

The authors used fluorescence recovery after photobleaching (FRAP) to confirm that these effects were due to slowed protein movement. Basically they zapped a small portion of a cell to burn out the fluorescence of the Hog1p-GFP in that region and then timed how long it took fluorescing Hog1p-GFP from other parts of the cytoplasm to diffuse into that area. To prevent the complication of nuclear translocation in these experiments, they used a pbs2 mutant strain, in which the HOG pathway is blocked and Hog1p stays in the cytoplasm.

They found that fluorescence recovery took less than one second in normal media, about five seconds in 1 M sorbitol, and never happened at 2 M sorbitol. Clearly the protein’s movement was slowed with increasing osmotic pressure. But the effects weren’t permanent: once the cells were put back into normal media, the protein returned to its original kinetics.

The authors expanded their studies to look at proteins that shuttle between the nucleus and cytoplasm in other pathways, including Msn2p, Yap1p, Crz1p, and Mig1p, and obtained similar results. All four proteins took longer to reach the nucleus after being exposed to high osmotic stress. They also looked at other cellular processes where protein movement is critical, such as endocytosis, and again found that increased osmotic stress led to slower moving proteins.

So it looks like molecular crowding can definitely slow down protein movement and affect how a cell functions. For example, the time it takes to respond to environmental stimuli could be significantly slowed, affecting the cell’s chances for survival.  Since yeast cells in the wild encounter high-sugar environments (like rotting grapes), their protein density must be regulated so that they can get through stresses like this. They are ultimately much better adapted than that goldfish in the bowl of Jell-O.

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

YeastMine Upgrade

May 28, 2013

YeastMine, SGD’s powerful search and retrieval tool, has been upgraded to use InterMine version 1.1 software. Highlights of this release include a new format for the template results page, the addition of PantherDB and Homologene homolog data, an improved representation of Gene Ontology (GO) information, the ability to set background population within the GO enrichment widget, and an option to share lists with other users. In addition to the existing video tutorials, a new Help document describes some common queries. See an overview of these new features in the video below, New, Fun YeastMine 1.1!:

New, Fun YeastMine 1.1! from yeastgenome on Vimeo.

A Radical Discovery About a Well-Known Enzyme

May 22, 2013

Living your life puts a lot of wear and tear on you. A big reason is that as your cells go about their business, they churn out lots of damaging chemicals. 

One of the worst offenders is the free radical superoxide, O₂^-. Cells can’t help producing this powerful oxidant during normal metabolism, but it’s so toxic that it can destroy proteins and damage DNA.

Cells have come up with a two-step process to deal with this toxic waste. In the first step, they use the enzyme superoxide dismutase (Sod1p is the cytosolic form in yeast) to convert superoxide into the less harmful hydrogen peroxide (H₂O₂) and water. The cells then use catalases to take care of the H₂O₂, converting it to water and molecular oxygen. 

We’ve known about the first enzyme, superoxide dismutase, for decades. It has always been thought to have a simple role, sitting in the cytoplasm and detoxifying O₂^-. But new research shows that its job is considerably more interesting than that: it also has a role in a regulatory process known as the Crabtree effect.

The Crabtree effect is named after the scientist who first described it way back in 1929. Some types of cells are able to produce energy by either fermentation or respiration in the presence of oxygen. Since these two processes have different metabolic costs and consequences, which one to use is a critically important choice.

If lots of glucose is around, yeast cells choose fermentation. They prevent respiration by repressing production of the necessary enzymes, and this glucose-dependent repression is the Crabtree effect. It happens not only in yeast, but also in some types of proliferating cancer cells.

A new study by Reddi and Culotta shows that Sod1p is actually a key player in the Crabtree effect. In response to oxygen, glucose, and superoxide levels, it stabilizes two key kinases that are involved in glucose repression.

It was recently found that the sod1 null mutant can’t repress respiration when glucose is around.  This is different from the wild type, which is subject to the Crabtree effect. 

Reddi and Culotta started by investigating this observation and found that SOD1 is part of the glucose repression pathway that also involves the two homologous protein kinases Yck1p and Yck2p. They found that Sod1p binds to Yck1p, which wasn’t totally unexpected since this interaction had been seen before in a large-scale screen. The unexpected part was that Sod1p binding actually stabilizes Yck1p and Yck2p.  These stabilized kinases can now phosphorylate targets that propagate the glucose signal down the pathway and ultimately repress respiration.

Now the question is why does Sod1p binding stabilize the kinases? It turns out that its enzymatic activity is crucial for stabilization. One idea is that the hydrogen peroxide that Sod1p makes in the neighborhood of the kinases could inactivate ubiquitin ligases that would target them for degradation. Ubiquitin ligases are rich in cysteine residues, and so could be especially sensitive to oxidation by H₂O₂.

This regulation might also feed into other pathways: these kinases are also involved in response to amino acid levels, and the sod1 null mutant was seen to affect the amino acid sensing pathway in this study.

Most excitingly, this mechanism is not just a peculiarity of yeast Sod1p. The authors mixed and matched yeast, worm, and mammalian superoxide dismutases and casein kinase gamma (the mammalian equivalent of Yck1p/Yck2p), and found that binding and stabilization works in the same way across all these species.

Superoxide dismutases may have been drafted into this regulatory role during evolution because they are the only molecules that sense superoxide, whose levels reflect both glucose and oxygen conditions. A radical idea indeed!

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

Codependent Genes

May 16, 2013

When a gene is duplicated, one copy usually dies. It is battered by harmful mutations until it eventually just fades into background DNA.

But this isn’t the fate of all duplicated genes.  Sometimes they can survive by gaining new, useful functions.  The genes responsible for snake venom proteins are a great example of this.

Another way for a duplicated gene to live on is when both copies get different mutations that confer different functions, so that a cell needs both to survive.  Two examples of this type of codependent gene survival are highlighted in a new study by Marshall and coworkers.  They compared various fungal species and identified cases where two functions were carried out by either one gene or by two separate genes.  Surprisingly, these cases involve alternative mRNA splicing, which is a rare process in fungi.

The first gene pair they focused on was SKI7 and HBS1 from Saccharomyces cerevisiae.  In this yeast these two genes exist as separate entities, but in other yeasts like Lachancea kluyveri they exist as a single gene which the authors have called SKI7/HBS1. 

The SKI7/HBS1 gene makes two differently spliced mRNAs, each of which encodes a protein that matches up with either Ski7p or Hbs1p.  In addition, the SKI7/HBS1 gene can rescue a S. cerevisiae strain missing either or both the SKI7 and HBS1 genes.  Taken together, this is compelling evidence that SKI7 and HBS1 existed as a single gene in the ancestor of these two fungal species. In S. cerevisiae, after this gene was duplicated each copy lost the ability to produce one spliced form.

The second gene Marshall and coworkers looked at experienced the reverse situation during evolution.  PTC7 exists as a single gene that makes two mRNA isoforms in S. cerevisiae: an unspliced form that generates a nuclear-localized protein, and a spliced form that produces a mitochondrial protein. 

But in Tetrapisispara blattae, these two forms exist as separate genes.  The PTC7a gene is similar to the unspliced form in S. cerevisiae and the protein ends up in the nucleus, while the PTC7b gene is similar to the spliced S. cerevisiae version and its product is mitochondrial.  

Because an ancestor of S. cerevisiae had every one of its genes duplicated about 100 million years ago, yeasts have been a great system to study the fate of duplicated genes.  This study shows that even though gene duplication is widespread in fungi and alternative splicing is rare, these mechanisms are actually interrelated and each can increase the diversity of the proteins produced by a species.

Fun fact: 544 genes survived duplication in S. cerevisiae.  That is around 10%.

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