New & Noteworthy
December 18, 2014
Yeast need translation and organelle tethering to sequester misfolded proteins away from the cytoplasm:
Sometimes parents feel like they’re constantly nagging their kids to clean things up. Eventually, some parents just give in and do it themselves. It turns out that yeast cells aren’t all that different.
And it is even more important for cells to clean up their cellular trash than it is for that sullen teenager. If trash like misfolded or damaged proteins is not sequestered from the rest of the cytoplasm, it can cause other proteins to change their conformation or interfere with metabolic processes. This is obviously much worse than having a smelly room!
One way that cells bin their trash is by compacting it into globs, or aggregates. While this works in the short run, as cells age these aggregates build up, and in human cells, they’re hallmarks of diseases like ALS and Alzheimer’s.
In a new study published in Cell, Zhou and colleagues looked at the process of protein aggregation in S. cerevisiae. A number of other studies had already suggested that just like human parents, mother yeast cells clean up trash for their daughters. The researchers confirmed this and also made a couple of very surprising findings.
Zhou and coworkers discovered that although aggregates are mostly composed of previously translated proteins, they don’t form without new translation. And they found that aggregates aren’t littered around the cytoplasm, but instead are collected in very specific trash bins located on the surface of cellular organelles.
To do these studies, the scientists assembled a toolkit of ways to induce and visualize protein aggregation. They used stresses like heat shock or various chemicals to stimulate aggregate formation.
They visualized the aggregates by using GFP-labeled Hsp104p, which binds to them specifically. They also had some thermally unstable reporter proteins fused to different colored fluorescent markers that helped them track aggregation. And they created time-lapse videos to watch the whole process.
They first looked in detail at aggregate formation, creating two different kinds of cellular trash (aggregates of distinct sets of marker proteins) at different times to ask whether they would all end up in the same aggregates or in separate ones. These experiments showed that in general, newly aggregated proteins will join existing aggregates rather than creating new ones. And intriguingly, these results also hinted that new translation was needed to start the aggregation process.
Zhou and coworkers tested this directly by adding cycloheximide, an inhibitor of translation, to their aggregation experiments. Sure enough, cycloheximide prevented all of the treatments from causing aggregation, and other treatments and conditions that blocked translation did the same thing.
So just as the threat of taking away the car keys may get that sullen teenager off the couch to start cleaning up, newly synthesized polypeptides are the inducer for the cellular clean-up. Without them, all the trash just stays littered around the cell.
The researchers guessed that if aggregation starts at sites where translation is occurring, it might be concentrated at the surface of the endoplasmic reticulum (ER), where a large proportion of ribosomes are bound. They used several different sophisticated microscopy techniques to confirm that most aggregates were in fact associated with the ER.
But they also got a surprise: in addition to the ER, many aggregates were associated with the mitochondrial surface and some even formed directly on it.* The authors also observed aggregates that formed at the ER but migrated along it to ER-mitochondrial contact points and eventually to mitochondria. So, cells don’t litter their trash just anywhere; they start specific collection points on the surfaces of organelles. And much of the trash seems to end up attached to mitochondria.
The researchers noticed something else when they looked at aggregates in cells that were dividing. When a bud forms, mitochondria are actively transported into it. However, the aggregates that were on mitochondria didn’t go into buds. They stayed on mitochondria that remained in the mother cell. Mom protects her daughter from any trash that mom created!
To figure out what controls this asymmetric segregation, Zhou and colleagues tested a panel of 72 mutant strains, each with a deletion in a mitochondrial outer membrane protein. One strain, the fis1 null mutant, was markedly defective: aggregates often went into the bud. Fis1p is known to be involved in mitochondrial fission, but this result suggests it may have an entirely separate role in making sure that trash-bearing mitochondria stay in the mother cell.
And finally, the authors saw that as mother cells got older, they got less and less able to keep aggregates out of their daughters’ cytoplasm. Towards the end of the mothers’ lives, aggregates were distributed more or less randomly between mother and daughter.
Trash build up is a big problem in teenagers’ rooms and in cells. Just like mom, the cell packs the trash away into bins where it will do less harm. Unfortunately, as the cell (and mom) get older, this gets harder and harder to do. In both cases, the daughter is saddled with more and more trash as mom struggles to keep up. And this is bad for the daughter as well as for the mom.
So, there’s actually a very good reason behind all that nagging to clean up your room. The secret to a long life is to always pick up your trash!
*New data from the Weissman lab, described here in a recent blog post, dovetail nicely with this finding since they establish that a lot of translation takes place on the mitochondrial surface.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
December 10, 2014
A simple tool for adding telomeres to linear DNA:
Instead of being a homicidal robot from the future bent on killing Sarah Connor, the telomerator is a tool that lets scientists easily turn circular DNA into stable chromosomes in the yeast Saccharomyces cerevisiae. While less splashy, this bit of synthetic biology is definitely cool in its own way (and much less dangerous!).
The system that Mitchell and Boeke created is very clever. They first inserted the intron from the ACT1 gene into the middle of the URA3 gene. The URA3 gene was still functional, as ura3 mutants could use it to grow on medium lacking uracil.
They next inserted a sequence into the middle of the intron that consisted of an 18 base pair I-SceI cleavage site flanked on each side by around 40 base pairs of yeast telomere repeats (called Telomere Seed Sequences or TeSSs). This construct still allowed ura3 mutants to grow in the absence of added uracil.
The final step was to introduce the homing endonuclease I-SceI to the cell so that it cut the circular DNA precisely between the two TeSSs. The idea is that when you add the homing endonuclease, the newly linearized piece of DNA ends up with the telomere seeds on each end. Telomerase adds more repeats to the seeds until the DNA has proper telomeres. Voilà, a chromosome is born.
The URA3 gene part of the plasmid is important for selecting cells with the linearized DNA. Basically a circularized DNA will grow on medium lacking uracil but fail to grow on medium with 5-FOA, while the linearized DNA will do the opposite. In other words, the process of linearization should destroy the URA3 gene. And that’s just what they found.
Previous work had shown that to be stable in yeast, a chromosome needs to be at least 90 kilobases (kb) or so long. This is why they tested their new telomerator in synIXR, a synthetic yeast chromosome that is about 100 kb in length. This chromosome has 52 genes from the right arm of chromosome 9, two genes from the left arm, around 10 kb of nonessential BAC DNA, the native centromere CEN9, and a LEU2 marker.
Mitchell and Boeke inserted the telomerator sequence into two different locations in the BAC part of the circularized synIXR and found that adding I-SceI appeared to linearize the DNA. In both cases they found that around 100 out of 200 cells were resistant to 5-FOA and unable to grow in the absence of uracil but could still grow in the absence of leucine. This is just what we would predict if we cut the DNA in the middle of the URA3 gene and created a stable piece of linear DNA.
They next wanted to use this tool to study the effects of telomeric DNA on nearby genes. We would predict that because of telomeric silencing, genes near a telomere will be downregulated. Any genes that affect growth when turned down should quickly become evident.
To accomplish this they inserted the telomerator three base pairs downstream of each of the 54 genes on synIXR, generating 54 new plasmids. After activating the telomerator by expressing the I-SceI nuclease, they used pulsed field gel electrophoresis to confirm that 51 of the 54 synthetic chromosomes had indeed been linearized.
As expected, they found that putting a telomere near a gene sometimes has profound effects. For example, when they linearized DNA where the telomerator was 3’ of either YIR014W, MRS1, or YIR020C-B, they got no growth. They also found many more effects on the growth rate at both 30° C and 37° C at many different, “telomerized” genes. The implication is that when these genes are near telomeric DNA, they no longer function at a high enough level for the yeast to grow well or in some cases to even survive.
To confirm that the effects they saw were due to telomeric silencing, Mitchell and Boeke tested each linearized DNA in a sir2 mutant, a key player in this form of silencing. Mutating sir2 reversed the effects of placing a telomere near the gene, further supporting the idea that the newly created chromosome ends are like normal telomeres because they undergo the same Sir2-mediated silencing.
Finally, the researchers tested the stability of the newly created chromosomes by selecting for Ura+ revertants from six individual cultures with different linearized molecules. They failed to select any revertants in which the DNA had recircularized, showing that the linear chromosomes are stable.
So in contrast to the Terminator, who sliced and diced his victims randomly, the telomerator will allow synthetic biologists to create linear chromosomes with precisely positioned telomeres. This study proved the concept, and this tool will be incredibly useful in the future, both in yeast and potentially in other eukaryotes. Both the Terminator and the telomerator can say, “I’ll be back”!
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
December 4, 2014
A collapsible protein complex makes sure that dividing cells get the right number of chromosomes:
The invention of collapsible tent poles was a boon to backpackers everywhere. These long, rigid poles provide strong support for a tent, but when it’s time to pack up and go, they fold up into a short package. The secret? Flexible regions in between the rigid sections.
Although the inventors of these poles couldn’t have known it, something similar already existed in nature. In a new paper in GENETICS, Tien and colleagues used the awesome power of yeast genetics, along with some cool biochemistry, to look at the shape of the S. cerevisiae Ndc80 complex in vivo. They found that, just like a tent pole, it bends sharply at a flexible region to fold two rigid sections next to each other. And this ability to fold is critical for accurate chromosome segregation.
During cell division, chromosomes must be correctly attached to the mitotic spindle so that the mother and daughter cells each get one, and only one, copy of each. If a yeast cell doesn’t get this process right, it can become sick or even die. And if it happens in an animal cell, it can lead to cancer.
The Ndc80 complex, which is conserved from yeast to humans, is an integral part of this process because it connects the chromosomes to the spindle during mitosis. It consists of four subunits and has an elongated middle and globular parts on each end, sort of like a dumbbell. The Ndc80 protein, one of the subunits, has an unstructured “loop” region in the middle of its elongated section.
Previous work had shown that the loop region of Ndc80 is flexible in vitro, and in vivo experiments had shown that the whole complex can change its conformation. Tien and colleagues wanted to know whether the Ndc80 loop region was important for the shape of the complex during mitosis, and whether flexibility in this region was important for function.
They started with a genetic approach, and isolated mutations in NDC80 that caused heat sensitivity. One particular allele, ndc80-121, was especially interesting. The mutant protein had two amino acid changes, near each other and near the loop region. The Ndc80 complex containing the mutant protein was just as stable, and bound to microtubules just as tightly, as the wild-type complex. So why did the cells die at higher temperatures?
Tien and colleagues visualized mitosis in the mutant cells using fluorescence microscopy. They could see that when they raised the temperature, dividing mutant cells had lots of aberrant attachments between chromosomes and the spindle. Because of these attachments, proceeding through mitosis caused their spindles to break—a lethal event.
However, if they timed the temperature shift to happen later in the cell cycle, the ndc80-121 mutant cells were fine. If chromosomes had already been lined up correctly on the spindle before the temperature was raised, then the rest of mitosis could go on without a problem.
Tien and coworkers wondered whether the mutation might disrupt the binding of some other protein to the complex at high temperatures. To look for interactions, they selected mutations that suppressed the heat-sensitive phenotype of ndc80-121. But they didn’t find any suppressor mutations in other genes. However, they did find an intragenic suppressor mutation within the ndc80-121 gene.
Interestingly, this mutation affected a residue that was on the other side of the loop relative to the original two changes. If the Ndc80 complex is a dumbell, imagine that the dumbell is collapsible like a two-segment tent pole, with the loop region of Ndc80 as the elastic between the sections. If you folded the complex in this way, the amino acids changed in the ndc80-121 mutant protein would be positioned close to the amino acid that the suppressor mutation affected—an intriguing explanation for how these mutations might affect each other.
Of course, genetic interactions don’t prove a direct physical interaction. So the researchers looked to see whether they could detect physical interactions between these regions. They treated the complex with a reagent that would permanently cross-link amino acids that were close to each other. Then they chopped the complex into smaller peptides using a protease, and analyzed the cross-linked peptides using mass spectrometry to locate the linked residues.
Sure enough, they were able to detect multiple cross-links within the complex, and their locations confirmed that the complex folds much like a tent pole. Based on their mutant phenotypes, the researchers think it’s likely that the original ndc80-121mutation destabilizes folding of the complex and that the intragenic suppressor mutation makes folding tighter. Consistent with this idea, the intragenic suppressor mutation alone confers a slow-growth phenotype, as if it makes the complex fold just a little too tightly to support vigorous growth.
These experiments as a whole establish that the Ndc80 complex folds tightly early in mitosis. So, creative inventors and Mother Nature have arrived at similar solutions for the tent pole and for this important complex. And just as collapsible tent poles have become ubiquitous in the backpacking world, so too has the collapsible Ndc80 complex been conserved throughout evolution: even the specific residues that mediate the folding are highly conserved. Since this work has shown that correct folding of the yeast complex is necessary for its role in helping chromosomes to line up accurately on the spindle, the same is almost certainly true in mammalian cells.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
November 24, 2014
If you want to see what animals really do out in the wild, first you need to hide a camera and a trip-wire so well that the jungle seems totally undisturbed. Then, if you’re lucky, you’ll be able to catch them in the middle of the night as they pass by. Now you can surprise that tiger and find out what he is doing at that specific spot.
In two companion Science articles from the Weissman group at UCSF, Jan et al. and Williams et al. did essentially the same thing to S. cerevisiae ribosomes. They hid a molecular tag and the enzyme that recognizes it at various interesting places within yeast cells, so cleverly that the cells had no idea anything was different. Instead of a flash of light, they used a pulse of the small molecule biotin to find out which mRNAs were being translated at specific locations in the cell.
What they found was that when ribosomes are translating proteins that are targeted to a particular organelle, they hang around the surface of that organelle—way more frequently than was previously thought. And the exquisite specificity of this technique, allowing them to pinpoint one particular mRNA within the cell, uncovered a fascinating case of dual protein localization.
Pinpointing Translation Locations
The researchers needed to develop a technique for catching ribosomes in the act of translation. One part of this had already been worked out in the same group: ribosomal profiling, a method that allows you to map very precisely the positions of ribosomes on mRNAs.
Briefly, cells are lysed and translating ribosomes are treated with nucleases that nibble away mRNAs, except for the 30 nucleotides or so that are protected within the ribosome. Then those protected fragments are analyzed by deep sequencing. This shows, at the single nucleotide level, where ribosomes are sitting on each individual mRNA.
Ribosomal profiling tells us where translating ribosomes are in relation to mRNAs, but not where they are in relation to the rest of the cell. To get this location information, the researchers came up with a clever tagging strategy.
They started with a bacterial gene, E. coli BirA, that encodes a biotin ligase—an enzyme that can attach biotin to specific acceptor peptides. They fused BirA to various yeast genes in order to target biotin ligase to different places in the cell.
Next they tagged ribosomes by putting a biotin acceptor, called the AviTag, on ribosomal proteins such that the tag would be sticking out on the ribosomal outer surface. They tested both the BirA and AviTag fusions to make sure that they didn’t interfere with the functions of any proteins. Just like the camera hidden in the jungle, the tags didn’t perturb yeast cells in the least.
Now the researchers were set to surprise ribosomes with a pulse of biotin. Any ribosomes that were close to BirA would become biotinylated. The tagged ribosomes could then be isolated, and the mRNA sequences being translated in those ribosomes could be identified. The method as a whole is termed proximity-specific ribosomal profiling.
Jan and coworkers set up and validated this method in their paper, and used it to look at translation of secretory proteins at the surface of the endoplasmic reticulum (ER), while Williams and colleagues used the method to look closely at translation at the mitochondrial surface. Import into both of those organelles has previously been studied intensively, but often in vitro and mostly for just a few model protein substrates. In contrast, proximity-specific ribosomal profiling gives us the ability to look at translation of the entire proteome in vivo.
While it was known before that proteins targeted towards a certain organelle tended to be translated near that organelle, these researchers found that it was much more common than previously believed. For example, they found that most mitochondrial inner membrane proteins were translated at the mitochondrial surface and imported cotranslationally, in contrast to the previous view that mitochondrial import is predominantly posttranslational.
Both studies discovered many more details than we can summarize here. But the comparison between the ER and mitochondrial studies led to a special insight about one protein.
Osm1p Goes Both Ways
Osm1p, fumarate reductase, was thought to be a mitochondrial protein (although results from a few high-throughput studies had hinted at a link to the ER). But proximity-specific ribosomal profiling showed very clearly that it was translated at both the ER and mitochondrial surfaces. Williams and coworkers went on to confirm by fluorescence microscopy of an Osm1p-GFP fusion that Osm1p is indeed present in both ER and mitochondria.
Both of these organelles have pretty strict criteria for the signal sequences of proteins they import, so how could it be possible that the same protein goes to both locations? The researchers found that in fact, it’s not! They repeated the ribosomal profiling on the OSM1 mRNA, this time adding the drug lactimidomycin which makes ribosomes pile up at translational start sites. This showed that OSM1 actually has two start codons and produces two different proteins targeted to the two locations.
The OSM1 methionine codon currently annotated as the start would produce a protein with an ER targeting signal. Ribosomes piled up there, but also at another methionine codon 32 codons downstream. Starting translation at this codon would produce a protein with a mitochondrial targeting signal. Williams and colleagues confirmed this idea by showing that mutating the first Met codon made all of the Osm1p go to mitochondria, while mutating the Met codon at position 32 sent all of it to the ER.
The mutant form of Osm1p that couldn’t go to the ER conferred an intriguing phenotype: the inability to grow in the absence of oxygen. Osm1p generates oxidized FAD, which is necessary for oxidative protein folding, and it also interacts genetically with ERO1, which is involved in this process. Taken together, this all suggests that Osm1p activity drives oxidative protein folding in the ER.
The traditional ways of determining where a protein is in the cell, microscopic visualization or physical fractionation, can both be difficult and imprecise. Proximity-specific ribosomal profiling gets around those challenges, and gives a very precise picture of exactly where proteins are being created and how ribosomes are oriented with respect to organelles.
The example of Osm1p localization gives just a hint of the insights that are waiting for scientists who exploit this technique further. And we’re not just talking about yeast: the authors tested and validated the method in mammalian cells. Just like that tiger, surprised ribosomes in many different cell types will be giving up their secrets about where they roam and what they do.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD