New & Noteworthy

Unfrying An Egg

January 20, 2016


Unlike the proteins in this egg, most aggregated yeast proteins get back to their normal shape after a heat shock. Image from Wikimedia Commons.

Eggs start out as slimy and awful, but can end up warm, firm and wonderful. All it takes is some heat to denature the egg proteins and voilà, a tasty breakfast.

Not that anyone would want to do it, but of course it is impossible to do the reverse. You can’t take a fried egg and turn it back into a raw one. The denaturation is pretty much permanent.

When a cell is hit with high temperatures, its proteins start to denature as well. And scientists thought that most of the denaturation of many of these proteins was as irreversible as the eggs. The thought was that many or most of these denatured proteins were “eaten” through proteolytic degradation. Although cellular chaperones are capable of disaggregating and refolding some heat-denatured proteins, it wasn’t known which aggregated proteins met which fate in a living cell.

A new study out in Cell by Wallace and colleagues shows that at least in yeast, most eggs get unfried. After a heat shock, aggregated proteins in the cell return to their unaggregated form and get back to work.

Now those earlier scientists weren’t crazy or anything. The proteins they looked at did indeed clump up and get broken down by the cell after a heat shock. But these were proteins introduced to the cell.

In the current study, Wallace and colleagues looked at normal yeast proteins being made at their normal levels. And now what happens after a brief heat shock is an entirely different story.

The first experiment they did looked at which endogenous yeast proteins aggregated after they were shifted from their normal 30 to 46 degrees Celsius for 2, 4, or 8 minutes. The researchers detected aggregation using ultracentrifugation—those proteins that shifted from the supernatant to the pellet after a spin in the centrifuge were said to have aggregated.

Using stable isotope labeling and liquid chromatography coupled to tandem mass spectroscopy (LC-MS/MS), they were able to detect 982 yeast proteins easily. Of these, 177 went from the supernatant to the pellet after the temperature shift. (And 4 did the reverse and went from the pellet to the supernatant!)

After doing some important work investigating these aggregated proteins, the researchers next set out to see what happened to them when the cells are returned to 30 degrees Celsius. Are they chewed up and recycled, or nursed back to health and returned to the wild?

To figure this out they did an experiment where proteins are labeled at two different times using two different labels. The researchers first grew the yeast cells at 30 degrees Celsius in the presence of arginine and lysine with a “light” label. This labels all of the proteins in the cell that have an arginine and/or lysine.

Then the cells are washed and a new media is added that contains “heavy” labeled arginine and lysine. The cells are shifted to 42 degrees Celsius for 10 minutes and then allowed to recover for 0, 20, or 60 minutes.

After 60 minutes of recovery, the ratio of light to heavy aggregated proteins looked the same as proteins that hadn’t aggregated. In other words, aggregation did not cause proteins to turn over more quickly.

It looks as if aggregated proteins are untangled and allowed to go about their business. So after a heat shock the cell doesn’t throw its hands in the air and simply start things over.

Other experiments done by Wallace and coworkers in this study, that we do not have the space to tackle here, suggest that the cell has an orderly process for dealing with heat stress. After a heat shock, certain proteins aggregate with chaperones in specific areas of the cell. Once the temperature returns to normal, these stress granules disassemble and the aggregated proteins are released intact.

None of this will help us unfry an egg — a denatured egg protein is obviously significantly different than an aggregated protein protected by chaperones in a stress granule. But this study does help us better understand how our cells work. And that’s a good thing.

Unlike Mr. Bill’s dog, most aggregated yeast proteins can return from a heat shock.

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

Categories: Research Spotlight

Tags: protein aggregates , protein aggregation , heat shock , chaperones

Mother Yeast Keeps Daughters Tidy

December 18, 2014


A mess in a room is annoying, but a mess in a cell may be lethal. Image by Tobin via Flickr

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

Categories: Research Spotlight

Tags: mitochondria , protein aggregation , Saccharomyces cerevisiae

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