September 30, 2015
Stinging wasps get our attention, and with good reason—getting stung hurts a lot! If you see wasps going into a nest within the walls of your house, you’ll likely try to block their access.
But this could backfire: instead of being able to peacefully go into their nest, a swarm of angry wasps could be buzzing around looking for trouble. It might get so bad that you’ll need to call in an exterminator to take care of the problem.
Mitochondrial proteins might seem a lot less scary than wasps, but it turns out that they can also cause trouble if they can’t get into mitochondria. In two new letters to Nature, Wrobel and colleagues and Wang and Chen used complementary approaches to ask what happens when dysfunctional mitochondria aren’t able to import all of the proteins that are waiting to get in.
What they both found was that these piled up proteins cause real problems for the cells. In desperation, the cells slow down protein synthesis to reduce the excess, and also turn to their own exterminator, the proteasome, to keep these proteins under control.
This is a paradigm shift in thinking about how poorly functioning mitochondria cause disease. In the past, almost everyone focused on how damaged mitochondria couldn’t make enough energy for a cell. Now it looks like there are other ways for a nonworking mitochondria to do a cell in. And new targets for scientists to go after in treating mitochondrial disease.
As we all know, mitochondria are the powerhouses of the cell, where energy is generated, and they’re also the site of many other essential biochemical reactions. They’re composed of about 1,000 proteins, and nearly all of those are synthesized in the cytoplasm and then imported into mitochondria by an intricate system of transporters.
In order to find out what happens when these 1,000 proteins don’t get into mitochondria as efficiently as they should, both groups created strains whose mitochondrial import was impaired.
Wrobel and colleagues used the temperature-sensitive mia40-4int mutation, affecting an essential component of the mitochondrial import system. Wang and Chen started with the aac2-A128P mutation in PET9 (which is also known as AAC2), an ADP/ATP carrier of the mitochondrial inner membrane. Overexpression of the aac2-A128P allele causes mitochondrial dysfunction and eventual cell death.
Wrobel and colleagues decided to get a comprehensive look at what happens in the mia40-4int mutant by assaying its transcriptome and proteome, using RNA-seq and stable isotope labelling by amino acids in cell culture (SILAC), respectively. Surprisingly, one of the biggest differences from wild type that they saw in the import-defective mutant was a decrease in cytoplasmic translation. Whether they looked at the mRNAs encoding ribosomal proteins, the proteins themselves, or the polysome content and translational activity of the cells, everything pointed to down-regulation of translation. And at the same time, the proteasome—the molecular machine that breaks down unwanted proteins—was activated.
To verify that what they were seeing wasn’t peculiar to the mia40-4int mutant, Wrobel and colleagues slowed down mitochondrial import in several other ways: using different mia40 mutant alleles, other import mutants, or treatment with a chemical that destroys the mitochondrial membrane potential required for import. Under these different conditions causing the accumulation of mitochondrial precursor proteins in the cytoplasm, they still saw decreased cytoplasmic translation and increased proteasome activity.
Wang and Chen took a different approach, looking to see whether over-expression of any other genes could compensate for the lethality of overexpressing the aac2-A128P allele. The researchers transformed the mutant with a library of yeast genes on a multicopy plasmid, and found 40 genes whose expression could keep it alive.
The suppressor genes found by Wang and Chen were all involved in some aspect of synthesis or degradation of cytoplasmic proteins, just like the genes found by Wrobel and colleagues whose expression was altered in the mia40 mutant. And Wang and Chen also verified that these suppressors weren’t specific to the aac2-A128P mutation: they suppressed a variety of other mutations that decreased import.
Both groups observed precursors of mitochondrial proteins accumulating in the cytosol of the mutant strains they studied. Wang and Chen saw a couple other very interesting proteins increase in abundance: Gis2 and Nog2. These proteins are involved in regulating ribosome function, and the researchers speculate that their stabilization during this stress response contributes to the translational down-regulation. Intriguingly, their human orthologs are implicated in neuromuscular degenerative disease.
So, using orthogonal approaches, the two groups converged on the same model: a newly discovered cellular pathway that regulates cytosolic translation and protein degradation in order to deal with the stress of inefficient mitochondrial import. Wrobel and colleagues have named it UPRam, for Unfolded Protein Response activated by mistargeting of proteins, while Wang and Chen call it mPOS, mitochondrial Precursor Over-accumulation Stress.
Before this work, it was unknown whether cytosolic pathways were even affected by mitochondrial dysfunction. Now we know that the cell has a specific response when mitochondrial precursor proteins begin swarming in the cytosol, unable to get into their home: it slows down the production of those proteins and calls in the proteasome exterminator to take care of them.
We usually think of mitochondrial disease symptoms as being caused by the reduced energy generation of sick mitochondria, or by the lack of other key events that happen in mitochondria—for example, the synthesis of the iron-sulfur clusters that some vitally important enzymes need. Now, these findings raise the possibility that proteostatic stress on the cell caused by the accumulation of mitochondrial precursors could also lead to impaired cell function and disease.
Perhaps drugs that inhibited cytoplasmic translation, or activated the proteasome exterminator, would be helpful in reducing the buzzing swarm of mitochondrial precursor proteins. Wouldn’t it be wonderful if this knowledge suggested new avenues of treatment to take some of the sting out of human mitochondrial disease?
by Maria Costanzo, Ph.D., Senior Biocuration Scientist, SGD
Categories: Research Spotlight
Tags: mitochondria, proteasome, proteostatic stress, Saccharomyces cerevisiae
May 07, 2014
In the Sherlock Holmes mystery story “The Adventure of the Crooked Man,” a man and his wife are heard having an argument behind closed doors. There is a crash, and the doors are opened to reveal that the man is dead in a pool of blood and his wife has fainted. No one else is nearby, and it seems beyond any doubt that the wife has murdered her husband…of course, until Sherlock Holmes delves into the details and uncovers the real story.
Something similar is going on in the nucleus behind those nuclear pores. The proteasome is a huge molecular machine that recognizes and degrades ubiquitinated proteins. Scientists have seen key parts of the proteasome in the nucleus, and nuclear proteins are degraded by this complex. Seems like an open and shut case that the proteasome degrades nuclear proteins in the nucleus. But like our Sherlock Holmes story, things aren’t always as they appear.
Chen and Madura applied some detective work to the details of our nuclear protein degradation mystery in a new study published in GENETICS and found that it probably doesn’t work this way at all. Nuclear proteins need to be exported out of the nucleus to be degraded by the proteasome.
The researchers first confirmed earlier work showing that the Sts1 protein is responsible for escorting proteasomes to the nucleus. In the temperature-sensitive sts1-2 mutant at the restrictive temperature of 37 degrees, two proteasome subunits from different subcomplexes of the proteasome (Rpn11p from the regulatory particle and Pup1p from the catalytic particle) didn’t make it into the nucleus.
They then looked at two nuclear proteins, Rad4p and Pol1p (also known as Cdc17p) that are substrates of proteasomal degradation. When the proteasome subunits Rpn11p and Pup1p didn’t make it into the nucleus because of the sts1-2 mutation, Rad4p and Pol1p were not degraded.
So the proteasome needs to get into the nucleus in order for nuclear proteins to get degraded. Sounds like the proteasome is guilty of degrading proteins in the nucleus. But like a good mystery novel, the story takes an interesting twist here.
Chen and Madura found that when they raised the temperature of the sts1-2 mutant cells, Rad4p and Pol1p were stabilized immediately. This didn’t really make sense though. Even if the temperature-sensitive mutation blocked import of proteasomes into the nucleus as soon as the temperature increased, the proteasomes already inside the nucleus should have been able to continue degrading their substrates.
Wondering whether the substrates might be exported from the nucleus to be degraded elsewhere, they tested what happened to Rad4p and Pol1p when nuclear export of proteins was blocked. Using a few different ways to prevent nuclear export (combinations of mutations, chemicals, and temperature), they showed that if Rad4p and Pol1p could not get out of the nucleus, they were not degraded by the proteasome.
So it’s clear that nuclear export is part of the degradation process for at least these two nuclear proteins. Chen and Madura also detected a general increase in multi-ubiquitinated proteins (tagged for proteasomal degradation) in the nucleus under conditions where export was blocked, suggesting that this mechanism may apply to other proteins as well. And it’s been shown in human cells that several specific proteins, including the tumor suppressor p53, need to get out of the nucleus to be degraded.
There are still a lot of details to be filled in about where degradation is really happening. An intriguing clue comes from the fact that Sts1p is distantly related to the Schizosaccharomyces pombe protein Cut8, which is a nuclear envelope protein that tethers the proteasome to the nuclear membrane. Might nuclear proteasomes work on the outside of the nuclear envelope?
More detective work is needed to answer this question. But it’s clearly a very important one. Nuclear export and protein degradation are highly conserved processes, and both are currently under study as potential targets of cancer treatment.
Let this be a warning to all of us not to take everything at face value. Just because someone is holding the bloody knife, that doesn’t mean he is the murderer. And just because subunits and subcomplexes of the proteasome machinery look to be in the nucleus, that doesn’t mean nuclear proteins are degraded there. It is elementary, my dear Watson.
by Maria Costanzo, Ph.D., Senior Biocurator, SGD
Categories: Research Spotlight
Tags: nuclear export, proteasome, Saccharomyces cerevisiae