September 25, 2014
One of the great joys of teaching can be found in the questions that students ask. Because they are unconstrained by previous knowledge, they can think outside of the box and ask questions that force the teacher to see a problem in a new light. Their unbiased questions often uncover aspects of a problem that a teacher didn’t think to look for or even consider.
The scientific enterprise can be very similar. Sometimes an unbiased search of a process will uncover hidden parts scientists were completely unaware of.
This is exactly what happened in a new study in Science by Foresti and coworkers. Using an unbiased proteomics approach they found a previously hidden part of the endoplasmic reticulum-associated degradation (ERAD) pathway in the inner nuclear membrane (INM) of the yeast Saccharomyces cerevisiae. No one knew it existed before and, frankly, no one even knew to look! By thinking outside of the box, these authors found that a novel protein complex in the INM targets certain proteins for degradation – both misfolded proteins, and some correctly folded proteins whose functions are no longer needed.
Scientists already knew that the ERAD pathway uses different protein complexes to target proteins for degradation, depending on where those proteins are located. For example, misfolded cytoplasmic proteins are targeted by a complex containing Doa10 (also known as Ssm4), while those in the membrane are targeted by the Hrd1 complex. However, degradation of both sets of proteins requires ubiquitination by the shared subunit Ubc7. In addition to targeting misfolded proteins, both of these complexes also target certain functional proteins in response to specific conditions.
In the first set of experiments, Foresti and coworkers looked at the proteomes of strains deleted individually for Doa10, Hrd1, or Ubc7. To their surprise, they found a set of proteins, including Erg11 and Nsg1, that are unaffected by the deletion of either Doa10 or Hrd1, but whose levels are increased in strains deleted for Ubc7. This suggested there is a branch of the ERAD pathway that involves Ubc7 but is independent of Doa10 and Hrd1. The authors set out to find this undiscovered third branch lurking somewhere within the yeast.
Some possible candidates for being part of the ERAD pathway were two paralog proteins Asi1 and Asi3, and their associated protein Asi2. Based on their sequences, Asi1 and Asi3 are putative ubiquitin-protein ligases like Doa10 and Hrd1. Interestingly, all three Asi proteins localize to the inner nuclear membrane, which connects to the ER at nuclear pores.
When Foresti and coworkers deleted any one of the three Asi proteins, degradation of Erg 11 and Nsg1, both involved in sterol synthesis, was blocked. However deletion of Asi1, Asi2, or Asi3 didn’t affect all proteins involved in sterol biosynthesis, since Erg1 was unaffected. Biochemical experiments confirmed that Erg11 binds to a complex composed of these three Asi proteins.
Since the ERAD pathway is important for degradation of misfolded proteins, the authors set out next to determine whether the Asi complex plays a role in this process as well. That would be a somewhat surprising finding, since misfolded proteins aren’t generally found near the INM. But through a complicated set of experiments summarized below, Foresti and coworkers confirmed that the Asi complex does also have a role in this process.
They first tested several proteins that are known ERAD substrates, but mutations in the ASI genes had no measurable effect on them. Because some misfolded proteins are targeted by more than one ERAD complex, the authors next looked to see whether the Asi pathway contributed to either the Hrd1 or the Doa10 pathways. Testing the accumulation of several substrates in strains with different combinations of asi, hrd1, and doa10 mutations, they found that one mutant protein that misfolds, Sec61-2, had high steady state levels in a hrd1 knockout, but even higher steady state levels in a double knockout of hrd1 and asi1 or hrd1 and asi3. So both the Asi and Hrd1 pathways appeared to work on this misfolded protein.
The researchers hypothesized that the Asi branch may target misfolded proteins for degradation as they travel through the inner nuclear membrane on the way to the ER. To test this idea, they compared the steady state levels and localizations of two differently mutated versions of the Sec61 protein – one that localized to the inner nuclear membrane and one that did not, in both wild-type cells and a variety of deletion strains.
The bottom line from these experiments was that the mutant protein that was located at the inner nuclear membrane was more dependent on the Asi complex than the mutant that wasn’t. Not only that, but the mutant Sec61 protein that was directed to the inner nuclear membrane changed its localization to the nuclear envelope in an asi1 deletion strain. Both of these results are consistent with a role for the Asi complex in targeting proteins for degradation while they are in the inner nuclear membrane.
The final set of experiments confirmed the importance of the Asi complex in ER protein quality control. Yeast responds to the presence of too many misfolded proteins in the ER with a signaling pathway called the unfolded protein response (UPR). Strains in which this pathway is compromised, for instance by deleting IRE1, need a functional ERAD to thrive. The authors found that deleting HRD1, IRE1, and ASI1 had a much more severe effect on viability than did just deleting HRD1 and IRE1. This supports the idea that the Asi complex is important in ER protein quality control.
Foresti and coworkers have thus uncovered a previously undiscovered branch of the ERAD pathway in yeast by doing a broad, unbiased proteomics study. The key proteins they identified, Asi1, Asi2, and Asi3, were originally discovered for their genetic effects on the transcriptional repression of amino acid permeases (hence their name, Amino acid Signaling Independent). Their detailed biochemical functions were unknown until now.
A lesson here is that just because a process looks like it is pretty well locked down, this doesn’t mean that there aren’t hidden parts yet to be discovered. And just because a gene is implicated in one process, don’t assume it isn’t also involved in other processes as well. Looking from a different angle can allow you to see things you had missed before.
by D. Barry Starr, Ph.D., Director of Outreach Activities, Stanford Genetics
Categories: Research Spotlight