New & Noteworthy

Sensing Disturbances in the Membrane

September 25, 2017


In the original Star Wars movie, Darth Vader knows Obi-Wan is aboard the Death Star because he feels a “disturbance in the force.” The highly conserved protein Ire1p does something similar with disturbances in the membrane of the endoplasmic reticulum (ER) in Saccharomyces cerevisiae.

Comic Con reveals Spangdahlem's true identity

He can sense disturbances in the force. Ire1p can sense disturbances in the ER membrane. https://media.defense.gov

Ire1p isn’t sensing the presence of a Jedi master of course. Instead, it is sensing when the membrane is under stress through either the buildup of unfolded proteins in the ER lumen or membrane and/or through changes in the membrane itself.

And unlike Darth Vader, who upon sensing the presence of Obi-Wan seeks him out and destroys his physical form with a light saber, Ire1p instead forms dimers and larger oligomers when it senses a disturbance. This activates the unfolded protein response (UPR), which results in the activation of a bunch of genes so that the cell can start to deal with its unfolded proteins and perturbed membrane mess. (And no, unfortunately, the cell does not do this with tiny light sabers!)

Previous work had established the part of Ire1p that responds to unfolded proteins. But scientists had yet to nail down what part of the protein senses membrane problems. Until now that is.

In a new study in Molecular Cell, Halbleib and coworkers have not only worked out what part of Ire1p senses disturbances in the ER membrane in our old friend S. cerevisiae, but they may also have figured out how the protein does it.

At first blush, it would seem to make sense that the single, highly conserved transmembrane helix (TMH) of Ire1p would be important for sensing membrane issues. After all, it is in intimate contact with the membrane!

Previous mutation studies showed that the TMH alone probably isn’t the key player. Halbleib and coworkers confirmed this result by replacing all thirteen amino acids of the TMH in Ire1p with leucines and showing that this mutated protein was still able to respond to membrane problems caused by either too much dithiothreitol (DTT) or too little inositol.

A closer look at the protein revealed a conserved region of Ire1p that overlaps with the TMH and is predicted to form an amphipathic helix (AH). CD spectroscopy showed that an oligopeptide of identical sequence to this region formed an alpha helix in micelles but not in aqueous solution—it needs a membrane environment to become an alpha helix.

These authors used mutational analysis to show that the hydrophobic side of this AH is the part of the Ire1p involved in sensing problems in the ER membrane. For example, two specific mutations on the hydrophobic side, F531R and V535R, negatively affected a yeast cell’s ability to respond to disturbances in the ER membrane. Yeast containing Ire1p with a mutation on the hydrophilic side, R537E, however, had no such problem and responded fine. Ire1p function was also compromised when the researchers extended their leucine replacement of the transmembrane domain by three more residues into the AH.

So Halbleib and coworkers have seemingly identified the key part of Ire1p that can sense changes in the membrane. Now, they set out to examine how this part of the protein actually does the sensing.

ForceChoke

I find your lack of oligomerization disturbing. http://starwars.wikia.com/wiki/Force_choke

Right away the authors could see that the mutants that disrupted the ability of Ire1p to sense membrane disturbances also did not oligomerize under membrane-perturbing conditions in a living yeast cell. This makes sense as oligomerization is required to marshal the cellular forces needed to deal with these ER problems.

They confirmed that oligomerization could be affected by membrane composition with some in vitro experiments. They first made what they called a minimal sensor construct—the amphipathic helix and the transmembrane helix attached to the maltose binding protein. They reconstituted this sensor into eight different liposomes with varying lipid compositions.

Using continuous-wave paramagnetic resonance spectroscopy they were able to show that the ability of their minimal sensor to oligomerize depended upon the membrane composition. The more similar the composition was to a stressed membrane, the more likely their sensor was to oligomerize. This is exactly what we would expect if the AH was the key player in sensing membrane problems.

So certain mutations and membrane situations can affect the oligomerization state. But this still doesn’t really get at why Ire1 proteins oligomerize in perturbed membranes.

To try to figure this out, they turned to molecular dynamic simulations.

They found that the AH tended to compress the lipid bilayer and that the more stressed the membrane, the more energy this compression costs. They also found that if two Ire1p molecules happened to bump into each other and dimerize, that the energy costs went down.

In other words, stressed membranes tend to force Ire1p molecules to oligomerize to reduce the energy cost of being in the membrane. And of course oligomerization triggers the unfolded protein response (UPR)!

Ire1p senses disturbances in the membrane because the energy cost of remaining a monomer in stressed membranes is too high. Much more plausible than the midi-chlorians that supposedly let Darth Vader sense disturbances in the force!

The unfolded protein response (UPR) is involved in many diseases including cancer and diabetes. 

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

Categories: Research Spotlight

Tags: UPR , membrane sensing , IRE1

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