A “door” in the mitochondrial membrane

Mitochondria—the organelles responsible for producing energy in human cells—were once free-living organisms that found their way into early eukaryotic cells a billion years ago. Since then, they have fused seamlessly with their hosts in a classic example of symbiotic evolution, and now rely on many proteins produced in their host cell’s nucleus to function properly.

Proteins in the outer membrane of mitochondria are particularly important. they allow mitochondria to communicate with the rest of the cell and play a role in immune functions and a type of programmed cell death called apoptosis. During evolution, cells evolved a specific mechanism by which these proteins—which are produced in the cell’s cytoplasm—are inserted into the mitochondrial membrane. But what that mechanism was, and which mobile players were involved, has long been a mystery.

A new paper from the labs of MIT professor Jonathan Weissman and Caltech professor Rebecca Voorhees provides a solution to this mystery. The work, published on October 21 in the magazine Sciencereveals that a protein called mitochondrial carrier homolog 2, or MTCH2 for short, which has been linked to many cellular processes and even diseases such as cancer and Alzheimer’s, is responsible for acting as a “doorway” for a variety of proteins to access in the mitochondrial membrane.

“Until now, nobody knew what MTCH2 really did—they just knew that when you lose it, all these different things happen in the cell,” says Weissman, who is also a member of the Whitehead Institute for Biomedical Research and a researcher at the Institute of Medicine. Howard Hughes. “It was a mystery why this protein affects so many different processes. This study provides a molecular basis for understanding why MTCH2 is involved in Alzheimer’s and lipid biosynthesis and mitochondrial fission and fusion: because it was responsible for bringing all these different types of proteins into the membrane.”

“The collaboration between our labs has been essential to understanding the biochemistry of this interaction and has led to a really exciting new understanding of a fundamental issue in cell biology,” says Voorhees.

The search for a door

In order to learn how proteins from the cytoplasm—specifically a class called tail-anchoring proteins—were inserted into the outer membranes of mitochondria, Weismann Lab postdoc and first author of the study Alina Guna, along with Voorhees Lab graduate student Taylor Stevens and H postdoc Alison Inglis, decided to use a technique called the used CRISPR interference screening (or CRISPRi) approach, which was devised by Weissman and colleagues.

“The CRISPR screen allows us to systematically knock out each gene and then see what happened [to one specific tail-anchored protein]”, says Guna. “We found a gene, MTCH2, where when we knocked it out there was a huge reduction in how much of our protein made it to the mitochondrial membrane. So we thought, maybe this is the door to get in.”

To confirm that MTCH2 was functioning as an entrance to the mitochondrial membrane, the researchers performed additional experiments to observe what happened when MTCH2 was not present in the cell. They found that MTCH2 was necessary and sufficient to allow tail-anchored membrane proteins to move from the cytoplasm to the mitochondrial membrane.

The ability of MTCH2 to transport proteins from the cytoplasm to the mitochondrial membrane is likely due to its specialized shape. The researchers ran the protein sequence through Alpha Fold, an artificial intelligence system that predicts the structure of a protein through its amino acid sequence, which revealed that it is a hydrophobic protein—ideal for insertion into the oil membrane—but with a single hydrophilic groove where other proteins could enter.

“It’s basically like a funnel,” says Guna. “Proteins come from the cytoplasm, slide into this hydrophilic groove, and then move from the protein to the membrane.”

To confirm that this groove was important in the function of the protein, Guna and her colleagues designed another experiment. “We wanted to play with the structure to see if we could change its behavior, and we were able to do that,” says Guna. “We went and made a single point mutation, and that point mutation was enough to really change the way the protein behaves and the way it interacts with substrates. And then we went on and found mutations that made it less active and mutations that made it extremely active.”

The new study has applications beyond answering a fundamental question of mitochondrial research. “There are so many things that come out of it,” says Guna.

First, MTCH2 induces proteins key to a type of programmed cell death called apoptosis, which researchers could potentially harness for cancer treatments. “We can make leukemia cells more sensitive to a cancer treatment by giving them a mutation that changes the activity of MTCH2,” says Guna. “The mutation makes MTCH2 act more ‘greedy’ and insert more things into the membrane, and some of these things that have inserts are like pro-apoptotic factors, so those cells are more likely to die, which is fantastic in its context a cure for cancer’.

The work also raises questions about how MTCH2 evolved its function over time. MTCH2 evolved from a family of proteins called solute carriers, which transport a variety of substances across cell membranes. “We’re really interested in this evolutionary question, how do you evolve a new function from an old, ubiquitous class of proteins?” says Weissman.

And researchers still have a lot to learn about how mitochondria interact with the rest of the cell, including how they respond to stress and changes within the cell and how proteins find their way to mitochondria in the first place. “I think that [this paper] it’s just the first step,” says Weissman. “This only applies to one class of membrane proteins – and it doesn’t tell you all the steps that happen after the proteins are made in the cytoplasm. For example, how are they transported to the mitochondria? So stay tuned — I think we’re going to learn that we now have a very nice system to open up this fundamental part of cell biology.”