A “door” into the mitochondrial membrane

Mitochondria — the organelles responsible for energy production in human cells — were once free-living organisms that made their way into early eukaryotic cells over a billion years ago. They have since seamlessly merged with their hosts in a classic example of symbiotic evolution, and now rely on many proteins made in their host cell’s nucleus to function properly.

Proteins on the outer membrane of the 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 have developed a specific mechanism to insert these proteins, which are made in the cell’s cytoplasm, into the mitochondrial membrane. But what this mechanism was and which cellular actors were involved has long been a mystery.

A new paper from the labs of Whitehead Institute member Jonathan Weissman and California Institute of Technology professor Rebecca Voorhees provides a solution to this conundrum. The work, published October 21 in the journal Scienceshows that a protein called Mitochondrial Carrier Homolog 2, or MTCH2 for short, which has been implicated in many cellular processes and even diseases such as cancer and Alzheimer’s, is responsible for allowing a variety of proteins to act as a “door” to gain access mitochondrial membrane.

“Until now, nobody knew what MTCH2 really does — they just knew that when you lose it, all these different things happen to the cell,” said Weissman, who is also a professor of biology at the Massachusetts Institute of Technology and an investigator at the Howard Hughes Medical Institute. “It was kind of a mystery why this one protein affects so many different processes. This study provides a molecular basis for understanding why MTCH2 was involved in Alzheimer’s and lipid biosynthesis and mitochondrial fission and fusion: because it was responsible for inserting all these different types of proteins into the membrane.”

“Collaboration between our laboratories has been essential in understanding the biochemistry of this interaction and has led to a really exciting new understanding of a fundamental question in cell biology,” said Voorhees.

The search for a door

To figure out how proteins from the cytoplasm — specifically a class called tail-anchored proteins — were introduced into the outer membranes of mitochondria, Alina Guna, a postdoctoral researcher and first author of the study at Whitehead Lab, worked with Taylor Stevens, a graduate student at Voorhees Lab, along with Taylor Stevens and postdoc Alison Inglis, adopted a technique called the CRISPR Interference (or CRISPRi) screening approach invented by Weissman and co-workers.

“The CRISPR screen had us systematically get rid of each gene and then look and see what happened [to one specific tail-anchored protein],” Guna said. “We found a gene, MTCH2, where when we got rid of it, there was a huge drop in how much of our protein got to the mitochondrial membrane. So we thought maybe that’s the door to get in.”

To confirm that MTCH2 acts as a gateway 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 both necessary and sufficient to allow tail-anchored membrane proteins to move from the cytoplasm to the mitochondrial membrane.

MTCH2’s ability 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 a protein’s structure based on its amino acid sequence, which revealed that it is a hydrophobic protein – perfect for inserting into the oily membrane – but with a single hydrophilic groove where other proteins might invade.

“It’s basically like a funnel,” Guna said. “Proteins come out of the cytosol, they slip into this hydrophilic groove, and then they move from the protein into the membrane.”

To confirm that this groove was important for the protein’s function, Guna and her colleagues designed another experiment. “We wanted to play around with the structure to see if we could change its behavior, and we were able to do that,” Guna said. “We went in and made a single point mutation, and that point mutation was enough to really change how the protein behaved and how it interacted with substrates. And then we went further and found mutations that made it less active and mutations that made it super active.”

The new study has applications beyond answering a fundamental question in mitochondrial research. “A lot of things come out of this,” Guna said.

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

The work also raises questions about how MTCH2 evolved its function over time. MTCH2 evolved from a family of proteins called solute carriers that transport a variety of substances across cell membranes. “We’re really interested in this evolutionary question, how do you engineer a new function from an old, ubiquitous class of proteins?” said 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 even make their way to the mitochondria. “I think that [this paper] is just the first step,” Weissman said. “This only applies to one class of membrane proteins – and it doesn’t tell you all the steps that occur after the proteins are made in the cytoplasm. For example, how are they transported to the mitochondria? So stay tuned – I think what we’re going to learn is that we now have a very nice system to open up this fundamental piece of cell biology.