Buttered cats, engineers, and membrane proteins

There’s an old joke about how to make an antigravity machine. See, cats always land on their feet. And, as the Victorian wag James Payn said,

“I’ve never had a piece of toast

particularly long and wide,

but fell upon a sanded floor,

and always on the buttered side.”

So, simply attach a piece of buttered toast to the back of a cat. The toast will want to hit the floor, but it can’t, because the cat’s feet also want to hit the floor. The cat and toast will hover, spinning as butter and paws both strive to make contact—antigravity and perpetual motion to boot!

An engineer would want to play with this, of course. It would be necessary to quantify just how big a piece of toast and how much butter would be required to offset so many grams of cat. One can imagine the white-coated experimenters with clipboards in hand, attaching precisely calibrated toasts to uncooperative moggies. Too much butter, the cat lands on its back. Not enough butter, the cat lands on its paws.

This is almost like an experiment recently described by a group of biochemists from Sweden. These researchers were looking at proteins instead of cats, and how these proteins land in a cell’s membrane. This is actually a significant problem, because many membrane proteins are like turnstiles. You could install a turnstile to only allow people out of a room, or only allow people to enter a room. Similarly, membrane proteins act like one-way gates for the cell, only allowing food in or only allowing toxins out. Getting one of these proteins into the membrane pointing the wrong way would be pretty bad—keeping food out, or importing toxins. The Swedes were looking at a protein in bacterial membranes that allows toxins to go out of the cell, and how it gets oriented the right way.

Going into this, the researchers already knew something about the problem. They knew that the proteins that sit in membranes tend to have more positively charged amino acids facing the inside of the cell than facing the outside of the cell (Proteins are basically long strings of amino acids that fold up into precise shapes; there are twenty different amino acids, some are positively charged, others negative or neutral). They also knew that the proteins needed help getting into the membrane, help that was provided by a group of proteins known as a “translocon,” already sitting in the membrane. What they thought they knew was that the translocon looked at just the first few amino acids in the string that made up the incoming protein, and made it point one way or the other based on that information.

The particular protein the Swedes looked at, called EmrE, was interesting because it was like a cat with just the right amount of buttered toast on its back—half the time it landed in the membrane pointing one way, half of the time pointing the other way. They worked on the assumption that the first few amino acids were important for letting the translocon set the protein’s orientation—so adding an extra very positively charged amino acid arginine at the very beginning of the protein made EmrE point one way, but putting an extra arginine in twenty bases further down the string made EmrE point the other way. So far, the hypothesis that orientation is determined by positively charged amino acids at the beginning of the protein seemed solid.

The biggest danger in research is falling in love with your hypothesis and not challenging it. The Swedes tried to prove themselves wrong by putting positively charged arginine amino acids throughout the EmrE protein. To their surprise (and pleasure, I imagine, since we get a buzz from finding the unknown), they found that it didn’t matter where in the protein the extra arginine was added. It could be at the beginning, the middle, or the end, and the extra arginine would always be facing the inside of the cell. It was as if the EmrE protein were like a cat with the perfectly balanced amount of buttered toast on its back—adding that extra arginine anywhere was like adding an extra little morsel of buttered toast anywhere. It was enough to absolutely determine which side would hit the floor.

Having found a system that was fun to play with, the Swedes played with it. Could the arginine be put at the very end of the protein, rather than the beginning? Yes. Would this still work if the protein were longer? The Swedes made the protein a full 20% longer, and put the positively charged arginine at the very end of the protein—like a cat with a veeeery long tail with toast on the end—and still the protein was oriented in the membrane so that the positively charged arginine faced the inside of the cell. Other amino acids have weaker positive charges than arginine—would they work? Yes, and like arginine they worked anywhere in the protein, just more of them were needed.

This was a bit more than idle play, however. It told us something we didn’t know about how the translocon works. Apparently, it sits in the membrane and accepts an entire protein, rather than just looking at the first bit. Once the whole protein is there, it checks to see which side has the most positive charges, points it in the right direction, and only then inserts it into the membrane. So, the translocon has to be much bigger than we thought—big enough to hold an entire protein. Also, it has to have some source of energy to flip proteins one way or the other—and we don’t have a clue about this energy source. This is also of interest to more than just those who study bacteria; our cells have membranes full of proteins, and those proteins are inserted in the proper orientation by a translocon very similar to the bacterial version. If we know how this machine works, we come closer to the ancient instruction “know thyself.” And, no cats were harmed, or even buttered, in this study.

Susanna Seppälä, Joanna S. Slusky, Pilar Lloris-Garcerá, Mikaela Rapp, Gunnar von Heijne (2010). Control of Membrane Protein Topology by a Single C-Terminal Residue. Science 328, 1698-1700.