Transporting a Fully Folded Protein across a Membrane

Transporting a fully formed protein across a membrane is difficult, so transmembrane protein tends to take place before the newly synthesized protein has folded into its final functional shape. However, mitochondria manage to transfer one important protein across their inner membrane while it is in its folded state. An LMU paper published last week in Nature Structural and Molecular Biology explains how.

The Rieske protein (Rip1 in baker’s yeast) is necessary for ATP generation. Rip1 is synthesized in a precursor form by cytoplasmic ribosomes and is transported in its unfolded state across the outer and inner membranes of the organelle into the matrix, where it is processed to yield the Rip1 protein. Incorporation of an “iron-sulfur center” then allows it to adopt its functional conformation. Now, however, this globular protein must be inserted back into the inner membrane.

“Earlier work by our partners Walter Neupert and Nikola Wagener had shown that, in yeast, an enzyme called Bcs1 is involved in this final step,” says first author Lukas Kater. “We have now determined the 3D structure of this enzyme with the aid of cryo-electron microscopy. In fact, this is the first high-resolution structure of a eukaryotic translocator for folded proteins yet published.”

With a detailed map of the enzyme’s configuration in hand, the team set out to decipher how Bcs1 manages to selectively mediate the passage of the folded form of Rip1 through the inner membrane without allowing other molecules or ions to slip through the pore.

Bcs1 belongs to a class of enzymes called AAA-ATPases. These typically consist of six identical subunits, which together form a ring-like pore. However, the structural data for Bcs1 revealed that it is comprised of seven subunits, and therefore forms a larger pore.

But that’s not all. The pore is divided into two clearly defined chambers. One of these is directly accessible from the matrix of the mitochondrion, while the other is located within the inner membrane. Crucially, they are linked by a central domain, which forms a seal between them that can be transiently opened.

“We therefore propose that transport through Bcs1 exploits the airlock principle,” says senior author Roland Beckmann. The Rieske protein first passes through the large pore and into the matrix chamber. This induces a conformational change in Bcs1, which causes the seal between the two chambers to open, allowing Rip1 to enter the airlock, the chamber within the inner membrane. In the next step, the seal that separates the two chambers forms again.