How large, fully folded proteins can pass through cell membranes without destroying them has long been one of the open questions in cell biology. Using cryo-electron microscopy (cryo‑EM), Leonid Sazanov and Ziyu Zhao at the Institute of Science and Technology Austria (ISTA) have now uncovered new details about a molecular "gate." Their findings were published in Molecular Cell.
Back in school, we all learned the central dogma of biology: DNA makes RNA, and RNA makes protein. These proteins perform countless vital functions in the body—but to do so, they often need to travel to other locations within the cell and cross membranes.
Professor Leonid Sazanov and postdoctoral researcher Ziyu Zhao at the Institute of Science and Technology Austria (ISTA) have now shown how the Tat system (twin‑arginine translocase) transports bulky proteins across membranes.
The mysterious Tat system
Inside every cell are many "rooms," or organelles, each enclosed by its own membrane. To reach their proper destination, proteins must sometimes cross these membrane walls.
In most cells, this usually happens through the Sec system, which pulls unfolded proteins through the membrane—like threading a rope through a hole.
It becomes much trickier when a protein is already fully folded and still needs to cross. In this case, the cargo is much larger and bulkier—it can't simply be threaded through.
In bacteria and chloroplasts (cells in plants that are known as the "powerhouses of photosynthesis"), there is a special solution to this problem: the Tat system. Scientists already knew that in E. coli it consists of three building blocks—TatA, TatB, and TatC—but exactly how the assembled system looks and how it manages to safely move large proteins through a membrane remained a mystery.
Sazanov and Zhao set out to visualize this molecular "gate" and find out how it manages to ferry its bulky cargo unharmed across the membrane.
Frozen for clarity
To uncover the details, Zhao isolated the Tat complex in vivo—directly from living E. coli cells.
"That was no easy task because the protein complex is very unstable," Zhao explains. "Only when I co‑expressed the Tat components and the cargo protein in the bacteria did the complex become stable enough to be isolated and visualized by cryo-EM."
The Tat complex with its bound cargo was then applied to electron microscopy grids and quickly dipped in cryogen. The rapid cooling causes the water in the sample to vitrify—turning glass‑like instead of forming ice crystals—thus preserving the delicate structural details.
Finally, the Tat system was imaged using cryo‑electron microscopy (cryo‑EM), a technique that produces extremely detailed images of proteins down to 2-3 angstrom resolution, needed to define atomic structures.
A look inside the "gate"
Three‑dimensional reconstruction of the data revealed a surprising picture: the Tat complex is made up of three TatB/C units that together float within the membrane. According to Sazanov, its shape is highly unusual.
"The Tat complex looks like a wide-open bowl with a very thin base," says the biochemist.
Interestingly and unexpectedly, the cargo interacts with the bowl at two specific sites on the TatB/C units. One site recognizes the cargo's signal peptide and anchors it like a glue. The second site seems to act as a checkpoint, verifying that the cargo is properly folded before transport begins.
The reconstruction showed that the Tat complex potentially also contains a pore at its base that can open and close. According to Sazanov and Zhao, this pore may open like a gate once the cargo has docked and passed inspection, allowing it to pass through the membrane. Exactly how this gating process works remains an open question and a focus of Zhao's future research.
A future target for new drugs
Because the Tat system does not exist in humans, it could serve as a potential target for antimicrobial interventions. In bacteria, the system is essential for metabolism and virulence—their ability to cause infection. The more we learn about the individual Tat components, the more opportunities will arise to design treatments that selectively disrupt this vital process in harmful bacteria.
