Wednesday, 19 February 2014 10:32

Of Carp and Protein Translocation

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RAPOPORT-345x239Tom Rapoport's latest discovery starts with a fish 30 years
ago and continues this month with the publication of
the structure of an open translocation channel.
Photo credit: Hadar Goren
For those who think scientific discoveries pop up overnight, consider Tom Rapoport's tale of the holiday carp and how it led him to study the translocation channel through which proteins, such as insulin, are secreted. Rapoport's latest discovery starts with a fish more than 30 years ago and ends, or at least continues, this month with a publication in Nature of the first structure of an open protein translocation channel.

The path to solving this difficult protein structure begins with the carp, a traditional German New Year's Eve dish, Silvesterkarpfen. Rapoport, who is now a professor at Harvard Medical School (HMS) and an HHMI investigator, decided over 30 years ago that carp would make an ideal source of material he needed to explore secretion. Carp was, for one thing, available.

Thirty years ago, Rapoport was a young investigator at the Institute for Molecular Biology of the Academy of Sciences in Berlin-Buch, which was still under the Communist-ruled German Democratic Republic (GDR). Rapoport's aim was to clone insulin mRNA. He knew that fish have bigger insulin producing parts (known as Brockmann bodies) than mammals, so he thought it would be easier to isolate their mRNA. He also knew that the German appetite for Silvesterkarpfen ensured a steady fish supply, even in the GDR. Rapoport started with some carp and a little help from his department.

On "fish days" his entire department would help kill, clean, and remove Brockmann bodies from up to 2,000 fish. What Rapoport called the leftovers (and the rest of us would call the good stuff), the lab sold cheaply to carp-loving locals.

Despite his best efforts, Rapoport was unable to isolate the insulin mRNA. Finally, in desperation, he took the ribosomes from the Brockmann bodies and translated them in vitro without isolating the mRNA. "And I noticed that there were two products and the products behaved differently from pro-insulin... I couldn't make any sense of my data," Rapoport said.

It wasn't until he was attending a conference in the GDR in the mid '70s that his results became clear. "The last speaker at the meeting was Günter Blobel, and he presented his signal hypothesis, which hadn't actually arrived in East Germany yet because we got the journals about a year late. So of course I immediately recognized what the reason was for my funny results. I realized that I had actually synthesized pre-pro-insulin," Rapoport said.

Back at the lab, Rapoport confirmed his suspicions. Indeed he had isolated pre-pro-insulin, meaning there was a signal sequence in front of the pro-insulin sequence that directs the protein to the channel. "I became immediately interested in the question of how the signal sequence is recognized and what the receptor is for the signal sequences," he said. Rapoport has been researching protein channels ever since.

These protein translocation channels or translocons, are essentially the same in all organisms. They sit in cell membranes and facilitate proteins crossing the membrane as they are being made by ribosomes, before they are folded into a 3D structure. They are essential for cells to secrete proteins, like insulin.

Rapoport came to the U.S. in 1995 to take a position at HMS. In 2004, Rapoport and collaborators, including his colleague Stephen Harrison, were the first to solve the x-ray structure of a closed protein translocation channel. "That was a real revelation because it really gave us a lot of ideas about how the channel would work," Rapoport said. "Ever since then the goal was to get the structure of the active channel."

Finally, after 10 years of work, he got it. The structure, published on February 6 in Nature, required a few biochemical tricks to solve, and Rapoport gives special credit to his former graduate student, Eunyong Park, who is the paper's first author. "Park was absolutely ingenious in designing these methods," Rapoport said.

"Our paper is remarkable in the sense that we developed a new method by which we can isolate in substantial amounts the active channel with the translocating chain present," Rapoport said. "The way you have to do this is by catching the active translating ribosome in the act... The trick is to use a stalling sequence, a sequence which makes the ribosome stop at a certain point, and then crosslink the polypeptide chain to the channel."

Rapoport also credits his long-term collaborator Chris Akey, Boston University professor and ASCB member, with the structural analysis of both conformations of the channel. "We've been doing it together for a long, long time," Rapoport explained. "He is the [structural] EM guy and we are the biochemists who provide him with the samples. It's a very close collaboration... Without him, even the best biochemistry wouldn't have given any results. He really did an amazing job."

But Rapoport is not yet done with the tale of the carp. "I should tell you the structure that we just published, as important as it is, I'm not happy with the resolution yet... So that's clearly the next goal."


Park E, Ménétret JF, Gumbart JC, Ludtke SJ, Li W, Whynot A, Rapoport TA, & Akey CW (2014). Structure of the SecY channel during initiation of protein translocation. Nature, 506 (7486), 102-6 PMID: 24153188

Christina Szalinski

Christina is a science writer for the American Society for Cell Biology. She earned her Ph.D. in Cell Biology and Molecular Physiology at the University of Pittsburgh.

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