Hsp70 cannot bind runs of glutamine acid (left) or glycine (right ).

Neupert/EMBO

Amotor protein, by its very name, should really be pulling on something or exerting force. But Koji Okamoto, Walter Neupert (Universität München, München, Germany), and colleagues claim that the mitochondrial import motor works not by active pulling but by using a Brownian ratchet.The motor in question is heat shock protein 70 (Hsp70) in the mitochondrial matrix. ATP-bound Hsp70 is bound to the import channel and the incoming protein. Some researchers have suggested that ATP hydrolysis drives an Hsp70 power-stroke that drags a protein into the mitochondrial matrix. But Neupert and colleagues show that the system can import a protein with titin immunoglobulin domains, which have been shown to require >200 pN of force to unfold. In general, molecular motors can only generate ∼5 pN of force, suggesting that Hsp70 cannot be using a simple power stroke to overcome this thermodynamic barrier.

Besides, when Hsp70 cannot get a grip on its substrate close to the channel, as would be needed for a power stroke, the motor still works. The researchers engineered a protein with a run of 50 glycine or glutamic acid residues—a stretch that does not bind Hsp70—and showed that it is still imported. Neupert therefore believes that the folded proteins in the cytoplasm “breathe,” and that the unfolded segments of the protein can slide through the channel by thermal fluctuations. Once through the channel, the protein segments are trapped in the mitochondrial matrix by binding to Hsp70. This is similar to the denaturing action of urea, which does not insert itself into folded proteins but binds to temporarily unfolded proteins, thus stabilizing their unfolded structure.

An active motor that pulls is, Neupert admits, “easier to understand. But those more in physical chemistry and equilibria, these people prefer the ratchet.” A ratchet that could tolerate such a long stretch of nonbinding sequence was a bit of a surprise. “There's always the possibility that you are missing something,” says Neupert. “But we think the breathing of the molecule is enough.” ▪

Reference:

Okamoto, K., et al. 2002. EMBO J. 21:3659–3671.