Mitochondria are cellular structures often referred to as the “powerhouses of the cell” due to their crucial role in generating ATP (adenosine triphosphate), the molecular energy source that fuels most cellular activities. These structures emerged over a billion years ago when a rudimentary archaeal cell formed a symbiotic partnership with an ancestral bacterium. Gradually, mitochondria became vital for metabolism and energy generation, transferring the majority of their genes to the host. Consequently, they now depend on the host cell for most of their proteins, which are produced by ribosomes located outside the organelle and must be accurately transported to mitochondria.
Recently, scientists at Caltech have revealed new insights into how mitochondrial proteins are delivered from ribosomes in the cytosol, the liquid surrounding the nucleus, to mitochondria. In an intriguing turn of events, this process is significantly influenced by the intricacies of protein folding.
“It appears that directing proteins to mitochondria entails a layered, intricate pathway that is connected to the biophysical principles governing protein folding,” comments Shu-ou Shan, the Altair Professor of Chemistry at Caltech.
For many years, the prevailing theory in biochemistry suggested that mitochondrial proteins are imported only after the completion of protein synthesis, or translation. (This ribosome-driven mechanism involves sequentially adding amino acids to a growing chain, adhering to the sequence dictated by the cell’s genetic instructions.) In a new study published in the journal Cell, Shan and her team propose an update to this model, indicating that as much as 20 percent of mitochondrial proteins can be cotranslationally imported, meaning they enter into mitochondria while translation is still underway as the proteins are being synthesized by the ribosome.
“Once we identified these mitochondrial proteins that are cotranslationally imported, we inquired, ‘What distinguishes this group of proteins?’” states Zikun Zhu (PhD ’24), Shan’s former graduate student and lead author of the study.
It turns out that the most notable characteristic of these proteins is their substantial size and complex folding patterns. Such topologically intricate proteins are abundant in residues—amino acids in the chain that forms the protein—which, while being far apart in linear sequence, must associate to enable the protein to adopt the correct three-dimensional structure. “That complicates the process significantly compared to simply folding through interactions between adjacent residues,” explains Shan.
Thus, the system for cotranslational import into mitochondria prioritizes these particularly challenging-to-fold proteins. This is logical when considering that these large structures must navigate narrow channels in the mitochondrial membrane during import. “There will be an issue if you allow these large, highly intricate proteins to complete translation in the cytosol,” notes Shan. “They will become trapped in irreversible configurations, which will not only obstruct import but also clog all the channels.”
So, how does the cell determine which proteins should be imported during translation?
The research team discovered that almost all of these proteins possess a mitochondrial targeting sequence, a signal that directs proteins toward mitochondria. Surprisingly, this alone is insufficient to indicate that this group of proteins should be delivered during translation. Zhu conducted experiments that demonstrated the system awaits an additional molecular signal to prompt early movement of a protein to the mitochondria. This signal manifests as the first substantial protein domain, or foldable structural unit within the sequence, exiting from the ribosome.
“It’s akin to having your boarding pass locked inside a suitcase,” Zhu explains. “The targeting sequence is the boarding pass, but to access it, you require the code to unlock the suitcase. In this scenario, the large domain is that code.”
The researchers even succeeded in transferring instances of such large protein domains to other mitochondrial proteins that typically undergo import after translation and demonstrated that these domains indeed acted as transferable signals, capable of rerouting proteins to be imported during translation.
“Cotranslational targeting to mitochondria proves to be entirely different from targeting to other organelles,” Zhu remarks. “Moving forward, it will be thrilling to unveil more mechanistic specifics and ultimately manipulate the timing of mitochondrial protein import. This will not only help us comprehend why cells have evolved such a sophisticated targeting mechanism for mitochondrial proteins but also pave the way for potential therapeutic applications.”
The paper is titled, “Principles of cotranslational mitochondrial protein import.” Additional Caltech contributors include Taylor A. Stevens (PhD ’24), a postdoctoral research associate in biology and biological engineering, and Riming Huang, a graduate student in biochemistry and molecular biophysics. Saurav Mallik from the Weizmann Institute of Science in Israel and Emmanuel D. Levy from the University of Geneva in Switzerland are also co-authors. The research received support from the National Institutes of Health and the Howard Hughes Medical Institute through a Freeman Hrabowski Scholar grant.