Fluorescence mapping of mitochondrial TIM23 complex reveals a water-facing, substrate-interacting helix surface

Area of Science:

• Mitochondrial biology
• Protein transport
• Membrane biophysics

Background:

• Protein translocation across the mitochondrial inner membrane is crucial for cellular function and is mediated by the TIM23 complex.
• The precise structure of the Tim23 subunit, particularly the regions forming the protein-conducting channel, remains largely unknown.

Purpose of the Study:

• To investigate the high-resolution structure and environment of the Tim23 subunit within the functional TIM23 complex in intact mitochondria.
• To identify the regions of Tim23 that interact with imported proteins and to understand channel gating mechanisms.

Main Methods:

• Utilized environment-sensitive fluorescent probes, each at a specific site on Tim23 derivatives, integrated into functional TIM23 complexes.
• Employed multiple spectral techniques to analyze probe environments within active mitochondria.
• Combined fluorescence data with crosslinking studies to map probe locations within the protein-conducting channel.

Main Results:

• Identified an alpha-helical structure within the transmembrane region of Tim23, situated in an amphipathic environment.
• Observed specific spectral changes in probes on the aqueous-facing helical surface during protein import, indicating dynamic structural alterations.
• Assessed probe accessibility to quenching agents, providing insights into the channel gating process.

Conclusions:

• Provided an unprecedented, high-resolution view of a translocon channel structure within a fully operational, membrane-embedded complex.
• Elucidated the structural basis for protein translocation through the TIM23 channel and offered insights into its dynamic regulation.
• Demonstrated the utility of site-specific fluorescent probes for studying membrane protein complexes in situ.