A small molecule dissociates the PAM motor from the TIM23 channel
Mitochondrial dysfunction is a contributing factor in various neural and muscular degenerative diseases. Modulation of mitochondrial protein pathways can have regulatory effects on mitochondrial function. Conventional methods using yeast genetics and RNAi approaches are powerful, but limited because RNAi requires several days and during this time, cells can alter metabolism. Therefore, we have developed several approaches to identify small molecule modulators for mitochondrial protein translocation.
This study utilizes a high-throughput small molecule screening approach to identify modulators of the TIM23 import pathway, which transports proteins across the inner membrane and into the matrix. This strategy will be useful in generating a toolbox of small molecule modulators for mitochondrial translocation to understand how defects in mitochondrial assembly contribute to disease.
To this end, we have identified and characterized a small molecule probe MitoBloCK-20 (MB-20) that binds specifically to Tim17 in the TIM23 complex. MB-20 inhibits the import of substrates that use the TIM23 translocon, but not the TIM22 translocon. MB-20 seems to target Tim17, because Tim17p, but not Tim23p and the PAM complex, was resistant to protease in the presence of MB-20. In pull-down experiments with His-tagged components, the interaction between Tim17 and Tim23 was stabilized but Tim44, Pam18 and Pam16 of the motor precipitated, suggesting the interaction between Tim17/Tim23 and the PAM complex was disrupted. Based upon biochemical analysis, MB-20 seems to target Tim17 and suggests that Tim17 plays a critical role in coordinating activities between the channel and the import motor. This modulator also showed a critical role for mitochondria in development of the zebrafish neural system.