Stoichiometry and Turnover of the Bacterial Flagellar Switch Protein FliN

Some proteins in biological complexes exchange with pools of free proteins while the complex is functioning. Evi- dence is emerging that protein exchange can be part of an adaptive mechanism. The bacterialflagellar motor is one of the most complex biological machines and is an ideal model system to study protein dynamics in large multimeric complexes. Recent studies showed that the copy number of FliM in the switch complex and the fraction of FliM that exchanges vary with the direc- tion offlagellar rotation. Here, we investigated the stoichiometry and turnover of another switch complex component, FliN, la- beled with the fluorescent protein CyPet, in Escherichia coli. Our results confirm that, in vivo, FliM and FliN form a complex with stoichiometry of 1:4 and function as a unit. We estimated that wild-type motors contained 120 26 FliN molecules. Motors that rotated only clockwise (CW) or counterclockwise (CCW) contained 114 17 and 14426 FliN molecules, respectively. The ratio of CCW-to-CW FliN copy numbers was 1.26, very close to that of 1.29 reported previously for FliM. We also measured the exchange of FliN molecules, which had a time scale and dependence upon rotation direction similar to those of FliM, consistent with an exchange of FliM-FliN as a unit. Our work confirms the highly dynamic nature of multimeric protein complexes and indicates that, under physiological conditions, these machines might not be the stable, complete structures suggested by aver- agedfixed methodologies but, rather, incomplete rings that can respond and adapt to changing environments. IMPORTANCE Theflagellum is one of the most complex structures in a bacterial cell, with the core motor proteins conserved across species. Evidence is now emerging that turnover of some of these motor proteins depends on motor activity, suggesting that turnover is important for function. The switch complex transmits the chemosensory signal to the rotor, and we show, by using single-cell measurement, that both the copy number and the fraction of exchanging molecules vary with the rotational bias of the rotor. When the motor is locked in counterclockwise rotation, the copy number is similar to that determined by averaged, fixed methodologies, but when locked in a clockwise direction, the number is much lower, suggesting that that the switch com- plex ring is incomplete. Our results suggest that motor remodeling is an important component in tuning responses and adapta- tion at the motor.