Stoichiometry and turnover in single, functioning membrane protein complexes Export

@article{2006Leake, abstract = {Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media1, 2, 3. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall4, 5. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body6, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains 22 copies of GFP–MotB, consistent with 11 stators each containing two MotB molecules. We also observed a membrane pool of 200 GFP–MotB molecules diffusing at 0.008 m2 s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine.}, author = {Leake, Mark C. and Chandler, Jennifer H. and Wadhams, George H. and Bai, Fan and Berry, Richard M. and Armitage, Judith P.}, citeulike-article-id = {842117}, citeulike-linkout-0 = {http://dx.doi.org/10.1038/nature05135}, citeulike-linkout-1 = {http://dx.doi.org/10.1038/nature05135}, citeulike-linkout-2 = {http://view.ncbi.nlm.nih.gov/pubmed/16971952}, citeulike-linkout-3 = {http://www.hubmed.org/display.cgi?uids=16971952}, day = {13}, doi = {10.1038/nature05135}, issn = {0028-0836}, journal = {Nature}, keywords = {coli, e, flagellum, in}, month = {September}, number = {7109}, pages = {355--358}, posted-at = {2009-11-01 14:01:26}, priority = {2}, publisher = {Nature Publishing Group}, title = {Stoichiometry and turnover in single, functioning membrane protein complexes}, url = {http://dx.doi.org/10.1038/nature05135}, volume = {443}, year = {2006} }

TY - JOUR ID - 2006Leake L3 - citeulike-article-id:842117 N2 - Many essential cellular processes are carried out by complex biological machines located in the cell membrane. The bacterial flagellar motor is a large membrane-spanning protein complex that functions as an ion-driven rotary motor to propel cells through liquid media1, 2, 3. Within the motor, MotB is a component of the stator that couples ion flow to torque generation and anchors the stator to the cell wall4, 5. Here we have investigated the protein stoichiometry, dynamics and turnover of MotB with single-molecule precision in functioning bacterial flagellar motors in Escherichia coli. We monitored motor function by rotation of a tethered cell body6, and simultaneously measured the number and dynamics of MotB molecules labelled with green fluorescent protein (GFP–MotB) in the motor by total internal reflection fluorescence microscopy. Counting fluorophores by the stepwise photobleaching of single GFP molecules showed that each motor contains 22 copies of GFP–MotB, consistent with 11 stators each containing two MotB molecules. We also observed a membrane pool of 200 GFP–MotB molecules diffusing at 0.008 m2 s-1. Fluorescence recovery after photobleaching and fluorescence loss in photobleaching showed turnover of GFP–MotB between the membrane pool and motor with a rate constant of the order of 0.04 s-1: the dwell time of a given stator in the motor is only 0.5 min. This is the first direct measurement of the number and rapid turnover of protein subunits within a functioning molecular machine. IS - 7109 JF - Nature SN - 0028-0836 EP - 358 TI - Stoichiometry and turnover in single, functioning membrane protein complexes PB - Nature Publishing Group VL - 443 SP - 355 KW - coli KW - e KW - flagellum KW - in AU - Leake, Mark AU - Chandler, Jennifer AU - Wadhams, George AU - Bai, Fan AU - Berry, Richard AU - Armitage, Judith PY - 2006/09/13/ UR - http://dx.doi.org/10.1038/nature05135 ER -