Abstract
A photoregulated reversible protein phosphorylation system controlled by the halobacterial rhodopsins was recently reported. The results presented in this paper identify the initial steps in the pathway from the absorption of light to the photoregulated protein phosphorylation and dephosphorylation reactions. Action spectrum, biochemical, and genetic analyses show that the proton pump bacteriorhodopsin mediates light-induced dephosphorylation of three photoregulated phosphoproteins. Light absorbed by bacteriorhodopsin is used to establish a proton efflux from the cells. The increase in the inwardly directed protonmotive force (pmf) from this efflux induces dephosphorylation of the three phosphoproteins, as demonstrated by the effects of the protonophore CCCP and of artificially imposed transmembrane pH gradients. Upon darkening the cells, cessation of the proton efflux through bacteriorhodopsin causes a decrease in pmf, which induces rephosphorylation of the proteins. Pmf appears to function as a regulator rather than a driving force in this system. Measurements of pmf-driven ATP synthesis in our conditions indicate the regulation of protein phosphorylation by pmf is probably not a consequence of proton flux through the H+ ATPase, a known energy coupling structure in these cells. The properties of this system may indicate the existence of a pmf detector which regulates kinase or phosphatase activity; i.e., a regulatory coupling device.