Selective inhibition of the spinach thylakoid LHC II protein kinase

Abstract

Treatment of spinach thylakoids with the adenosine affinity inhibitor 5′- p-fluorosulfonylbenzoyl adenosine (FSBA) resulted in at least 95% inhibition of phosphorylation of the light-harvesting protein complex of Photosystem II (LHC II), while the M r 10 000 polypeptide showed a 35% decrease in phosphorylation. This residual kinase activity after FSBA treatment appears to have the same properties as the control, since phosphorylation of the M r 10 000 polypeptide subsequent to FSBA treatment could be achieved with either light or reducing conditions in the dark. [ 14C]FSBA labelled several polypeptides, but only the M r 50 000 band was protected against the label by prior addition of ADP or adenosine, making it a possible candidate for the LHC II kinase. FSBA had no effect on electron transport, and [ 14C]FSBA did not label LHC II or the M r 10 000 polypeptide, indicating that the FSBA was not interfering with activation of the kinase or modifying the substrates, but rather acting at the level of the LHC II protein kinase. Inhibition of LHC II phosphorylation by FSBA resulted in the elimination of the slow ATP-induced decrease in variable fluorescence, a parameter believed to be associated with phosphorylation of the LHC II. The half-times and time-course for inhibition of LHC II phosphorylation and inhibition of the ATP-induced decrease of fluorescence yield were identical, consistent with the concept that LHC II phosphorylation plays a major role in this fluorescence change.