Characterization of a chloroplast inner envelope P-ATPase proton pump

Abstract

P-ATPases such as the plasma membrane proton pump are known to generate a phosphorylated intermediate as a step in their reaction mechanism; phosphoenzyme formation is a basis for classification of an ATPase as a member of this subfamily of ion pumps. The chloroplast inner envelope is known to contain a H+-ATPase which acts to maintain an alkaline stroma and, thus, optimal photosynthesis. Our characterization of this chloroplast envelope proton pump described in this report focused on determining whether purified chloroplast inner envelope membrane protein preparations containing this ATPase form a phosphorylated intermediate. Incubation of envelope membranes with [γ-³²P]ATP documented the formation of P-type ATPase phosphoenzyme intermediates by these membrane protein preparations. Our work cannot discount the possibility that more than one chloroplast inner envelope ATPase contributes to this phosphoenzyme formation. However, the kinetics of this phosphoenzyme formation, along with the sensitivity of phosphoenzyme formation to inhibitors and other assay conditions suggested that one of the envelope membrane proteins which is covalently radiolabeled by [γ-³²P]ATP is a P-type H+-ATPase. Autoradiography of chloroplast envelope membrane proteins size fractionated on lithium dodecyl sulfate-PAGE indicated that the phosphoenzyme intermediate corresponds to a 103 kDa polypeptide. P-type proton pumps are known to be comprised of a single type of ∼100 kDa subunit. Experimental evidence presented in this report is consistent with the classification of a chloroplast inner envelope H+-ATPase as a P-type proton pump.