Biochimica et Biophysica Acta, 980 (1989)241-247 241 Elsevier Lysosomal H+-translocating ATPase has a similar subunit structure to chromaffin granule H+-ATPase complex Yoshinori Moriyama and Nathan Nelson Roche Institute of Molecular Biology. Roche Research Center, Nutlev. NJ I U.S A. (Received 20 September 1988) (Revised manuscript received 13 December 1988) Key words: Lysosome: ATPase. H *-: Subunit structure: Vacuolar ATPa~ Subunit structure of the lysosomal H +-ATPase was investigated using cold inactivation, immunological cross-reactivity with antibodies against individual subunits of the H +-ATPase from chromaffin granules and chemical modification with N,N'-dieyclohexyl[ t4cIcarbodiimid¢. The lysosemal H +-ATPase was irreversibly inhibited when incubated at 0 °C in the presence of chloride or nitrate and MgATP. Inactivation in the cold resulted in the release of several polypeptides (72, 57, 41, 34 and 33 kDa) from the membrane, which had the same electrophoretie mobility as the corresponding subnnits of chromaffin granule H +,ATPase. Cross-reactivity of antibodies revealed that the 72, 57 and 34 kDa polypeptides were immunologieally identical to the corresponding subunits of chromaffin granule H +-ATPase. Di- cydohexylcarhedilmide, which inhibits proton translocation in the vacuolar ATPase, predominantly labeled two polypeptides of 18 and 15 kDa, which compose the membrane sector of the enzyme. These results suggest that the lysosomal H +-ATPase is a multimerlc enzyme, whose subunit structure is similar to the chromaffin granule H +-ATPase. The subunit structure of other vacuolar H +.ATPases, revealed by cold inactivation and immunological cross.reactivity, is also presented. Introduction The lysosomal H+-translocating ATPase (H ÷- ATPase) plays a crucial role in pH homeostasis of lysosomes, maintaining an acidic interior [1,2]. The H+-ATPase is electrogenic, sensitive to nitrate (Mo0yama, Y. and Nelson, N., unpublished data) and N-ethylmaleimide, and insensitive to oligomycin, azide and vanadate [3-8]. These observations indicate that the H÷-ATPase is of the vacuolar type, which has been recently classified as a third type of H +-ATPa~e [9-15]. However, no structural information on iysosomal H +- ATPase has been obtained mainly due to the extreme instability of the enzyme. Recently a few vacuolar H+-ATPases have been purified in a form which, upon reconstitution, are active as ATP-dependent proton pumps [16-19]. One of them, the H+-ATPase from ch,-omaffin granules, is composed of at most nine different polypeptides with apparent molecular mass of 115, 72, 57, 41, 39, 34, 20 and 17 kDa [16,23] *. H +-ATPases from clathrin-coated vesicles and kidney microsome have similar subunits [17-19]; except that the latter lacks the 115 kDa polypeptide. Every vacuolar H+-ATPase purified so far contains subunits equivalent to the 72, 57 and 17 kDa polypeptides. Gene sequencing analyses provided evidence that these three polypeptides contain sequences homologous to the/~, a and DCCD binding subunits of FoFt-ATPase, respec- tively [20-22]. It was found that vacuolar H+-ATPases exhibited sensitivity to cold under specific conditions such as presence of nitrate and MgATP. The inhibition is irre- versible and results in the release of the water-soluble sector of H +-ATPase from membranes [23]. Five poly- peptides with apparent molecular weights of 72 kDa (subunit A), 57 kDa (B), 41 kDa (C), 34 kDa (D) and 33 Abbreviations: Mops, 4-morpholinepropanesulfonic acid; NEM, N- ethylmaleimide; DCCD, N,N'-dicyclohexylcarbodiimide; DTT, di- thiothreitol; SDS, sodium dodecylsulfatc. Correspondencer N, Nelson, Department of Biochemistry. Roche Institute of Molecular Biology, Roche Research Center, Nutley, NJ 07110, U.S.A. * These values are apparent molecular weights of each of the sub- units, which are determined on SDS gels. The exact molecular weights of 39 kDa and 17 kDa polypeplide were calculated from the deduced amino acid sequenceas 31495 and 1.5849, respectively [21,4o1. 0005-2"/36/89/$03.50 © 1989 Elsevier Science Pubfishers B.V. (Biomedical Division)