The stoichiometry of c subunits in the H ؉ -transporting Fo rotary motor of ATP synthase is uncertain, the most recent suggestions varying from 10 to 14. The stoichiometry will determine the number of H ؉ transported per ATP synthesized and will directly relate to the P͞O ratio of oxidative phosphorylation. The experiments described here show that the number of c subunits in functional complexes of F oF1 ATP synthase from Escherichia coli can be manipulated, but that the preferred number is 10. Mixtures of genetically fused cysteine-substituted trimers (c 3) and tetramers (c 4) of subunit c were coexpressed and the c subunits crosslinked in the plasma membrane. Prominent products corresponding to oligomers of c 7 and c10 were observed in the membrane and purified F oF1 complex, indicating that the c10 oligomer formed naturally. Oligomers larger than c 10 were also observed in the membrane fraction of cells expressing c3 or c4 individually, or in cells coexpressing c 3 and c4 together, but these larger oligomers did not copurify with the functional F oF1 complex and were concluded to be aberrant products of assembly in the membrane. The ATP made during oxidative and photo phosphorylation is synthesized by closely related enzymes located in the inner membrane of mitochondria, the thylakoid membrane of chloroplasts, and the plasma membrane of eubacteria (1). Recent evidence supports a rotary mechanism for ATP synthesis in which proton-transport-coupled rotation of an oligomeric ring of c subunits in the membrane is coupled to rotary movement of subunit ␥ between alternating catalytic sites in the ␣ 3 ␤ 3 sector of the enzyme (refs. 2-8; Fig. 1). The proton-motive force is thought to drive rotation of the c-ring by a transport mechanism by using half channels that connect the aspartyl-61 protonbinding site of subunit c in the middle of the membrane to the aqueous compartments on either side of the membrane. After H ϩ binding from the entrance half channel at the a 1 b 2 stator, the c subunit carrying the proton would rotate nearly 360°before encountering the H ϩ exit channel on the opposite side of the membrane. In such a mechanism, the number of H ϩ transported per revolution of the c oligomer would equal the number of subunit c in the ring. The number of subunit c would also determine the H ϩ ͞ATP stoichiometry (i.e., H ϩ transported per ATP synthesized) and directly relate to the P͞O ratio for oxidative phosphorylation, a fundamental parameter of biology (9).The number of c subunits in F o is controversial. Direct measurements of subunit ratios in Escherichia coli F o F 1 , after growth of cells on radioactive amino acids, indicated a range of 10 Ϯ 1 c per 3 ␣␤ pairs in the purified F o F 1 complex or 12 c per 3 ␣␤ in the membrane fraction in which F o F 1 was overproduced (10). More recently, genetically fused subunit c were generated by insertion of a loop between the C-terminal residue of transmembrane helix-2 (TMH-2) of the first subunit and the N-terminal residue of TMH-1 of the next subunit (11). The geneti...

A B S T R A C T Two minor proteins of frog rod outer segments become phosphorylated when retinas are incubated in the dark with a2pi. The proteins, designated component I (13,000 dahons) and component II (12,000 daltons), are dephosphorylated when retinas are illuminated. The dephosphorylation is reversible; the two proteins are rephosphorylated when illumination ceases. Each outer segment contains -10 n molecules of components I and II. These remain associated with both fragmented and intact outer segments but dissociate from the outer segment membranes under hypoosmotic conditions. The extent of the light-induced dephosphorylation increases with higher intensities of illumination and is maximal with continuous illumination which bleaches 5.0 X 105 rhodopsin molecules/outer segment per second. Light which bleaches 5.0 • 103 rhodopsin molecules/outer segment per second causes approximately half-maximal dephosphorylation. This same intermediate level of illumination causes half-suppression of the light-sensitive permeability mechanism in isolated outer segments (Brodie and Bownds. 1976. J, Gen. Physiol. 68:1-11) and also induces a half-maximal decrease in their cyclic GMP content (Woodruff et al. 1977.J. Gen. Physiol. 69:667-679). The phosphorylation of components I and II is enhanced by the addition of cyclic GMP or cyclic AMP to either retinas or isolated rod outer segments maintained in the dark. Several pharmacological agents which influence cyclic GMP levels in outer segments, including calcium, cause similar effects on the phosphorylation of components I and II and outer segment permeability. Although the cyclic nucleotide-stimulated phosphorylation can be observed either in retinas or isolated rod outer segments, the lightinduced dephosphorylation is observed only in intact retinas.

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We have previously shown that the E31C-substituted ⑀ subunit of F 1 can be cross-linked by disulfide bond formation to the Q42C-substituted c subunit of F 0 in the Escherichia coli