AtpI, a membrane protein encoded by many bacterial atp operons, is reported to be necessary for c-ring oligomer formation during assembly of some ATP synthase complexes. We investigated chaperone functions of AtpI and compared them to those of AtpZ, a protein encoded by a gene upstream of atpI that has a role in magnesium acquisition at near-neutral pH, and of SpoIIIJ and YqjG, two YidC/OxaI/Alb3 family proteins, in alkaliphilic Bacillus pseudofirmus OF4. A strain with a chromosomal deletion of atpI grew nonfermentatively, and its purified ATP synthase had a c-ring of normal size, indicating that AtpI is not absolutely required for ATP synthase function. However, deletion of atpI, but not atpZ, led to reduced stability of the ATP synthase rotor, reduced membrane association of the F 1 domain, reduced ATPase activity, and modestly reduced nonfermentative growth on malate at both pH 7.5 and 10.5. Both spoIIIJ and yqjG, but not atpI or atpZ, complemented a YidC-depleted Escherichia coli strain. Consistent with such overlapping functions, single deletions of spoIIIJ or yqjG in the alkaliphile did not affect membrane ATP synthase levels or activities, but functional specialization was indicated by YqjG and SpoIIIJ showing respectively greater roles in malate growth at pH 7.5 and 10.5. Expression of yqjG was elevated at pH 7.5 relative to that at pH 10.5 and in ⌬spoIIIJ strains, but it was lower than constitutive spoIIIJ expression. Deletion of atpZ caused the largest increase among the mutants in magnesium concentrations needed for pH 7.5 growth. The basis for this phenotype is not yet resolved.T he F 1 F o ATP synthase is a large membrane complex in bacteria, mitochondria, and chloroplasts which synthesizes ATP by a rotary mechanism that is energized by a transmembrane protonor sodium-motive force (1-3). The bacterial enzyme consists of a soluble F 1 domain (subunits ␣ 3  3 ␥␦ε) and a membrane-embedded F o domain (subunits ab 2 c 10-15 ) (4-6). In most bacteria, the atp operon contains nine open reading frames: eight structural genes (atpBEFHAGDC) of ATP synthase preceded by a ninth atpI gene, which encodes a membrane protein and is not a structural gene (7-9). When atpI, originally called uncI, was first reported (10), it was suggested that it encoded a pilot protein with a role in assembly of the membrane sector of the synthase, although AtpI clearly did not have an essential role, since deletion of the atpI gene from Escherichia coli only slightly reduced the growth yield and ATPase activity (11). Similarly, we recently showed that atpI, as well as the atpZ gene that is found upstream of atpI in many low %GC bacteria, could be deleted from the chromosome of alkaliphilic Bacillus pseudofirmus OF4 without apparent loss of the capacity to grow nonfermentatively (8, 12).More recently, these earlier findings have been challenged by the studies of Yoshida and colleagues (13), who have shown that AtpI plays a necessary and sufficient chaperone-like role in assembly of a hybrid Na ϩ -coupled ATP synthase containing all ...