Bacterial F-type ATP synthases follow a well-choreographed assembly pathway

F-type ATP synthases are multiprotein complexes composed of two separate coupled motors (F(1) and F(O)) generating adenosine triphosphate (ATP) as the universal major energy source in a variety of relevant biological processes in mitochondria, bacteria and chloroplasts. While the structure of many ATPases is solved today, the precise assembly pathway of F(1)F(O)-ATP synthases is still largely unclear. Here, we probe the assembly of the F(1) complex from Acetobacterium woodii. Using laser induced liquid bead ion desorption (LILBID) mass spectrometry, we study the self-assembly of purified F(1) subunits in different environments under non-denaturing conditions. We report assembly requirements and identify important assembly intermediates in vitro and in cellula. Our data provide evidence that nucleotide binding is crucial for in vitro F(1) assembly, whereas ATP hydrolysis appears to be less critical. We correlate our results with activity measurements and propose a model for the assembly pathway of a functional F(1) complex.