Pozidis, CChalkiadaki, AGomez-Serrano, AStahlberg, HBrown, ITampakaki, APLustig, ASianidis, GPolitou, ASEngel, APanopoulos, NJMansfield, JPugsley, APKaramanou, SEconomou, A2020-02-132020-02-132020-02-132003-05-0610.1074/jbc.M301903200https://infoscience.epfl.ch/handle/20.500.14299/165427Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F-1/V-1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa ( monomer); II, similar to300 kDa ( putative hexamer); III, 575 kDa ( dodecamer); and IV, similar to3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (> 700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.text::journal::journal article::research article