Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization
Pozidis, Charalambos × Chalkiadaki, Aggeliki Gomez-Serrano, Amalia Stahlberg, Henning Brown, Ian Tampakaki, Anastasia P Lustig, Ariel Sianidis, Giorgos Politou, Anastasia S Engel, Andreas Panopoulos, Nickolas J Mansfield, John Pugsley, Anthony P Karamanou, Spyridoula Economou, Anastassios #
American Society for Biochemistry and Molecular Biology
Journal of Biological Chemistry vol:278 issue:28 pages:25816-24
Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-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, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 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.