Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization.
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.
Institute of Molecular Biology and Biotechnology, FORTH and Department of Biology, University of Crete, P.O. Box 1527, GR-711 10 Iraklio, Crete, Greece.
Charalambos
C
Pozidis
Aggeliki
A
Chalkiadaki
Amalia
A
Gomez-Serrano
Henning
H
Stahlberg
Ian
I
Brown
Anastasia P
AP
Tampakaki
Ariel
A
Lustig
Giorgos
G
Sianidis
Anastasia S
AS
Politou
Andreas
A
Engel
Nickolas J
NJ
Panopoulos
John
J
Mansfield
Anthony P
AP
Pugsley
Spyridoula
S
Karamanou
Anastassios
A
Economou
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000502:physiology*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry*; D002462:Cell Membrane / Q000201:enzymology / Q000378:metabolism; D002845:Chromatography; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D003902:Detergents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007477:Ions; D007700:Kinetics; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000502:physiology*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011549:Pseudomonas / Q000201:enzymology*; D013347:Subcellular Fractions; D013696:Temperature; D014867:Water / Q000378:metabolism
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D003902:Detergents; D007477:Ions; D026901:Membrane Transport Proteins; D014867:Water; D000251:Adenosine Triphosphatases