title: Alternating translocation of protein substrates from both ends of ClpXP protease.
abstract: In ClpXP protease complexes, hexameric rings of the ATP-dependent ClpX chaperone stack on one or both faces of the double-heptameric rings of ClpP. We used electron microscopy to record the initial binding of protein substrates to ClpXP and their accumulation inside proteolytically inactive ClpP. Proteins with N- or C-terminal recognition motifs bound to complexes at the distal surface of ClpX and, upon addition of ATP, were translocated to ClpP. With a partially translocated substrate, the non-translocated portion remained on the surface of ClpX, aligned with the central axis of the complex, confirming that translocation proceeds through the axial channel of ClpXP. Starting with substrate bound on both ends, most complexes translocated substrate from only one end, and rarely (<5%) from both ends. We propose that translocation from one side is favored for two reasons: initiation of translocation is infrequent, making the probability of simultaneous initiation low; and, further, the presence of protein within the cis side translocation channel or within ClpP generates an inhibitory signal blocking translocation from the trans side.
authors.affiliation: Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases and Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Ortega
authors.forename: Hyun Sook
authors.initials: HS
authors.lastname: Lee
authors.forename: Michael R
authors.initials: MR
authors.lastname: Maurizi
authors.forename: Alasdair C
authors.initials: AC
authors.lastname: Steven
mesh_terms: D000074183:ATPases Associated with Diverse Cellular Activities; D000251:Adenosine Triphosphatases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D049071:Endopeptidase Clp; D029968:Escherichia coli Proteins; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000378:metabolism; D046911:Macromolecular Substances; D018832:Molecular Chaperones / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure; D011485:Protein Binding; D021381:Protein Transport / Q000502:physiology*; D012329:RNA, Bacterial / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D012697:Serine Endopeptidases / Q000737:chemistry / Q000378:metabolism* / Q000648:ultrastructure
chemical_list: D029968:Escherichia coli Proteins; D008164:Luminescent Proteins; D046911:Macromolecular Substances; D018832:Molecular Chaperones; D012329:RNA, Bacterial; D011993:Recombinant Fusion Proteins; C096222:tmRNA; D049452:Green Fluorescent Proteins; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D012697:Serine Endopeptidases; D049071:Endopeptidase Clp; D000251:Adenosine Triphosphatases; C088072:ClpX protein, E coli; D000074183:ATPases Associated with Diverse Cellular Activities