Alternating translocation of protein substrates from both ends of ClpXP protease

Joaquin Ortega; Hyun Sook Lee; Michael R Maurizi; Alasdair C Steven

doi:10.1093/emboj/cdf483

Alternating translocation of protein substrates from both ends of ClpXP protease

EMBO J. 2002 Sep 16;21(18):4938-49. doi: 10.1093/emboj/cdf483.

Authors

Joaquin Ortega¹, Hyun Sook Lee, Michael R Maurizi, Alasdair C Steven

Affiliation

¹ 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.

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.

Publication types

Research Support, Non-U.S. Gov't
Research Support, U.S. Gov't, P.H.S.

MeSH terms

ATPases Associated with Diverse Cellular Activities
Adenosine Triphosphatases / chemistry
Adenosine Triphosphatases / metabolism*
Adenosine Triphosphatases / ultrastructure
Adenosine Triphosphate / analogs & derivatives*
Adenosine Triphosphate / metabolism
Amino Acid Motifs
Binding Sites
Endopeptidase Clp
Escherichia coli Proteins
Green Fluorescent Proteins
Luminescent Proteins / metabolism
Macromolecular Substances
Molecular Chaperones / chemistry
Molecular Chaperones / metabolism
Molecular Chaperones / ultrastructure
Protein Binding
Protein Transport / physiology*
RNA, Bacterial / genetics
RNA, Bacterial / metabolism
RNA, Bacterial / ultrastructure
Recombinant Fusion Proteins / metabolism
Serine Endopeptidases / chemistry
Serine Endopeptidases / metabolism*
Serine Endopeptidases / ultrastructure

Substances

Escherichia coli Proteins
Luminescent Proteins
Macromolecular Substances
Molecular Chaperones
RNA, Bacterial
Recombinant Fusion Proteins
tmRNA
Green Fluorescent Proteins
adenosine 5'-O-(3-thiotriphosphate)
Adenosine Triphosphate
Serine Endopeptidases
Endopeptidase Clp
Adenosine Triphosphatases
ClpX protein, E coli
ATPases Associated with Diverse Cellular Activities