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Alternating translocation of protein substrates from both ends of ClpXP protease

2002; Springer Nature; Volume: 21; Issue: 18 Linguagem: Inglês

10.1093/emboj/cdf483

1460-2075

Joaquı́n Ortega,

Signaling Pathways in Disease

Article16 September 2002free access Alternating translocation of protein substrates from both ends of ClpXP protease Joaquin Ortega Joaquin Ortega Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, 20892 USA Search for more papers by this author Hyun Sook Lee Hyun Sook Lee Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Michael R. Maurizi Corresponding Author Michael R. Maurizi Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Alasdair C. Steven Alasdair C. Steven Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, 20892 USA Search for more papers by this author Joaquin Ortega Joaquin Ortega Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, 20892 USA Search for more papers by this author Hyun Sook Lee Hyun Sook Lee Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Michael R. Maurizi Corresponding Author Michael R. Maurizi Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA Search for more papers by this author Alasdair C. Steven Alasdair C. Steven Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, 20892 USA Search for more papers by this author Author Information Joaquin Ortega1, Hyun Sook Lee2, Michael R. Maurizi 2 and Alasdair C. Steven1 1Laboratory of Structural Biology, National Institute of Arthritis, Musculoskeletal and Skin Diseases, Bethesda, MD, 20892 USA 2Laboratory of Cell Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD, 20892 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:4938-4949https://doi.org/10.1093/emboj/cdf483 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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 ( 70% of the remaining ClpX–ClpP interfaces showed evidence of partially translocated substrate. That observation, together with the greater recovery of ClpXPin complexes when substrate was present, suggests that having partially translocated substrate bridging the ClpX surface and the ClpP chamber may help hold the complex together. In this case, dissociation of ClpX would mean that no translocation occurred from that end of the complex, further supporting the interpretation of unilateral translocation. The results obtained in the presence of ATPγS are consistent with this interpretation. Further more, in 32% of the stable complexes obtained in ATPγS, the non-translocated substrate remained bound to the other end of the 2:1 complexes. Since translocation is fast (<2 min in ATP and <20 min in ATPγS), the low frequency of translocation from both ends suggests that there is an inhibitory effect on further translocation once a substrate has been translocated. We conclude that the presence of protein in the translocation channel or within the ClpP chamber sends an inhibitory signal to the other end of the ClpXP complex, preventing further translocation. Between 15 and 20% of the complexes had no detect able end-associated density after incubation, although they had stain-occluded cores indicative of translocation (Figure 4A and B). Such complexes were unexpected because no degradation of the GFP moieties has been observed with active ClpXP. Possibly, some of the GFP might not have been well folded, in which case it would have been translocated along with the λO162. In addition, poorly folded GFP would be less visible in the negative stain. It is also possible that a few ClpXPin complexes retained a minor amount of active ClpP that cleaved between the λO162 and the GFP allowing release of the GFP moiety. Further analysis will be needed to test these alternatives. Translocation of λO–GFP–SsrA We constructed a fusion, λO162–GFP–SsrA, which we found provided a high proportion of doubly loaded complexes and could be translocated from the N- or C-terminus. With active ClpXP, λO162–GFP–SsrA is completely degraded when translocation is initiated from the C-terminal SsrA tag, and a fraction of the fusions are degraded from the λO side, resulting in transient accumulation of the GFP portion (H.S.Lee and M.R.Maurizi, manuscript in preparation). We assembled complexes of λO162–GFP–SsrA and ClpXPin in the presence of ATPγS and examined them by EM. More than 80% of the complexes had extra density at both ends. λO162– GFP–SsrA bound in two modes: the 'λO mode', with a broad, diffuse density extending out from the surface of ClpX (Figure 5A, top); and the 'SsrA mode', with a compact density apposed to the surface of ClpX (Figure 5A, bottom). More than half of the doubly loaded complexes (46% of the total) had one λO162–GFP–SsrA bound in the SsrA mode and one in the λO mode (Figure 5A, middle); the remainder had both molecules bound in one or the other mode (Figure 5A, top and bottom left). A minority of 2:1 complexes had a single λO162– GFP–SsrA bound, and they were about evenly split between the two modes (Figure 5A, right, top and bottom). This mixture of complexes was a good starting point for translocation assays. Figure 5.Binding and translocation of a substrate with N-terminal and C-terminal recognition tags. (A) λO162–GFP–SsrA bound to ClpXPin in the presence of 1 mM ATPγS. Particles were subjected to classification according to the density associated with the outer surface of ClpX, and averages were obtained of five different classes. λO162–GFP–SsrA can interact with ClpXPin either by its N-terminal λO recognition motif (λO mode) or by its C-terminal SsrA motif (SsrA mode). The latter class was identifiable because of the intense, axially aligned density observed when SsrA-tagged proteins bind to ClpX (see Figure 1). Complexes with λO162–GFP–SsrA bound to one or both faces of ClpXPin in the λO mode (A1 and A2) and in the SsrA mode (A4 and A5) were found. About half of the complexes had substrate molecules in the λO mode and the SsrA mode (A3) on either side. (B) After translocation of λO162–GFP–SsrA in 8 mM ATP or in ATPγS for 20 min electron micrographs were recorded and scanned. Particles with translocated density in ClpP were further classified according to the appearance of the residual density bound to the surface of ClpX, and average images of each class were generated. The majority of particles (B1, B2, B4 and B5) had one side with a condensed axially aligned density similar to the non-translocated GFP domain seen previously with λO162–GFP, indicative of translocation from the λO end. With either ATP or ATPγS, about half of these particles retained unaltered substrate bound to ClpX in the λO mode (B1 and B4), and in half, the substrate had dissociated from the other surface (B2 and B5). A minority of particles (12% with ATP, and 7% with ATPγS) appeared to have a reduced amount of substrate associated with ClpX on both ends of the complex (B3 and B6), implying that translocation occurred from both sides. Each average image combined between 150 and 350 particles for resolutions of between 27 and 32 Å. The scale bar represents 100 Å. Download figure Download PowerPoint After incubation for 20 min with ATPγS or 2 min with ATP, the centers of the complexes filled with stain-occluding protein and there were changes in the bound substrate. With either nucleotide, a small, axially aligned density, similar to the compact GFP domain remaining after translocation of the λO moiety of λO162–GFP, remained on one end of ∼90% of the particles (Figure 5B, left and middle). In about half of these, the other end of the post-translocation complexes had density very similar to the original substrate density (Figure 5B, left), suggesting little, if any, translocation from that side. In the other particles, clearly evident in the ATPγS-treated samples, there was little or no density visible on the other end (Figure 5B, middle). A faint density was visible on the left side of the images of ATP-treated complexes (Figure 5B, bottom middle), but examination of individual particles showed no discrete densities that we could identify. We believe this density may result from the N-terminal domains of ClpX that are occasionally visible in side views or may be a result of misclassification of a small number of particles. The absence of significant density on the non-translocating side in half of the complexes reflects a greater tendency of substrates to dissociate from filled complexes, as was also seen with the λO162–GFP complexes (Figure 4). Another 7% of post-translocation complexes with ATPγS and 12% with ATP appeared to have translocated substrate from both ends (Figure 5B, right).