Blm10 binds to pre-activated proteasome core particles with open gate conformation - PubMed (original) (raw)

Blm10 binds to pre-activated proteasome core particles with open gate conformation

Andrea Lehmann et al. EMBO Rep. 2008 Dec.

Erratum in

Abstract

Blm10 is bound to the yeast proteasome core particle, a crucial protease of eukaryotic cells [corrected]. Two gates, at both ends of the CP, control the access of protein substrates to the catalytic cavity of the CP. Normally, substrate access is auto-inhibited by a closed gate conformation unless regulatory complexes are bound to the CP and translocate protein substrates in an ATP-dependent manner. Here, we provide evidence that Blm10 recognizes pre-activated open gate CPs, which are assumed to exist in an equilibrium with inactive closed gate CP. Consequently, single-capped Blm10-CP shows peptide hydrolysis activity. Under conditions of disturbed CP assembly, as well as in open gate mutants, pre-activated CP or constitutively active CP, respectively, prevail. Then, Blm10 sequesters disordered and open gate CP by forming double-capped Blm10(2)-CP in which peptide hydrolysis activity is repressed. We conclude that Blm10 distinguishes between gate conformations and regulates the activation of CP.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Figure 1

Figure 1

Pre-activated core particles are stabilized by Blm10 in _ump1_Δ cells. (A) Affinity-purified wild-type proteasomes (Lehmann et al, 2002) contain Blm10 and Ecm29, as identified by finger print mass spectroscopy; RP and CP subunits are embraced. (B–E) GFP-labelled CPs of isogenic wild-type (wt; lane 1), _blm10_Δ (lane 2), _ump1_Δ (lane 3) and _blm10_Δ _ump1_Δ cells (lane 4) were resolved by native PAGE; CP configurations are assigned. (B) CP distributions were visualized by GFP imaging. (C) Blm10-associated CPs were detected by Western blot. (D) By using Suc-LLVY-AMC (Y) as a peptide substrate, in-gel CP activity was detected in the absence and (E) in the presence of 0.02% SDS. The lower part of (D) is shown with high contrast. (F) Blm10 is neither associated with half-CP (Fehlker et al, 2003) nor completely recruited to CP. To visualize half-CP by GFP imaging, the contrast below the dotted line is increased. Blm10-associated CPs are detected by immunoblot. (G) GFP-labelled CP were affinity-purified from wild-type (lane 1), _ump1_Δ (lane 2), _blm10_Δ (lane 3) and _blm10_Δ _ump1_Δ (lane 4) cells, resolved by native PAGE, visualized by GFP imaging, probed for Blm10, and assayed for Y-activity in the absence and presence of SDS. Owing to the strong stimulation of CP activity by 0.02% SDS, the exposure time of the in-gel activity assays had to be reduced. The histogram shows arbitrary AMC/GFP ratios of Blm102-CP, Blm10-CP and CP. (H) Blm102-CP, Blm10-CP and CP of _ump1_Δ cells were extracted from the native gel, subjected to SDS–PAGE and analysed by Western blot as indicated; lysate acted as a control. Pac1 to Pac4 were not identified in mature CP (data not shown). AMC, 7-amino-4-methyl-coumarin; CP, core particle; GFP, green fluorescent protein; RP, regulatory particle; SDS–PAGE, SDS–polyacrylamide gel electrophoresis.

Figure 2

Figure 2

Constitutively active core particles are capped by Blm10 or RP. (A) Extracts of wild-type (wt; lane 1), α3ΔN (lane 2), α7ΔN (lane 3) and α3ΔN/α7ΔN (lane 4) cells were analysed by native PAGE and GFP imaging. Equal amounts of protein were loaded. (B) Blm10-bound CPs were identified by Western blot. (C) In-gel assays for Y-activity were performed in the absence of SDS or (D) in the presence of SDS. (E) Blm10 and β5 levels of wild type (lane 1), α3ΔN (lane 2), α7ΔN (lane 3) and α3ΔN/α7ΔN (lane 4) were determined by Western blot. (F) Extracts of wild-type cells (lane 1) and α4ΔN mutants (lane 2) expressing GFP-tagged α4 were analysed by native PAGE and GFP imaging. Blm10 and β5 levels were determined by Western blot. (G) Extracts of _pac1_Δ _pac2_Δ (upper panels), _pac3_Δ _pac4_Δ (lower panels) and isogenic wild-type cells, respectively, were analysed by native PAGE and GFP imaging. Y-activity assays were performed in the absence and presence of SDS. To visualize Blm10-CP activity, the upper in-gel activity assay was overexposed. Equal amounts of protein were loaded. (H) Native PAGE of α4ΔN _blm10_Δ cell extracts (GFP-tagged α4) were analysed by GFP imaging (lane 1) and Y-activity assay (lane 2). Native PAGE of α3ΔN/α7ΔN _blm10_Δ cell extracts (GFP-tagged β5) were analysed by GFP imaging (lane 3) and Y-activity assay (lane 4). (I) Extracts from wild-type (lane 1) and α3ΔN/α7ΔN _blm10_Δ (lane 2) cells were subjected to native PAGE, blotted and probed for Rpt1 (RP base). CP, core particle; GFP, green fluorescent protein; PAGE, polyacrylamide gel electrophoresis; RP, regulatory particle.

Figure 3

Figure 3

Model of Blm10 function. For a description of the model, see the Speculation section. CP, core particle; RP, regulatory particle.

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