Zn2+-induced reversible dissociation of subunit Rpn10/p54 of the Drosophila 26 S proteasome

Abstract

In the presence of Zn2+, the Drosophila 26 S proteasome disassembles into RP (regulatory particle) and CP (catalytic particle), this process being accompanied by the dissociation of subunit Rpn10/p54, the ubiquitin receptor subunit of the proteasome. The dissociation of Rpn10/p54 induces extensive rearrangements within the lid subcomplex of the RP, while the structure of the ATPase ring of the base subcomplex seems to be maintained. As a consequence of the dissociation of the RP, the peptidase activity of the 26 S proteasome is lost. The Zn2+-induced structural and functional changes are fully reversible; removal of Zn2+ is followed by reassociation of subunit Rpn10/p54 to the RP, reassembly of the 26 S proteasome and resumption of the peptidase activity. After the Zn2+-induced dissociation, Rpn10/p54 interacts with a set of non-proteasomal proteins. Hsp82 (heat-shock protein 82) has been identified by MS as the main Rpn10/p54-interacting protein, suggesting its role in the reassembly of the 26 S proteasome after Zn2+ removal. The physiological relevance of another Rpn10/p54-interacting protein, the Smt3 SUMO (small ubiquitin-related modifier-1)-activating enzyme, detected by chemical cross-linking, has been confirmed by yeast two-hybrid analysis. Besides the Smt3 SUMO-activating enzyme, the Ubc9 SUMO-conjugating enzyme also exhibited in vivo interaction with the 5'-half of Rpn10/p54 in yeast cells. The mechanism of 26 S proteasome disassembly after ATP depletion is clearly different from that induced by Zn2+. Rpn10/p54 is permanently RP-bound during the ATP-dependent assembly-disassembly cycle, but during the Zn2+ cycle it reversibly shuttles between the RP-bound and free states.

Figure 1. Zn²⁺-binding sites on proteasomal subunits

Lane 1, silver-stained one-dimensional SDS/PAGE pattern of highly purified Drosophila 26 S proteasome; lane 2, proteasomal subunits recovered with 0.2 M imidazole from Zn²⁺-charged fractogel metal chelate column.

Figure 2. Zn²⁺-induced changes in proteasomal subunit interactions, detected by chemical cross-linking and immunoblot analysis

Highly purified Drosophila 26 S proteasome was preincubated in the presence of ATP, with or without 200 μM ZnCl₂, and cross-linked with disuccinimidyl suberate. The cross-linking patterns, representing subunit interactions, were analysed in immunoblots with subunit-specific mAbs. Changes in the subunit interactions of Rpn7/p42A and Rpn9/p39A lid subcomplex subunits were analysed with mAb 123 and mAb 50 respectively. The Rpt5/p50 ATPase subunit and the ubiquitin receptor subunit Rpn10/p54 were analysed with mAb 112 and mAb 170 respectively. The asterisk denotes subunit interactions not influenced by Zn²⁺ treatment. Arrow-heads mark the non-cross-linked subunits.

Figure 3. Zn²⁺-induced structural and functional changes in the 26 S proteasome, analysed by native PAGE

(A) The DEAE-fractogel fraction of the Drosophila 26 S proteasome was fractionated on 1 mM ATP-containing native polyacrylamide gel and analysed by immunoblotting with mAb 170 (lane 1) and mAb IIG7 (lane 2), or its peptidase activity was tested in a fluorigenic overlay assay (lane 3). (B) The DEAE-fractogel fraction of the Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂ and fractionated on 1 mM ATP and 200 μM ZnCl₂-containing native polyacrylamide gel. Immunoblot analysis was performed with mAb 170 (lane 1), mAb IIG7 (lane 2), mAb 112 (lane 3) and mAb 50 (lane 4). The peptidase activity of the Zn²⁺-treated proteasomes was tested in a fluorigenic overlay assay (lane 5). (C) Reversibility of Zn²⁺-induced changes: Zn²⁺ was removed with 1,10-phenanthroline from the Zn²⁺-treated proteasome shown in (B), and analysed on 1 mM ATP-containing native gel. Immunoblot analysis was performed with mAb 170 (lane 1), mAb IIG7 (lane 2), mAb 112 (lane 3) and mAb 50 (lane 4). The peptidase activity was tested in a fluorigenic overlay assay (lane 5). (D) Reassembly of the 26 S proteasome after ATP depletion. Lanes 1–3: the ATP-depleted DEAE-fractogel fraction of the Drosophila 26 S proteasome was fractionated on native polyacrylamide gel prepared without ATP. Immunoblot analysis was performed with mAb 170 (lane 1) and mAb IIG7 (lane 2); the peptidase activity was tested in a fluorigenic overlay assay (lane 3). Lanes 4–5: the DEAE-fractogel fraction of the Drosophila 26 S proteasome was analysed on 1 mM ATP-containing native polyacrylamide gel. Immunoblots were tested with mAb 170 (lane 4) and mAb IIG7 (lane 5). Lanes 6–8: the ATP-depleted DEAE-fractogel fraction was incubated in the presence of ATP and fractionated on 1 mM ATP-containing native polyacrylamide gel. Immunoblot analysis was performed with mAb 170 (lane 6) and mAb IIG7 (lane 7); the peptidase activity was tested in a fluorigenic overlay assay (lane 8). (E) Integrity of the RP after zinc treatment. RP of control and zinc-treated DEAE-fractogel fraction was purified on a Superose 6 sizing column and their subunit pattern was analysed on silver-stained denaturing gel (lanes 1 and 2) and by immunoblotting (lanes 3 and 4) with a mixture of mAbs specific for the subunits indicated on the right side.

Figure 4. Detection of Rpn10/p54-interacting proteins

(A) The DTT-free DEAE-fractogel fraction of the Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂ for the time periods indicated. The preincubation was followed by cross-linking with disuccinimidyl suberate. Rpn10/p54-interacting proteins were detected by immunoblotting with mAb 170. (B) The DEAE-fractogel sample preincubated for 20 min with 200 μM ZnCl₂ (A) was supplemented with 200 μM 1,10-phenanthroline, incubated for an additional 10 min, cross-linked and analysed as described in (A). Arrowhead marks the non-cross-linked Rpn10/p54 protein.

Figure 5. Purification of Rpn10/p54-interacting proteins by immunoprecipitation

The cross-linked DEAE-fractogel sample shown in Figure 4(A) (20 min) was immunoprecipitated with a mixture of mAb 28 and mAb 170. mAb developed against potato leghaemoglobin was used for control immunoprecipitation. Aliquots of the precipitated materials were analysed by immunoblotting. Lane 1: the immunoprecipitate obtained using potato leghaemoglobin antibody was analysed with mAb 170. The immunoprecipitate produced with a mixture of mAb 28 and mAb 170 was analysed with mAb 170 (lane 2), mAb IIG7 (lane 3), mAb 50 (lane 4), mAb 112 (lane 5) and mAb 123 (lane 6).

Figure 6. Hsp82 supports the reassembly of zinc-treated highly purified 26 S proteasome

(A) Highly purified Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂. After zinc removal, incubation was continued for an additional 20 min and the proteasomes were fractionated on Superose 6 sizing column. Column fractions (200 μl) were concentrated by precipitation with 7 vol. of acetone, run on 10% (w/v) SDS gel and immunoblotted with a mixture of five mAbs specific for subunits indicated on the left. (B) Highly purified Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂. After the removal of zinc, incubation was continued with 5-fold molar excess of purified Hsp82 protein for an additional 20 min and the proteasomes were fractionated on Superose 6 sizing column. Column fractions (200 μl) were concentrated by precipitation with 7 vol. of acetone, run on 10% SDS gel and immunoblotted with a mixture of five mAbs specific for subunits indicated on the left. (C) DEAE-fractogel fraction of Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂ and fractionated on Superose 6 sizing column in the presence of 200 μM ZnCl₂. Aliquots (20 μl) of the column fraction were run on 10% SDS gel and immunoblotted with a mixture of five mAbs specific for subunits indicated on the left. (D) DEAE-fractogel fraction of Drosophila 26 S proteasome was preincubated in the presence of ATP with 200 μM ZnCl₂. After zinc removal, incubation was continued for an additional 20 min and the proteasomes were fractionated on Superose 6 sizing column. Aliquots (20 μl) of the column fraction were run on 10% SDS gel and immunoblotted with a mixture of five mAbs specific for subunits indicated on the left.

Figure 7. In vivo interaction of the 5′-half of Rpn10/p54 with the Smt3 SUMO-activating and the DmUbc9 SUMO-conjugating enzymes

Single- and double-transformant yeasts were grown on minimal medium and tested for Lac Z activity.

Figure 8. In vitro interaction of the Smt3 SUMO-activating enzyme, the DmUbc9 SUMO-conjugating enzyme and the Hsp82 protein with the full-length Rpn10/p54

Uncharged anti-FLAG M2 affinity column (lane 1), or anti-FLAG M2 affinity columns charged with FLAG–Smt3 SUMO-activating enzyme (lane 2), FLAG–DmUbc9 SUMO-conjugating enzyme (lane 3) or FLAG–Hsp82, were loaded with Rpn10/p54. The columns were eluted with an excess of FLAG peptide and analysed by immunoblotting with mAb 170.