Two canonical subunits of the 26S proteasome, Rpn10 and Rpn13, function as ubiquitin (Ub) receptors. The mutual arrangement of these subunits-and all other non-ATPase subunits-in the regulatory particle is unknown. Using electron cryomicroscopy, we calculated difference maps between wild-type 26S proteasome from Saccharomyces cerevisiae and deletion mutants (rpn10Δ, rpn13Δ, and rpn10Δrpn13Δ). These maps allowed us to localize the two Ub receptors unambiguously. Rpn10 and Rpn13 mapped to the apical part of the 26S proteasome, above the N-terminal coiled coils of the AAA-ATPase heterodimers Rpt4/Rpt5 and Rpt1/Rpt2, respectively. On the basis of the mutual positions of Rpn10 and Rpn13, we propose a model for polyubiquitin binding to the 26S proteasome.EM single-particle analysis | protein degradation | ubiquitin-proteasome pathway | subunit localization | quantitative mass spectrometry
Recognition of polyubiquitylated substrates by the proteasome is a highly regulated process that requires polyubiquitin receptors. We show here that the concentrations of the proteasomal and extraproteasomal polyubiquitin receptors change in a developmentally regulated fashion. The stoichiometry of the proteasomal p54/Rpn10 polyubiquitin receptor subunit, relative to that of other regulatory particle (RP) subunits falls suddenly at the end of embryogenesis, remains low throughout the larval stages, starts to increase again in the late third instar larvae and remains high in the pupae, adults and embryos. A similar developmentally regulated fluctuation was observed in the concentrations of the Rad23 and Dsk2 extraproteasomal polyubiquitin receptors. Depletion of the polyubiquitin receptors at the end of embryogenesis is due to the emergence of a developmentally regulated selective proteolytic activity. To follow the fate of subunit p54/Rpn10 in vivo, transgenic Drosophila melanogaster lines encoding the N-terminal half (NTH), the C-terminal half (CTH) or the full-length p54/Rpn10 subunit were established in the inducible Gal4-UAS system. The daughterless-Gal4-driven whole-body expression of the full-length subunit or its NTH did not produce any detectable phenotypic changes, and the transgenic products were incorporated into the 26S proteasome. The transgene-encoded CTH was not incorporated into the 26S proteasome, caused third instar larval lethality and was found to be multi-ubiquitylated. This modification, however, did not appear to be a degradation signal because the half-life of the CTH was over 48 hours. Accumulation of the CTH disturbed the developmentally regulated changes in subunit composition of the RP and the emergence of the selective proteolytic activity responsible for the depletion of the polyubiquitin receptors. Build-up of subunit p54/Rpn10 in the RP had already started in 84-hour-old larvae and reached the full complement characteristic of the non-larval developmental stages at the middle of the third instar larval stage, just before these larvae perished. Similar shifts were observed in the concentrations of the Rad23 and Dsk2 polyubiquitin receptors. The postsynthetic modification of CTH might be essential for this developmental regulation, or it might regulate an essential extraproteasomal function(s) of subunit p54/Rpn10 that is disturbed by the expression of an excess of CTH.
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.
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