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. 2015 Oct 9;5:14909. doi: 10.1038/srep14909

Figure 6. The Rpt6 tail-α3 interaction decreases in the proteasome holoenzyme and is less nucleotide-dependent than the Rpt3 tail-α2 interaction.

Figure 6

(a) Rpt3 tail-α2 binding is strongly ATP-dependent in the proteasome holoenzyme, but is Nas6-independent. Wild-type and nas6Δ cells each harbor rpt3-K428C and α2-A79C-HA6 alleles; K428 is the last residue of Rpt3. Proteasomes were immunoprecipitated via α2-HA6 in the presence of ATP (1 mM) or ADP (2 mM), and then incubated with a chemical crosslinker BMOE (0.1 mM), or its solvent, DMF for 1 hour at 4 °C and subjected to SDS-PAGE. Rpt3-α2HA crosslinks were detected by immunoblotting (IB) for Rpt3 and α2HA. Molecular weight markers are at left in kDa. Asterisk (*) indicates a non-specific signal. Crosslinked products remain stable during our analysis since BMOE is an irreversible crosslinker. (b) Rpt6 tail-α3 binding occurs in both ATP and ADP in the proteasome holoenzyme. Experiments were conducted as in (a) in wild-type and nas6Δ cells, each harboring rpt6-K405C and α3-T81C-HA6 alleles; K405 is the last residue of Rpt6. Rpt6-α3HA crosslinks were detected by immunoblotting for Rpt6 and α3HA. Asterisk (*) indicates a non-specific band. (c) Rpt6-α3 interaction decreases in the proteasome holoenzyme whereas Rpt3-α2 interaction increases. The Rpt3-α2 crosslinks and Rpt6-α3 crosslinks as in (a,b) were quantified using ImageJ software and shown as mean + SEM (n = 6, Rpt3-α2, ATP; n = 3, Rpt3-α2, ADP and Rpt6-α3, ATP; n = 4, Rpt6-α3, ADP). The ratio on the Y axis was obtained by normalizing the intensities of Rpt-αHA crosslinked bands to corresponding uncrosslinked Rpt bands on the same immunoblot, for example, [Rpt6-α3HA band (80 kDa)]/[Rpt6 band (45 kDa)] (Fig. 6b, lane 2, top). Note that 43.6 + 0.6% decrease in Rpt6-α3HA ratio (ATP) is relative to Rpt3-α2HA ratio (ATP). (d) Rpt6-α3 interaction occurs in ADP whereas Rpt3-α2 interaction severely decreases. The Y axis indicates percent decrease of Rpt-αHA ADP samples relative to their corresponding ATP samples from (c). (e) The Rpt6 tail is crucial for Rpt3-α2 binding (arrow heads) in the proteasome holoenzyme. Experiments were conducted as in (a) in rpt6-Δ1 and wild-type cells, each carrying rpt3-K428C and α2-A79C-HA6 alleles. Rpt6 levels remain unchanged.