Ubiquitinated polyglutamine aggregates do not choke the 26S proteasome in vitro. (A) In vitro ubiquitination of radiolabeled Sic1 substrate. ³⁵S–PY-Sic1 purified from E. coli was ubiquitinated in vitro as described in Materials and methods. Aliquots of the reaction were removed at the indicated times, separated on a 4–20% gradient gel, and visualized by autoradiography. Mobilities of unmodified Sic1 and polyubiquitinated Sic1 are indicated. (B) Kinetic analysis of Sic1(Ub_n) degradation. 100 nM ³⁵S-Sic1(Ub_n) was incubated in the presence of 10 nM 26S proteasomes, and degradation kinetics were assessed by SDS-PAGE (Fig. S4 A) or release of TCA-soluble ³⁵S radioactivity (Fig. S4 B). Initial rates of substrate degradation were determined from the kinetics of ³⁵S radiolabel release (Fig. S4 B) and fitted to the Michaels–Menten equation by least-squares analysis assuming a K_m of 50 nM. The data shown are from a single representative experiment out of three independent repeats. (C) Preparation of ubiquitinated MPC. MPC, consisting of an N-terminal maltose binding protein (MBP) fused to a fragment of Xenopus cyclin (cyclin N100) and a T7 epitope tag, was purified from E. coli and was ubiquitinated in vitro as described in Materials and methods. Aliquots of the reaction were removed at the indicated times, separated on a 4–20% gradient gel, and visualized by immunoblotting with an anti-T7 HRP conjugate. (D) Ubiquitinated MPC is a competitive inhibitor of Sic1(Ub_n) degradation. Dependence of the initial rate of ³⁵S-Sic1(Ub_n) degradation on the concentration of MPC or ubiquitinated MPC (MPC(Ub_n)). 10 nM proteasomes were incubated with 100 nM substrate, and initial rates were determined by a linear fit to soluble TCA radioactivity as in Fig. S4 B. Initial rates are expressed as a percentage of the control reaction without MPC or MPC(Ub_n). The data were fit by least-squares analysis as described in Materials and methods. The data shown are from a single representative experiment out of two independent repeats. (E) Preparation of ubiquitinated N-htt fragments. GST–PY-N-htt(Qn) containing a C-terminal S tag was purified from E. coli and ubiquitinated in vitro as described in Materials and methods. Aliquots of the reaction were removed at the indicated times, treated as indicated with the deubiquitinating enzyme Usp2-cc, separated on a 4–20% gradient gel, and visualized by immunoblotting with an anti-T7 horse HRP conjugate. (F) Competitive inhibition of Sic1(Ub_n) degradation by ubiquitinated N-htt is independent of polyglutamine length or aggregation state. Dependence of the initial rate of ³⁵S-Sic1(Ub_n) degradation on the concentration of N-htt(Q18)(Ub_n) (top) or N-htt(Q51)(Ub_n) (bottom). Analysis was performed with nonaggregated (uncleaved) N-htt(Q51)(Ub_n) (closed circles) or after TEV cleavage and aggregation of the ubiquitinated N-htt(Q51)(Ub_n) (open circles). 10 nM proteasomes were incubated with 100 nM substrate, and initial rates were determined by a linear fit of soluble TCA radioactivity as in Fig. S4 B. Initial rates are expressed as a percentage of the control reaction without inhibitor present. The data were fit by least-squares analysis as described in Materials and methods. The data shown are from a single representative experiment out of two independent repeats. (G) N-htt(Q51)(Ub_n) aggregates are insoluble. N-htt(Q51)(Ub_n) was aggregated and filtered through a 0.2-µm cellulose acetate filter as described in Materials and methods. The blot was probed with anti-Ub (FK2) monoclonal antibody (top) or S protein–HRP to detect polyubiquitinated trapped N-htt aggregates.