The plasmids expressing GFP-CL1, N-htt–GFP, and N-htt exon 1 were described previously (Bence et al., 2001
). In brief, the GFP-CL1 plasmid was created by ligating an oligonucleotide encoding ACKNWFSSLSHFVIHL into the GFP-C1 plasmid (Takara Bio Inc.). N-htt–GFP encodes htt exon 1 and contains mixed CAG/CAA repeats fused to a C-terminal GFP tag in pcDNA3.1 (Invitrogen). N-htt exon 1 was created by removing GFP from N-htt–GFP and ligating a linker between the BamHI and XbaI sites. Plasmids expressing Ub-R-GFP and UbG76V
-GFP were gifts from N. Dantuma (Karolinska Institutet, Stockholm, Sweden). The cODC-GFP–expressing plasmid was created by E. Bennett (University of California, San Diego, San Diego, CA) by amplification of the C-terminal 37 amino acids of ODC by PCR and cloned into pEGFP1 (Takara Bio Inc.) using the HindIII and BamHI sites. Plasmids expressing N-htt(Q25)–chFP and N-htt(Q91)–chFP were created by inserting chFP (a gift from R. Tsien, University of California, San Diego, La Jolla, CA) into the BamHI site of the N-htt exon 1 plasmids described in this paragraph. The chFP-CL1 plasmid was created by insertion of chFP and the CL1 sequence into pcDNA3.1 (Invitrogen). pET3a-Ub and pET15b-Ubc4 were provided by C. Pickart (Johns Hopkins University, Baltimore, MD) and D. Rotin (The Hospital for Sick Children, Toronto, Canada), respectively. pHUE and pHUsp2-cc were gifts from R. Baker (Australian National University, Canberra, Australia). Plasmids for GST-Rsp5 and GST-ΔC2Rsp5 were obtained from J. Huibregtse (University of Texas, Austin, TX). The ΔC2Rsp5 coding sequence was PCR amplified and cloned into a pET28a vector (EMD) to yield a His6
-tagged construct (pET28a-ΔC2Rsp5). The plasmid for expressing His6
–PY-Sic1 was obtained from R. Deshaies (Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA). The PY-Sic1 coding sequence was PCR amplified and cloned into the pHUE vector as previously described (Catanzariti et al., 2004
) to create the pHUE–PY-Sic1 plasmid. A plasmid containing the cyclin B N100 fragment (Chen and Fang, 2001
) was obtained from G. Fang (Stanford University, Stanford, CA). The cyclin B N100 coding sequence was PCR amplified and cloned into the pMAL-c2X plasmid (New England Biolabs, Inc.). Site-directed mutagenesis was used to introduce a Pro-Pro-Pro-Tyr sequence (PY motif) in front of the cyclin N100 sequence and a T7 tag at the end of cyclin N100 to create pMAL-PY-cyclinN100-T7 (MPC). Plasmids expressing the original GST–N-htt fusion constructs containing either Q18 or Q51 were obtained from E. Wanker (Max Delbrück Center, Berlin, Germany). These constructs were modified by the addition of a TEV protease cleavage site between the GST and htt exon 1 coding regions (GST–N-htt(Qn)-ΔS) or by the addition of a TEV protease cleavage site between the GST and htt exon 1 coding regions and the addition of an S tag C terminal to the htt exon 1 coding region (GST–N-htt(Qn); Bennett et al., 2005
). Within these constructs, site-directed mutagenesis was used to insert a PY motif immediately after the TEV protease site to create plasmids encoding for GST–PY-N-htt(Q18)-S and GST–PY-N-htt(Q51)-S.
The GFP-CL1 (Bence et al., 2001
), CFTRΔF508-GFP (Johnston et al., 1998
), and TCR-α–GFP (DeLaBarre et al., 2006
) cell lines were previously described. These cell lines were prepared by transfection of HEK293 cells followed after 48 h by selection of transformed cells by growth in G418. Also, the other stable HEK293 cell lines expressing the constructs described in this study were created by transfection, selected with G418, and cloned by limiting dilution. The temperature-sensitive ts20 Balb/C 3T3 clone A31 fibroblast cell line (Kulka et al., 1988
) was a gift from D.T. Madden (The Buck Institute for Research on Aging, Novato, CA). All cells were grown in DME with 10% animal serum complex, l
-glutamine, and antibiotics.
Flow cytometry and cell sorting
Unless indicated, cells were harvested 72 h after transfection and analyzed with a flow cytometer (LSR II; BD) with a 488- and 535-nm laser (BD). To ensure a sufficient number of cells with elevated levels of the transfected protein, >200,000 cells were analyzed per condition in a typical experiment. To plot the level of the reporter protein versus the level of the transfected protein (described in Fig. S2), a set of 41 gates of equal width (on a logarithmic scale) was set up in the channel for the transfected protein. The mean compensated fluorescence of the reporter protein in each of these gates was calculated and plotted on the ordinate with the gate number (corresponding to the log of fluorescence intensity of the transfected protein) plotted on the abscissa. Each construct and condition in singly transfected HEK293 cells was used as a single-color control to compensate the spillover between chFP and GFP individually for each gate. Gates with <100 events were not included in the analysis. The data shown in , , and are from single representative experiments out of a minimum of two independent repeats. Raw flow cytometry data were analyzed using FlowJo (version 8.8.6; Tree Star) software.
To isolate high and low N-htt(Q91)–GFP–expressing populations, cells were harvested 72 h after transient transfection and sorted according to N-htt(Q91)–chFP intensity using a cytometer (Digital Vantage; BD) with a 80-µm nozzle and 570–595-nm tunable laser. To define the LS and HS populations, the photomultiplier tube voltage was set to center the nonexpressing population over the 102-a.u. intensity mark, and the LS gate was defined to include cells in the 102–103-a.u. interval, whereas the HS gate included cells with >103 a.u. 1–2 × 106 cells were sorted from each gate, and pellets were flash frozen in liquid N2.
Recombinant human E1 enzyme was purchased from Boston Biochem. Recombinant Ub was expressed and purified as previously described (Kaiser et al., 2011
). In brief, Ub was expressed from the pET3a-Ub expression vector in BL21 (DE3) pLysS RIL cells upon induction with IPTG. Bacterial cells were harvested, resuspended in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 10% [vol/vol] glycerol, 1% [vol/vol] Triton X-100, 1 mM EDTA, and protease inhibitor cocktail [Complete; Roche]), lysed by french press, and clarified by centrifugation (SS-34 rotor; Sorvall; 18,500 rpm for 60 min). Glacial acetic acid was added dropwise with mixing on ice until the solution reached pH 4. The precipitate was pelleted by centrifugation (SS-34 rotor; 13,000 rpm for 20 min), and the supernatant was dialyzed against 25 mM sodium acetate, pH 4.5. The dialysate was purified using a column (HiTrap SP XL; GE Healthcare) and eluted with a 0–500 mM NaCl linear gradient. Elution fractions containing Ub were pooled and further purified by gel filtration chromatography on a column (Sephacryl S-200; GE Healthcare) with 50 mM Tris, pH 7.5, 500 mM NaCl, and 1 mM DTT. Eluted fractions were dialyzed into 25 mM Hepes, pH 7.5, aliquoted, and stored at −80°C.
Recombinant Ubc4 was expressed and purified as previously described (Kaiser et al., 2011
). In brief, Ubc4 was expressed from the pET15b-Ubc4 plasmid in BL21 (DE3) cells upon induction with IPTG. Bacterial cells were harvested by centrifugation, resuspended in lysis buffer (50 mM Tris, pH 7.5, 300 mM NaCl, 20 mM imidazole, 10% glycerol, 0.2% Triton X-100, 1 mM PMSF, and EDTA-free protease inhibitor cocktail [Complete]), and lysed by french press. The lysate was clarified by centrifugation, and the supernatant was batch bound to Ni-nitrilotriacetic acid resin for 2 h. The resin was washed five times with 10 bed vol lysis buffer, and Ubc4 was eluted with lysis buffer supplemented with 250 mM imidazole. The eluate was dialyzed into 50 mM Tris, pH 7.6, 150 mM NaCl, 10% glycerol, and 1 mM DTT, aliquoted, and stored at −80°C.
Recombinant Usp2-cc was expressed from the pHUsp2-cc plasmid in BL21 (DE3) as an N-terminal His6-tagged fusion protein. Expression was induced with 0.4 mM IPTG for 4 h at 37°C. Cells were harvested by centrifugation, resuspended in buffer A (50 mM Hepes, pH 7.5, 300 mM NaCl, 30% glycerol, 0.1% Triton X-100, and 2 mM β-mercaptoethanol), and lysed by french press. The lysate was clarified by centrifugation, and the supernatant was bound to Ni-nitrilotriacetic acid beads. The beads were washed with buffer A without Triton X-100 and eluted with 400 mM imidazole. The eluted protein was dialyzed into 50 mM Hepes, pH 7.5, 150 mM NaCl, 30% glycerol, and 5 mM DTT, aliquoted, and stored at −80°C.
-tagged ΔC2Rsp5 protein was expressed from the pET28a-ΔC2Rsp5 plasmid in E. coli
and purified by using metal affinity chromatography (TALON; Takara Bio Inc.). In brief, cell pellets were resuspended in 40 vol of 50 mM Tris-HCl, pH 7.6, 300 mM NaCl, 5 mM β-mercaptoethanol, and 1 mM PMSF and lysed by sonication. The lysate was clarified at 10,000 g
for 30 min before being loaded onto a gravity column packed with 3 ml of TALON beads. The column was washed with 20 column vol resuspension buffer without β-mercaptoethanol and eluted with 3 column vol of 50 mM Tris, pH 7.6, 300 mM NaCl, 200 mM imidazole, and 10% glycerol. The eluted protein was dialyzed into 50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1 mM DTT, and 10% glycerol. GST-Rsp5, GST–N-htt(Q51)-ΔS, GST–PY-N-htt(Q18)-S, and GST–PY-N-htt(Q51)-S were expressed in BL21 (DE3) cells and affinity purified with glutathione–Sepharose 4b resin (GE Healthcare) as recommended by the manufacturer. PY-Sic1 was expressed as an N-terminal His6
-tagged Ub fusion protein using the pHUE–PY-Sic1 plasmid and metabolically labeled in BL21 (DE3) cells grown in minimal media supplemented with [35
S]methionine (Zhang et al., 2003
). In brief, a 100-ml culture of E. coli
was grown at 37°C in M9 minimal media to OD600
= 0.5, washed once with M9 media, and resuspended in 80 ml M9 media containing 0.4% glucose and 0.063% methionine assay media. After further incubation at 37°C for 30 min, protein expression was induced with 1 mM IPTG. After a 1-h incubation, 1 mCi [35
S]methionine was added. After 10 min, 1 mM of unlabeled methionine was added to the culture and grown for an additional 10 min. Cells were harvested by centrifugation and lysed by 3× freeze/thaw steps in the presence of 1 mg/ml lysozyme and DNase I (Sigma-Aldrich). Clarified lysate was then bound to TALON metal affinity resin for 1 h at RT. Resin-bound protein was incubated with His-tagged Usp2-cc. PY-Sic1 was released from the TALON beads upon cleavage with Usp2-cc, collected in the unbound fraction, and supplemented with 0.2 mg/ml BSA as a carrier. The final protein concentration was measured using a bicinchoninic acid protein assay (Thermo Fisher Scientific) relative to a BSA standard. MPC was expressed in BL21 (DE3) cells and affinity purified with amylose resin according to the manufacturer’s instructions (New England Biolabs, Inc.). 26S proteasomes were purified from Sprague-Dawley rats as previously described (Zhang et al., 2003
). In brief, frozen rat livers were resuspended in 3 ml/g of tissue with buffer A (20 mM Tris, pH 7.5, 2 mM ATP, 5 mM MgCl2
, 1 mM DTT, 0.1 mM EDTA, 50 mM NaCl, and 20% [vol/vol] glycerol), dounce homogenized, and clarified by centrifugation (SS-34 rotor; 9,300 rpm for 80 min). From the clarified supernatant, S100 extracts were prepared by centrifugation (Ti45 rotor; Beckman Coulter; 30,000 rpm for 58 min). The 26S proteasome within the S100 extract was enriched by sedimentation centrifugation (Ti45 rotor; 35,000 rpm for 18 h). The resulting pellet was resuspended in buffer A without glycerol and fractionated by centrifugation using a 15–40% glycerol gradient (SW27 rotor; Beckman Coulter; 25,000 rpm for 16 h). Gradient fractions containing peptidase activity were further purified using a column (Mono Q HR 5/50; GE Healthcare) equilibrated with 20 mM Tris, pH 7.5, 1 mM DTT, 50 mM NaCl, and 10% (vol/vol) glycerol. Proteasomes were eluted using a 50–800-mM NaCl linear gradient. Active fractions were pooled, enriched by sedimentation centrifugation as described in this paragraph, and finally resuspended in 20 mM Tris, pH 7.5, 1 mM DTT, 50 mM NaCl, and 10% (vol/vol) glycerol. The concentration of 26S proteasomes was measured using a Bradford assay relative to a BSA standard curve.
0.05–0.2 mg/ml 35S–PY-Sic1 and MPC were ubiquitinated for 16 h in the presence of 50 nM E1 (Boston Biochem), 2.4 µM E2 (His6-Ubc4), 2 µM E3 (GST-Rsp5), 300 µM Ub, and ATP buffer (40 mM Tris-HCl, pH 7.2, 2 mM ATP, 5 mM MgCl2, and 1 mM DTT). The reaction was then mixed with 0.5 vol glutathione–Sepharose beads for 3 h at RT to remove contaminating autoubiquitinated GST-Rsp5. Similarly, 0.05–0.2 mg/ml GST–PY-N-htt(Qn)-S was ubiquitinated, except that 2 µM E3 (ΔC2Rsp5) was used, and 1 vol TALON metal affinity resin was needed to remove any autoubiquitinated E3 enzyme. 35S–PY-Sic1 ubiquitination (35S-Sic1(Ubn)) was monitored by SDS-PAGE and autoradiography. The ubiquitination of MPC (MPC(Ubn)) was monitored by SDS-PAGE and detected with T7-HRP conjugate (Thermo Fisher Scientific) followed by chemiluminescence. The ubiquitination of GST–PY-N-htt(Qn)-S was monitored by separation on SDS-PAGE and detection with S-HRP conjugate (EMD) followed by chemiluminescence.
In vitro Sic1 degradation assay
The substrate degradation reaction was performed at RT in 50–100 µl of total volume containing 10 nM 26S, ATP buffer, and 100 nM 35
). 5-µl reaction aliquots were removed at various time points and separated on a 4–20% gradient gel followed by autoradiography. Alternately, aliquots were added to 10% TCA or double-distilled H2
0 on ice for 30 min and 2 mg/ml BSA as a carrier. Samples were centrifuged at 12,000 g
for 30 min, and the supernatant was removed for analysis by scintillation spectrometry. The percentage of degradation is determined by dividing the measured counts after TCA precipitation by the total counts measured in double-distilled H2
0. To assess the Ub dependence of degradation, 35
) was pretreated with 10 nM Usp2-cc (Baker et al., 2005
) for 30 min at 37°C before the addition of proteasomes. To inhibit substrate degradation, 26S proteasomes were pretreated with 10 µM MG132 (Enzo Life Sciences) before the addition of substrate. The initial rate of degradation was determined by least-squares linear regression analyses of 5-, 10-, and 20-min time points using SigmaPlot (Systat Software, Inc.). The dependence of the initial rate of degradation on substrate concentration was plotted, and the data were fit to the Michaelis–Menten equation using SigmaPlot.
Proteasome inhibition assay
The proteasome degradation inhibition assay was set up in 50 µl total volume containing 10 nM 26S proteasomes, 100 nM 35
), and 0–500 nM MPC(Ubn
) in ATP buffer. 5-µl aliquots were removed at 5, 10, and 20 min for TCA precipitation, and scintillation counting was performed as described in the previous paragraph. The initial rate of degradation as a percentage of the control reaction is reported as the percent ratio of the initial rate of degradation of 35
) in the presence of varying concentrations of MPC(Ubn
) and the initial rate of degradation for 35
) in a control reaction with no MPC(Ubn
). The dependence of the initial rate of degradation of 35
) on the concentration of MPC(Ubn
) was plotted, and the data fit to the following equation for competitive inhibition using SigmaPlot: v0
]), in which K0.5
= (1 + [S]/KM
is the concentration of ubiquitinated inhibitor, [S] is the concentration of 35
), and vi
is the initial rate in the presence of inhibitor (Thrower et al., 2000
In vitro formation of ubiquitinated N-htt aggregates
The TEV protease-induced cleavage and aggregation of GST–N-httEx1(Q51)-S and filter trap analysis were performed as previously described (Bennett et al., 2005
). Aggregation of 0.15 mg/ml ubiquitinated GST–PY-N-htt(Q51)-S was initiated by the addition of 1:1 (wt/wt) TEV protease and 1:1 (wt/wt) GST–N-htt(Q51)-ΔS in aggregation buffer (40 mM Tris-HCl, pH 7.2, 1 mM DTT, and 0.5 mM EDTA). The aggregation reaction was incubated at 37°C and considered complete after 24-h filter trap analysis of ubiquitinated aggregates was performed using both the anti-Ub antibody (clone FK2; Enzo Life Sciences) and an S tag HRP conjugate followed by chemiluminescence. The degradation of 100 nM 35
) in the presence of 0–625 nM ubiquitinated aggregates was assessed, and the Ki
was determined as described in the previous paragraph.
HEK293 cells were transfected with the indicated N-htt–chFP plasmid and grown on poly-d-lysine–coated coverslips. 72 h after transfection, cells were fixed with 4% paraformaldehyde. Cells were imaged by epifluorescence on a microscope (Axiovert 200M; Carl Zeiss) with a 100× oil lens, NA 1.4 (Carl Zeiss). Digital (12 bit) images were acquired with a cooled charge-coupled device (CoolSNAP HQ; Photometrics) and processed using MetaMorph software (Universal Imaging).
For live-cell imaging of transiently transfected cells U2OS cells, cells were plated in DME with 10% FCS and 5% CO2 on glass-bottom 35-mm plates (MatTek) or on 8-well cover glass-bottom plates. Cells were transfected the next day with N-htt(Q91)–chFP or N-htt(Q47)–chFP with FuGENE 6 (Roche) according to the manufacturer’s instructions. Imaging of cells was started 16 h after transfection when most of the cells expressed the fluorescent protein at low but detectable levels.
For imaging of cells expressing YFP-Ub and UbG76V-GFP, cells were stably cotransfected with pEF-YFP-Ub or UbG76V-GFP with a vector that confers G418 resistance. Drug-resistant colonies were pooled, and expressing cells were sorted by FACS and further propagated. These cells were then transiently transfected with N-htt(Q91)–chFP as described in the previous paragraph.
Live-cell imaging was performed on an inverted microscope (Axiovert 200M) encased in a perspex chamber that was heated to 37°C. Plates were placed in a smaller internal chamber that was continuously perfused with humidified 5% CO2. The set up included a motorized stage (before) that enabled multiple simultaneous imaging of multiple fields. Digital (12 bit) images were acquired with a cooled charge-coupled device camera (CoolSNAP HQ) with an exacte light source (X-Cite; Lumen Dynamics), filter cubes for visualizing mCherry, YFP, or GFP, and phase contrast with a 20× NA 0.8 air objective. The entire set up was controlled by MetaMorph software, and ImageJ (National Institutes of Health) was used for image processing, analysis, and assembly.
Poly-Ub affinity capture and MS
For poly-Ub affinity capture, HEK293 cells stably expressing UbG76V
-GFP were transfected with N-htt(Q91)–chFP. Cells were then sorted into low (102
a.u.) and high (>103
a.u.) N-htt(Q91)–chFP fluorescence as described in Flow cytometry and cell sorting. Cells were lysed in 20 mM Hepes, pH 7.2, 150 mM NaCl, 10% glycerol, 1% Triton X-100, 5 mg/ml N
-ethylmaleimide, 2% SDS, and EDTA-free protease inhibitors (Complete) followed by 3× sonication for 10–15 s (Kaiser et al., 2011
). Extracts were centrifuged at 20,000 g
in a microcentrifuge (Centrifuge 5424; Eppendorf) for 10 min, and supernatants were quantified by bicinchoninic acid before enrichment of Ub species by affinity capture with human P2UBA resin (Ub association domain from human ubiquilin 2, UBQLN2, also known as PLIC-2; 1:1 slurry).
MS was performed as previously described (Bennett et al., 2007
; Riley et al., 2011
). In brief, trypsin-digested samples were analyzed on a liquid chromatography–MS system that consisted of a capillary HPLC system (Agilent Technologies) coupled to a mass spectrometer (mitrOTOF system [Bruker Daltonics]; LTQ Orbitrap XL [Thermo Fisher Scientific]). Peptide mixtures were separated on a C-18 reversed-phase column using a linear gradient of acetonitrile (0–45%). Relative intensities of the isotope-labeled standards and analyses were obtained by measuring the intensities of the monoisotopic molecular ions after averaging ~10 scans across the elution profile of the standard. Absolute quantification was performed using the ion intensities of the tracked endogenous peptides relative to the spiked labeled peptides. Ub ions measured included LIFAGK-GG-QLEDGR (UbK48) isopeptide unlabeled (mass/charge [m/z] = 487.60) and heavy isotope labeled (m/z = 489.94), ESTLHLVLR (ESTL) peptide unlabeled (m/z = 356.55) and heavy isotope labeled (m/z = 358.88), TLTGK-(GG)-TITLEVEPSDTIENVK (UbK11) isopeptide unlabeled (m/z = 801.43) and heavy isotope labeled (m/z = 803.43), and TLSDYNIQK-(GG)-ESTLHLVLR (UbK63) isopeptide unlabeled (m/z = 561.81) and heavy isotope labeled (m/z = 563.56). In addition, ions were measured for chFP tryptic peptide LSFPEGFK (m/z = 462.74) and its heavy isotope-labeled standard (m/z = 466.25) and for N-htt tryptic peptide AFESLK (m/z = 347.69) and its heavy leucine-labeled standard (m/z = 351.20). The chFP peptide is unique to chFP and is not found in GFP. To calculate the percentage of Ub-associated N-htt, the amount of P2UBA-associated N-htt(Q91)–-chFP (as measured by chFP) was divided by the total amount of N-htt(Q91)–chFP (as measured by chFP) using Orbitrap analysis. The chFP and N-htt peptides gave similar results.
Online supplemental material
Fig. S1 shows that the chFP tag does not alter the aggregation or UPS impairment phenotypes of htt. Fig. S2 explains the method of transformation of FACS data used to generate the plots in , , and . Fig. S3 shows experiments further characterizing the reporter lines used in this study. Fig. S4 accompanies and shows the kinetics of radiolabeled Sic1 degradation by 26S proteasomes and controls for the production of ubiquitinated and aggregated htt(Q51) in vitro. Fig. S5 shows MS data accompanying and characterizes the cell line used in and . Video 1 shows aggregation of N-htt(Q91)–chFP in U2OS cells. Video 2 shows that N-htt(Q91)–chFP aggregates recruit Ub. Video 3 shows that N-htt(Q91)–chFP aggregates form many hours before impaired proteasomal degradation. Table S1 shows the event log for time-lapse video microscopy. Online supplemental material is available at http://www.jcb.org/cgi/content/full/jcb.201110093/DC1