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1.
FIGURE 2.

FIGURE 2. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

DOPA-Rpt3 C-terminal peptide cross-links to a specific α subunit of 20 S proteasome. The indicated concentrations of biotin-DOPA-Rpt3 or biotin-DOPA-Rpt3(−3C) (A) or fluorescein-DOPA-Rpt3 or fluorescein-DOPA-Rpt3(−3C) (B) peptides were used for cross-linking assays with 20 S proteasome as described under “Experimental Procedures.” Proteasome subunits were separated by SDS-polyacrylamide gel and detected by Coomassie Blue staining, as indicated, Western blotting with α-neutravidin (A), or fluorescence scanning (B). C, cross-linking was conducted as above with fluorescein-DOPA-Rpt3. The sample was divided and subjected to either SDS-PAGE (left) or two-dimensional PAGE (right), as described under “Experimental Procedures.” Cross-linked product was detected by fluorescence scanning. D, 20 S proteasome was subjected to two-dimensional PAGE and stained with Coomassie Blue. Similar results for experiments in all panels were obtained in at least four separate experiments. IEF, isoelectric focusing.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
2.
FIGURE 4.

FIGURE 4. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

The C terminus of Rpt3 does not influence 20 S proeasome activation. A, 20 S proteasome (12 nm) activity against the indicated peptide substrates was determined in the presence and absence of peptides corresponding to the last 10 residues of Rpt3 (400 μm) or Rpt5 (200 μm). B, 20 S proteasome (12 nm) activity against Suc-LLVY-AMC substrate was determined in the absence (Con) and presence of the indicated C-terminal Rpt peptides (400 μm). C, 20 S proteasome (30 nm) activity against [methyl-14C]casein substrate was determined in the presence of the indicated Rpt peptides. For each experiment, activity in the absence of Rpt peptides was assigned a value of 1.0, and other activities are expressed as relative values. Data represent mean values ± S.D. of triplicate assays. Similar results were obtained in at least four separate experiments.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
3.
FIGURE 5.

FIGURE 5. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

Structural determinants of Rpt C-terminal peptides on proteasome binding and activation. A, structures of C-terminal Rpt peptides used for proteasome binding and activation assays. B, His-tagged SUMO proteins containing the C-terminal fusions of the indicated peptides were expressed, purified, and used for pull-down assays with 20 S proteasome, as described under “Experimental Procedures.” Lane 1, assay containing 20 S proteasome but no SUMO-Rpt protein. Relative intensities of the bands in lanes 1–10, as determined by densitometry, are 0, 1.0, 0.01, 0.13, 0.29, 0.02, 1.14, 0.89, 0.01, and 0.88, respectively. C, 20 S proteasome activity against Suc-LLVY-AMC substrate was measured in the presence of the indicated SUMO-Rpt C-terminal peptide fusion proteins (100 μm). Proteasome activity in the absence of SUMO-Rpt protein was assigned a value of 1.0, and all other activities are expressed as relative values. Data represent mean values of triplicate assays ± S.D. Similar results were obtained in at least three separate experiments. D, peptides were synthesized corresponding to either the C-terminal 10 residues of each indicated Rpt subunit (RptX peptide; black bars) or the C-terminal three residues of each indicated Rpt subunit (RptX) and the adjacent seven residues of Rpt5 (Rpt5 head-RptX tail; gray bars). 20 S proteasome (12 nm) activity was assayed in the absence (Control) or presence of these peptides (400 μm) using Suc-LLVY-AMC substrate. Activity in the absence of Rpt peptides was assigned a value of 1.0, and all other activities are expressed as relative values. Data represent mean values of triplicate assays ± S.D. (error bars). Similar results were obtained in at least four separate experiments. WB, Western blot.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
4.
FIGURE 7.

FIGURE 7. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

The C terminus of Rpt3 is required for assembly of 26 S proteasome. HEK293 cells were transfected with expression vectors without insert (Mock) or with inserts for either FLAG-tagged wild-type Rpt3 (Rpt3) or FLAG-tagged Rpt3 lacking the last three C-terminal residues (Rpt33C), as described under “Experimental Procedures.” A, whole cell extracts were Western blotted for the indicated proteins (top) and assayed for hydrolysis of Suc-LLVY-AMC (bottom). Activity assays were normalized for total extract protein content and represent mean values of triplicate assays ± S.D. B, extracts from non-transfected cells (Control) and from indicated Rpt3-expressing cells were subjected to glycerol density gradient centrifugation as described under “Experimental Procedures.” Fractions were Western blotted for the indicated proteins. The arrows indicate the normal peak of sedimentation profile for purified PA700 and 26 S proteasome (data not shown). C, extracts of the indicated cells were subjected to immunoprecipitation with anti-FLAG beads as described under “Experimental Procedures.” Immunoprecipitates were subjected to the following: Western blotting (WB) for the indicated antigens, including FLAG, β5 subunit of 20 S proteasome, Rpt2, Rpt5, and Rpn12 (left); native PAGE, followed by silver staining (middle; arrows indicate known migration positions of purified singly and doubly capped 26 S proteasome); and proteasome activity assays using Suc-LLVY-AMC as substrate (right). Data represent mean values of triplicate assays ± S.D. (error bars) and were normalized for FLAG content. Similar results for data in each panel were obtained in three separate experiments.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
5.
FIGURE 1.

FIGURE 1. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

The C terminus of Rpt3 binds to the 20 S proteasome. Fusion proteins of SUMO and the C termini of the indicated Rpt proteins were expressed, purified, and used for pull-down assays of purified 20 S proteasome as described under “Experimental Procedures.” A, pull-down assays with the indicated His-tagged SUMO-Rpt peptide fusion peptides (lanes 2–7) were performed as described under “Experimental Procedures.” Lane 1 shows an assay with 20 S proteasome but no SUMO-Rpt protein. Relative intensities of the bands in lanes 1–8, as determined by densitometry are 0, 0, 1.0, 4.32, 0, 1.49, 0, and 0.96, respectively. B, pull-down assays of 20 S proteasome with indicated SUMO-Rpt peptide fusion proteins were conducted in the absence (lanes 2 and 5) or presence (lanes 3, 4, 6, and 7) of the indicated Rpt C-terminal peptides. 20 S proteasome, SUMO-Rpt protein, and Rpt peptides were present in relative concentrations of 240 nm, 100 μm, and 1 mm, respectively. Relative intensities of the bands in lanes 1–8, as determined by densitometry, are 0, 1.0, 0.01, 0.80, 0.16, 0.19, 0.03, and 0.30, respectively. C, pull-down assays of 20 S proteasome with the indicated SUMO-Rpt peptide fusion proteins and Rpt C-terminal peptides as in B. Relative intensities of the bands in lanes 1–5, as determined by densitometry, are 0, 1.0, 1.1, 0.01, and 1.06, respectively. D, pull-down assays were conducted with the indicated His-tagged SUMO-Rpt3 C-terminal peptide fusion proteins. −3C, −2C, and −1C denote deletions of the last 3, 2, and 1 C-terminal Rpt3 residues, respectively. Relative intensities of the bands in lanes 1–6, as determined by densitometry, are 0, 1.0, 0.01, 0, 0.44, and 1.93, respectively. E, the indicated SUMO-Rpt C-terminal peptide fusion proteins (300 μm) were preincubated with 650 nm 20 S proteasome for 15 min at 37 °C, subjected to native PAGE, and visualized after overlay of Suc-LLVY-AMC fluorescent peptide substrate. Similar results for experiments in all panels were obtained in at least four separate experiments. WB, Western blot.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
6.
FIGURE 3.

FIGURE 3. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

Identification of the 20 S proteasome subunit cross-linked to the C-terminal peptide of Rpt3. Chemical cross-linking of 20 S proteasome with fluorescein-DOPA-Rpt3 (A) or fluorescein-DOPA-Rpt3(−3C) (B) and subsequent HPLC purification were conducted as described under “Experimental Procedures.” HPLC fractions were subjected to SDS-PAGE and either stained for protein with silver (top) or scanned for fluorescence (bottom). AS, sample applied to the column. Fractions containing the major fluorescent (36–39 min) (left) and equivalent fractions from unlabeled control samples (right) were pooled, concentrated, and subjected to two-dimensional PAGE (C and D, respectively). The area of the gel containing the fluorescent spot (denoted by an oval, upper left) and the corresponding area of the non-fluorescent control gel (upper right) were excised and processed for mass spectrometric identification of proteins, as described under “Experimental Procedures.” Gels were Western blotted for proteasome subunit α1 (lower right and left). Grids indicate relative registration of gels based on the position of common markers. The spot marked by the asterisk is an imaging artifact unique to this individual experiment. Similar results for experiments in all panels were obtained in at least four separate experiments. E and F, chemical cross-linking of 20 S was performed with biotin-DOPA-Rpt3 or biotin-DOPA-Rpt3(−3C), and the samples were enriched on monoavidin beads as described under “Experimental Procedures.” Enriched samples were subjected to Western blotting after SDS-PAGE (E) or two-dimensional PAGE (F) using either Streptavidin-IR or antibodies against individual 20 S proteasome subunits, as indicated. Untreated 20 S proteasome (20S) was used as a control for each blot (E). Two-dimensional gels of the indicated samples were blotted for both the α1 subunit (F, top, green channel) and biotin (F, middle, red channel). F, bottom, merged image (yellow) for the two blots. The arrows point to cross-linked products. IEF, isoelectric focusing; WB, Western blot.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.
7.
FIGURE 6.

FIGURE 6. From: The C Terminus of Rpt3, an ATPase Subunit of PA700 (19 S) Regulatory Complex, Is Essential for 26 S Proteasome Assembly but Not for Activation.

The C-terminal peptide of Rpt3 inhibits 26 S proteasome assembly and activation in vitro. A and B, in vitro 26 S proteasome assembly and activation from purified 20 S proteasome and PA700 was conducted as described under “Experimental Procedures.” A, 20 S proteasome (15 nm) and PA700 (75 nm) were preincubated in the presence or absence of the indicated Rpt3 C-terminal peptide or Rpt3(−3C) C-terminal peptide. After 30 min, proteasome activity was measured using Suc-LLVY-AMC substrate. 20 S proteasome activity in the absence of PA700 was assigned a value of 1.0, and all other activities are expressed as relative values. B, 20 S proteasome (75 nm) and PA700 (200 nm) were preincubated in the presence or absence of Rpt3 C-terminal peptide or Rpt3(−3C) C-terminal peptide, as indicated. After 30 min, samples were subjected to native PAGE. The gel was incubated with a solution of Suc-Leu-Leu-Val-Tyr-AMC, incubated for 15 min at 37 °C, and exposed to UV light. The arrows indicate established migration positions of 26 S proteasome and 20 S proteasome, respectively. Relative fluorescence intensities of the bands in lanes 1–4 were quantified as 0/1.0, 1.0/0.57, 0.40/0.89, and 1.01/0.34, respectively, for 26 S/20 S proteasome bands, respectively. Similar results were obtained in four separate experiments. C, purified 26 S proteasome (10 nm) was assayed against Suc-LLVY-AMC substrate in the presence of the indicated concentrations of Rpt3 C-terminal peptide. Data points represent mean values of triplicate assays. Similar results were obtained in three separate experiments. D, purified 26 S proteasome (20 nm) was preincubated with indicated concentrations of Rpt3 C-terminal peptide for 30 min. Samples were then subjected to native PAGE and then assayed for in-gel proteasome activity by incubation with Suc-LLVY-AMC, as in B, or stained for protein with Coomassie Blue. Similar results were obtained in two separate experiments. E, 20 S proteasome (3 nm) was incubated in the absence (−) or presence (+) of 2 μg each of PA700 subcomplexes PS1, PS2, and PS3 and either 1 mm Rpt3 or Rpt3 (−3C) peptide, as indicated, or no peptide (−). Samples were incubated for 30 min at 37 °C in 45 mm Tris-HCl, pH 8.0, 5.6 mm DTT, 10 mm MgCl2, and 100 μm of ATP in a volume of 50 μl. Proteasome activity was assayed with 200 μm Suc-LLVY-AMC substrate peptide, as described above. Proteasome activity in the absence of subassemblies and Rpt peptide was assigned a value of 1.0, and all other activities are expressed as relative values. Data represent mean values of triplicate assays ± S.D. (error bars). Similar results were obtained in three separate experiments. AFU, arbitrary fluorescent units.

Brajesh Kumar, et al. J Biol Chem. 2010 December 10;285(50):39523-39535.

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