1. Volume 23, Issue 6, Pages 875-885 (September 2006)
An Arsenite-Inducible 19S Regulatory Particle-Associated Protein Adapts Proteasomes to Proteotoxicity 
Ariel Stanhill, Cole M. Haynes, Yuhong Zhang, Guangwei Min, Matthew C. Steele, Juliya Kalinina, Enid Martinez, Cecile M. Pickart, Xiang-Peng Kong, David Ron 
Molecular Cell 
Volume 23, Issue 6, Pages (September 2006)
DOI: /j.molcel
Copyright © 2006 Elsevier Inc. Terms and Conditions

2. Figure 1 AIRAP Association with Proteasomes
(A) Immunoblot of endogenous AIRAP, the 20S subunit PSMA1, and the 19S subunit PSMD7 in fractions of glycerol gradients prepared from mouse fibroblasts exposed to arsenite (ARS, 25 μM for 4 hr). The migration of complexes of known size on a parallel gradient is indicated.
(B) Immunoblot of AIRAP-GST and the 20S marker PSMA1 from fractions of glycerol gradients prepared from cells stably expressing an AIRAP-GST fusion protein (“input”). A PSMA1 immunoblot of proteins copurifying with AIRAP-GST is shown in the bottom panel (“GST purified”).
(C) Coomassie stain of SDS-PAGE of proteins purified by glutathione affinity chromatography from fractions 2–4 (lane 1) or 10–12 (lane 2) of a glycerol gradient loaded with lysate of cells transfected with AIRAP-GST. The bands identified by mass spectroscopy are indicated to the right of the gel.
(D) Immunoblot of endogenous AIRAP and PSMA1 from lysates of arsenite-exposed cells. Where indicated, the lysate had been depleted of its PSMA1 content by using a specific antiserum or nonimmune serum (NI). The depleted supernatant (“sup”) and pellet of the immunoprecipitation reaction were analyzed separately. The asterisk marks the immunoglobulin light chain.
(E) Representative image of the negatively stained particles purified by AIRAP-GST affinity chromatography and a schema of the 26S particle (top). Gallery of averaged images of doubly capped and singly capped AIRAP-GST-containing particles of the most populous orientations (bottom).
Molecular Cell  , DOI: ( /j.molcel )
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3. Figure 2
AIRAP Is Located in Proximity to PSMD2, a Subunit of the 19S Proteasome
(A) Cartoon depicting the domain organization of AIRAP and the deletion mutants used in the experiment shown in (B). The highly conserved Cys/His repeats are indicated.
(B) Immunoblots of PSMD7 (a 19S subunit) and PSMA1 (a 20S subunit) after glutathione affinity purification of lysates from cells transfected with the indicated AIRAP-GST fusion proteins (“GST purification”) and in the crude lysate that served as the input.
(C) Autoradiograph of a reducing SDS-PAGE of metabolically labeled proteins purified by glycerol gradient centrifugation followed by glutathione affinity chromatography from cells transfected with AIRAP-GST. The purified complex was displayed as is (lanes 1 and 10) or exposed to increasing amounts of diamide in vitro, followed by disruption in 2% SDS, dilution, and immunoprecipitation with anti-AIRAP serum or nonimmune serum (NI), as indicated (lanes 2–9). Note the diamide-dependent covalent crosslinking to AIRAP-GST of a ∼97 KDa protein that migrates at the same position as PSMD2.
(D) PSMD2 immunoblot of complexes crosslinked and immunopurified as in (C) (the anti-PSMD2 serum also reacts with the GST moiety of AIRAP-GST).
(E) Autoradiograph of metabolically labeled PSMD2 translated in vitro in a reticulocyte lysate and bound to full-length AIRAP-FL(1-171)-GST, AIRAP-ΔC(1-89)-GST, and GST purified from transfected cells by glutathione affinity chromatography. The stained gel (Coomassie) reveals the presence of the fusion proteins. Asterisks mark irrelevant proteins that copurify with the GST fusion proteins.
Molecular Cell  , DOI: ( /j.molcel )
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4. Figure 3 The Proteasome's AIRAP Binding Capacity
(A) Immunoblots of GST fusion proteins, coexpressed AIRAP (tagged with a myc epitope, AIRAP-myc) or endogenous PSMA1, purified by glycerol gradient centrifugation followed by glutathione affinity chromatography from transfected 293T cells expressing the indicated proteins. The panels below reveal the protein content in the input HMW fractions of the glycerol gradient.
(B) PSMA1 and AIRAP-myc immunoblots of material immunoprecipitated with anti-myc from the flowthrough of the glutathione affinity chromatography of the corresponding samples shown in (A).
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

5. Figure 4
Accumulation of Polyubiquitylated Proteins and an Enhanced Heat-Shock Response in C. elegans with Impaired aip-1 (AIRAP) Function
(A) Immunoblot of ubiquitylated proteins (poly-Ub) in lysates of wild-type and aip-1(RNAi) worms after exposure to sodium arsenite (3 mM).
(B) Anti-green fluorescent protein (GFP) and anti-HDEL (a loading control) immunoblots of lysates from wild-type, aip-1(RNAi), or hsf-1(RNAi) animals transgenic for a heat-shock reporter (hsp-16::gfp), after exposure to sodium arsenite (4.5 mM) for 4 hr.
(C) Time-dependent survival of a population of wild-type and aip-1(RNAi) worms exposed to 4.5 mM sodium arsenite (mean ± SEM survival of 50 worms per genotype, performed in triplicate).
(D) Anti-GFP and anti-HDEL immunoblots of lysates of wild-type and skn-1(RNAi) transgenic aip-1::gfp reporter worms exposed to arsenite, as in (B).
(E) Poly-Ub, GFP, and HDEL immunoblots of lysates of wild-type and skn-1(RNAi) animals transgenic for heat-shock reporter (hsp-16::gfp) after exposure to arsenite.
Molecular Cell  , DOI: ( /j.molcel )
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6. Figure 5 Proteasome Dysfunction in Mouse Fibroblasts Lacking AIRAP
(A) Poly-Ub, AIRAP, and eIF2α (a loading control) immunoblots of lysates from wild-type (AIRAP+/+), homozygous AIRAP−/− mutant mouse fibroblasts, and AIRAP−/− cells constitutively expressing a rescuing allele of AIRAP (AIRAP−/−[Rescue]) transiently exposed to arsenite (50 μM, for the first 2 hr of the 12 hr time course).
(B) Poly-Ub, PSMA1, and AIRAP immunoblots of lysates of wild-type and AIRAP−/− cells after exposure to arsenite (input) and in the complex immunoprecipitated with an antiserum to PSMA1 from these lysates (PSMA1-IP). Where indicated, the intensity of the signal of the blot has been estimated densitometerically (“signal”).
(C) Fluorescent image of a suc-LLVY-AMC zymogram of proteasomes in lysates from wild-type and AIRAP−/− cells exposed to arsenite (50 μM, for the first 2 hr of the 4 hr time course) and resolved by native acrylamide gel electrophoresis. The position of catalytic particles (CP) associated with two (RP2CP) or one (RP1CP) 19S regulatory caps is indicated to the left of the zymogram. Quantitation of the fluorescent signal is presented under the zymogram. The panels below are immunoblots of the PSMA1 and AIRAP content of the lysates used in zymography.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions

7. Figure 6
Discordant Effects of ATP on Conventional and AIRAP-Containing Proteasomes
(A) Immunoblots of endogenous AIRAP and PSMA1 (a 20S subunit) in fractions of glycerol gradients loaded with lysates of unexposed or arsenite-exposed mouse fibroblasts. The lysates were treated in vitro with hexokinase and glucose to deplete ATP (“ATP [Δ]”) or supplemented with ATP (5 mM, “ATP [+]”).
(B) Immunoblots of endogenous PSMD7 (a 19S subunit), PSMA1 (a 20S subunit), and AIRAP immunoprecipitated with anti-AIRAP, anti-PSMA1 (A1), or nonimmune serum (NI) from lysates of untreated mouse fibroblasts or cells exposed to arsenite. The lysates were depleted of ATP or supplemented with it, as in the experiments presented in (A).
(C) Silver-stained SDS-PAGE of proteins purified by glycerol gradient centrifugation, followed by glutathione affinity chromatography from lysates of 293T cells transfected with AIRAP-GST or PSMB2-GST (a 20S proteasome subunit). The lysates were supplemented with or depleted of ATP as indicated. The endogenous 20S and 19S subunits that associate with the transfected bait protein are indicated.
(D) Immunoblot of endogenous PSMA1 (a 20S subunit), PSMD7 (a 19S subunit), and coexpressed AIRAP-myc purified by glutathione affinity chromatography from lysates of 293T cells transfected with PSMB2-GST and supplemented or depleted of ATP during purification, as indicated. Note that the association of PSMB2-GST with endogenous PSMD7 is stabilized by AIRAP-myc in the ATP-depleted lysate.
Molecular Cell  , DOI: ( /j.molcel )
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8. Figure 7 Functional Characterization of AIRAP-Containing Proteasomes
(A) Degradation of a 32P-labeled polyubiquitylated protein substrate ([32P]Ub5-DHFR) by proteasomes purified by AIRAP-GST or PSMD14-GST affinity chromatography from transfected 293T cells. The reaction was performed in buffer containing an ATP regenerating system (+ATP) without or with the inhibitor MG132 (10 μM) or the stabilizing DHFR ligand methotrexate (MTX 50 nM) or in a buffer lacking ATP (−ATP). The results are expressed as a fraction of the [32P] counts released at the end time point.
(B) Digestion of a small, unstructured peptide, suc-LLVY-AMC monitored flurographically, in samples containing equal aliquots of proteasomes purified by AIRAP-GST (AIRAP-proteasome), PSMD14-GST (26S), PSMB2-GST (20S), or as a control GST affinity chromatography. Where indicated, the inhibitor MG132 was added to the sample before incubation with substrate.
(C) Digestion of a different peptide, Z-LLE-AMC, monitored flurographically as in (B).
(D) Immunoblot of the PSMA1 (a 20S marker), PSMD7 (a 19S marker), and fusion protein (PSMD14-GST or AIRAP-GST) of proteasome preparations, before or after incubation in the indicated assay buffers.
Molecular Cell  , DOI: ( /j.molcel )
Copyright © 2006 Elsevier Inc. Terms and Conditions