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Mol Cell Biol. 2003 Sep;23(18):6469-83.

Ataxin-3 interactions with rad23 and valosin-containing protein and its associations with ubiquitin chains and the proteasome are consistent with a role in ubiquitin-mediated proteolysis.

Doss-Pepe EW, Stenroos ES, Johnson WG, Madura K.

Source

Department of Biochemistry, Robert Wood Johnson Medical School, University of Medicine and Dentistry of New Jersey, Piscataway, NJ 08854, USA.

Abstract

Machado-Joseph disease is caused by an expansion of a trinucleotide CAG repeat in the gene encoding the protein ataxin-3. We investigated if ataxin-3 was a proteasome-associated factor that recognized ubiquitinated substrates based on the rationale that (i) it is present with proteasome subunits and ubiquitin in cellular inclusions, (ii) it interacts with human Rad23, a protein that may translocate proteolytic substrates to the proteasome, and (iii) it shares regions of sequence similarity with the proteasome subunit S5a, which can recognize multiubiquitinated proteins. We report that ataxin-3 interacts with ubiquitinated proteins, can bind the proteasome, and, when the gene harbors an expanded repeat length, can interfere with the degradation of a well-characterized test substrate. Additionally, ataxin-3 associates with the ubiquitin- and proteasome-binding factors Rad23 and valosin-containing protein (VCP/p97), findings that support the hypothesis that ataxin-3 is a proteasome-associated factor that mediates the degradation of ubiquitinated proteins.

PMID:: 12944474; [PubMed - indexed for MEDLINE]
PMCID:: PMC193705

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FIG. 1.

A sequence motif (UIM) in S5a that can bind ubiquitinated proteins is present in ataxin-3. (A) Alignment of conserved regions of S5a and ataxin-3. The position of the amino acid residue at the N terminus of the conserved UIM sequence is shown on the left. Amino acid residues in blue are identical or highly conserved. (B) Ataxin-3 can bind ubiquitinated proteins in yeast cell extracts. Protein extracts were prepared from a wild-type yeast strain, and 1 mg was incubated with various amounts of Thio-ataxin-3^Q20 that was purified from E. coli. After extensive washing, the proteins that were bound to Thio-ataxin-3 were separated in an SDS-12% polyacrylamide gel, transferred to nitrocellulose filters (Bio-Rad), and incubated with ubiquitin-specific antibodies (Sigma). The immunoblot was developed with enhanced chemiluminescence (Dupont, NEN). Lanes 1 to 4 contain, respectively, 1, 2, 5, and 10 μg of Thio-ataxin-3^Q20 on affinity beads. Lane 5, control in which Thio-Bond resin was incubated with a yeast cell extract. A sample of the yeast cell extract (10% input) is shown in lane 6. The positions of molecular size standards are shown in the left margin. Asterisks in panels B and C indicate a nonspecific band (from Thio-Bond resin) that cross-reacted with anti-ubiquitin antibodies. Ponceau staining shows the amount of ataxin-3 protein used in the pulldown assay. At-3, ataxin-3. (C) Ataxin-3 can bind ubiquitinated proteins from NT2 human cell extracts. Protein extracts were prepared from NT2 cells, applied to various amounts of Thio-ataxin-3^Q20 immobilized onbeads, and examined as described for panel B, but in an SDS-10% polyacrylamide gel. Lanes are as in panel B, except that lane 6 corresponds to 10% input of NT2 cell extract. Ubiquitinated proteins are indicated by the designation (Ub)_n in the right margin. Ponceau staining shows the amount of ataxin-3 protein used in the pulldown assay. (D) Ataxin-3 interacts with ubiquitinated proteins in vivo in yeast cells. Protein extracts were prepared from strains expressing FLAG-Rpn10, GST-ataxin-3^Q20, GST-ataxin-3^Q79, GST, FLAG-Rad23, or GST-Rad23 from the copper-inducible CUP1 promoter. Fusion proteins were isolated either by pulldown with glutathione Sepharose (Amersham Pharmacia Biotech) or by immunoprecipitation with FLAG-agarose (Sigma). The bound proteins were separated in an SDS-10% polyacrylamide gel and transferred to nitrocellulose membranes, and ubiquitinated proteins were detected. Lanes 1 and 9, mock reactions in which an extract derived from wild-type yeast that did not overexpress any of the fusion proteins was applied to either glutathione Sepharose or FLAG-agarose, respectively; lane 2, FLAG-Rpn10; lane 3, GST-ataxin-3^Q20; lane 4, GST-ataxin-3^Q79; lane 5, GST; lane 6, molecular weight (MW) markers; lane 7, FLAG-Rad23; lane 8, GST-Rad23. For lanes 2, 7, and 9, an arrowhead indicates the immunoglobulin heavy chain. Asterisk, immunoglobulin light chain detected as an ∼23-kDa band in lanes 2, 7, and 9 in the lower panel. Ponceau staining shows the purified proteins prior to incubation with anti-ubiquitin antibodies, with theoretical molecular sizes as follows: for Flag-Rpn10, ∼30 kDa; for GST-ataxin-3^Q20, ∼69 kDa; for GST-ataxin-3^Q79, ∼78 kDa; for GST, ∼28 kDa; for FLAG-Rad23, ∼43 kDa; and for GST-Rad23, ∼70 kDa. Note that apparent molecular sizes as determined from SDS-PAGE are slightly different from those determined theoretically.

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.

FIG. 2.

Ataxin-3 interacts with ubiquitinated proteins in wild-type and rad23Δ yeast strains. (A) The high-molecular-weight ubiquitin-cross-reacting material that is purified with GST-ataxin-3^Q20 and GST-ataxin-3^Q79 does not represent ubiquitinated ataxin-3. Protein extracts were prepared from yeast strains expressing either GST (lanes 1 and 4), GST-ataxin-3^Q20 (Q20) (lanes 2 and 5), or GST-ataxin-3^Q79 (Q79) (lanes 3 and 6) and were purified on glutathione Sepharose. The precipitated proteins were washed with buffer A, separated by SDS-10% PAGE, transferred to nitrocellulose membranes, and exposed to either GST-specific antibodies (lanes 1 to 3) or ubiquitin-specific antibodies (lanes 4 to 6). The anti-GST immunoblot demonstrates that the majority of high-molecular-weight ubiquitin-cross-reacting material is not a ubiquitinated GST-ataxin-3 species. The GST-ataxin-3 constructs appear to be conjugated to one or two ubiquitins (Ub-Q79 and Ub-Q20 in the left margin) based on the reduced mobility of GST-ataxin-3^Q20 and GST-ataxin-3^Q79. The bands are consistent with the addition of one ubiquitin moiety. In contrast, incubation of the same blot with anti-ubiquitin antibodies demonstrates that a large quantity of ubiquitinated proteins migrating at a high molecular weight is associated with the ataxin-3 constructs. The reaction against anti-GST and anti-ataxin-3 (data not shown) antibodies demonstrated that the high-molecular-weight ubiquitin-cross-reacting material is not ubiquitinated GST-ataxin-3. The designations Q20 and Q79 refer to full-length ataxin-3 proteins. (B) Protein extracts were prepared from wild-type (WT) or rad23Δ yeast strains expressing either GST (lanes 1 and 2), GST-ataxin-3^Q20 (Q20) (lanes 3 and 4), or GST-ataxin-3^Q79 (Q79) (lanes 5 and 6). Equal amounts of extract were applied to glutathione Sepharose to purify GST or the GST-ataxin-3 fusion proteins. Interacting ubiquitinated proteins were detected with ubiquitin-specific antibodies. Ubiquitinated proteins were bound to both GST-ataxin-3 constructs purified from wild-type and rad23Δ strains. Ponceau staining shows the amounts of proteins purified. (C) Ataxin-3 interacts with hHR23B. GST-hHR23B and Thio-ataxin-3^Q20 were purified from E. coli. Varying amounts of Thio-ataxin-3^Q20 (indicated below the gel) were incubated with either 1 μg of GST (lanes 3, 5, 7, and 9) or 2 μg of GST-hHR23B (R23B) (lanes 4, 6, 8, and 10). Interaction between GST-hHR23B and Thio-ataxin-3^Q20 was detected by immunoblotting using anti-Thio antibodies (Invitrogen). Lane 1, 10 μg of Thio-ataxin-3^Q20 alone; lane 2, GST-hHR23B without incubation with Thio-ataxin-3^Q20. The position of Thio-ataxin-3^Q20 (Q20) is indicated in the right margin.

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.

FIG. 4.

Ataxin-3 interacts with the 26S proteasome. (A) Ataxin-3 cofractionates with yeast proteasome subunits Pre1 and Rpn8 in a Superose 6 10/30 HR column. (Top set of panels) Fractions of protein extracts that contain GST, Pre1-FLAG, and Rpn8-V5. (Center set of panels) Fractions of protein extracts that contain GST-ataxin-3^Q20 (Q20), Pre1-FLAG, and Rpn8-V5. (Bottom set of panels) Fractions of protein extracts that contain GST-ataxin-3^Q79 (Q79), Pre1-FLAG, and Rpn8-V5. Proteins from fractions were precipitated with 10% trichloroacetic acid, washed with 100% acetone, separated by SDS-10% PAGE, transferred to nitrocellulose membranes, and incubated with appropriate antibodies. Specific proteins that were detected are indicated in the left margin. Antibodies that were used for detection in immunoblots are given in the right margin. Fraction numbers are given below the bottom gel. (B) Ataxin-3 can be immunoprecipitated with the proteasome. The upper set of panels represents the fractions of separated extracts containing ataxin-3^Q20, and the lower set represents ataxin-3^Q79. Fractions were incubated with FLAG-agarose or glutathione Sepharose to purify Pre1-FLAG or GST-ataxin-3, respectively. Proteins that were coprecipitated were then separated in an SDS-10% polyacrylamide gel. The immunoprecipitations (IP) or pulldowns (PD) and the subsequent immunoblots that were performed are indicated in the right margin. Fraction numbers are given below the bottom gel. GST-ataxin-3^Q20 and Rpn8-V5 were both copurified with Pre1-FLAG (top set, first panel). Similarly, Pre1-FLAG was detected in association with GST-ataxin-3^Q20 (top set, second panel). Immunoblotting with anti-GST antibodies confirmed that similar amounts of GST-ataxin-3^Q20 and GST-ataxin-3^Q79 were recovered (top set, third panel, and bottom set, second panel). Pre1-FLAG was immunoprecipitated from fractions that contained GST-ataxin-3^Q79, and both ataxin^Q79 and Rpn8-V5 could be detected. Asterisks indicate a cross-reaction against the immunoglobulin heavy chain. The graph shows chymotrypsin-like protease activities determined in the gel filtration fractions (▪, Q20; ▴, Q79) by using the fluorogenic substrate Suc-Leu-Leu-Val-Tyr-AMC (10 μM). The y axis represents arbitrary fluorescence signal units for raw fluorescence readings (with the value for the blank subtracted). The x axis represents the fractions assigned. Maximal activity was detected in fractions 6 to 13. The activity detected in fractions 15 to 17 may represent an undetermined protein that has chymotryptic activity. (C) Residues 1 to 150 of ataxin-3 are sufficient for the interaction with the proteasome. Protein extracts containing GST, GST-ataxin-3, or truncated constructs were prepared from yeast cells that also expressed Pre1-FLAG. Pre1-FLAG was immunoprecipitated with FLAG-agarose (Sigma), and Ponceau staining showed that equivalent amounts were purified from each extract (bottom panel). Lane 1, GST; lane 2, GST-ataxin-3^1-150; lane 3, GST-ataxin-3^1-200; lane 4, GST-ataxin-3^1-243 (UIM1); lane 5, GST-ataxin-3^1-263; lane 6, GST-ataxin-3^1-354; lane 7, full-length GST-ataxin-3^Q20; lane 8, full-length GST-ataxin-3^Q79; lane 9, mock reaction in which a wild-type yeast cell extract was applied to FLAG-agarose. The immunoblot filter was incubated with anti-GST antibodies, and all the ataxin-3 proteins were detected (top panel). (D) Rad23 is not necessary for the interaction of ataxin-3 with the proteasome. GST (lane 1), GST-ataxin-3^Q20 (lane 2), or GST-ataxin-3^Q79 (lane 3) was expressed in wild-type and rad23Δ yeast strains that expressed Pre1-FLAG. Pre1-FLAG or GST-ataxin-3 fusion proteins were purified with FLAG-agarose or glutathione Sepharose, respectively. Pre1-FLAG was immunoprecipitated, and the interaction between GST-ataxin-3 and the proteasome was determined by immunoblotting with anti-GST antibodies (top panel). The immunoglobulin heavy chain is indicated in the left margin (HC). The bottom panel shows that in a reciprocal experiment, extracts were incubated with glutathione Sepharose, and GST or GST-ataxin-3 was purified. Immunoblotting with anti-FLAG antibodies confirmed the interaction between ataxin-3 and the proteasome. A small amount of Pre1-FLAG interacted nonspecifically with glutathione Sepharose (lane 1).

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.

FIG. 5.

(A) Myc-tagged ataxin-3 cofractionates with the proteasome in 293T cell extracts. The association of ataxin-3^Q27 and ataxin-3^Q78 with the proteasome was examined. The top panel shows the fractionation of Rpt1 (an ATPase subunit in the 19S regulatory particle) in untransfected 293T cells. A pair of panels shows the cofractionation of ataxin-3^Q27 with Rpt1 (Q27) (middle panels). Similarly, ataxin-3^Q78 is present in fractions that contain Rpt1 (Q78) (lower panels). Fraction numbers are given at the bottom, and the antibodies used are given in the right margin. Specific proteins detected are shown in the left margin. (B) Validation of polyclonal ataxin-3 antibodies in an SDS-12% polyacrylamide gel. Polyclonal antibodies were generated against Thio-ataxin-3^Q20 that was purified from E. coli and affinity purified before use. Lane 1 contains purified Thio-ataxin-3^Q20; lanes 2 to 5 contain control yeast extract, GST-ataxin-3^Q20, GST-ataxin-3^Q79, and FLAG-ataxin-3^Q20, respectively. The immunoblot was incubated with affinity-purified anti-ataxin-3 antibodies, and proteins of the expected sizes were detected. (C) VCP, Rpt1, and 20S core subunits (α1, -2, -3, -5, -6, and -7) can be coimmunoprecipitated with ataxin-3 from 293T cell extracts. Two milligrams of cell lysate was applied to rPA-Sepharose beads (Repligen) that were coupled with 1, 2, and 5 μg of an affinity purified anti-Thio-ataxin-3^Q20 antibody/ml (lanes 2 to 4, respectively). Lane 1, 10% input, total 293T cell extract; lane 5, 293T cell extract applied to rPA-Sepharose; lane 6, anti-Thio-ataxin-3^Q20 antibody coupled to rPA-Sepharose beads with no extract applied. The 20S core showed a very weak nonspecific binding to the rPA-Sepharose beads (lane 5). The asterisk in the right margin indicates a reaction against the immunoglobulin heavy chain. (D) Both 19S (Rpt1) and 20S proteasome (α1, -2, -3, -5, -6, and -7) subunits can be purified with MBP-ataxin-3^Q27 (lane 2) and MBP-ataxin-3^Q79 (lane 3). Two milligrams of cell lysate was applied to beads that contained the MBP-ataxin-3 proteins. Immunoblotting showed that two bands, representing the 20S subunits at approximately 29 and 32 kDa, were detected. Similarly, Rpt1 was detected in lanes that contained MBP-ataxin-3 (lanes 2 and 3). Lane 1 contains 10% of the input extract, while lane 4 represents a reaction in which 2 mg of the extract was applied to amylose beads (lacking MBP-ataxin-3 proteins). (E) GST, GST-ataxin-3 truncation constructs, GST-ataxin-3^Q20, and GST-ataxin-3^Q79 were purified from yeast cells and incubated with 1 mg of 293T cell extract. Lane 1, 293T whole-cell extract (10% input); lane 2, GST; lane 3, GST-ataxin-3^1-150; lane 4, GST-ataxin-3^1-200; lane 5, GST-ataxin-3^1-243 (UIM1); lane 6, GST-ataxin-3^1-263; lane 7, GST-ataxin-3^1-354; lane 8, full-length GST-ataxin-3^Q20 (Q20); lane 9, full-length GST-ataxin-3^Q79 (Q79). The top panel shows Ponceau staining of the amounts of GST or GST-fusion proteins used in the reactions. The bottom panel shows that reactions with antibodies against Rpt1 and 20S core subunits were consistent with the in vivo experiments that were performed in yeast cells. Specifically, the GST-ataxin-3 construct harboring only residues 1 to 150 was sufficient for interaction with both 19S (Rpt1) and 20S (α1, -2, -3, -5, -6, and -7) proteasome subunits.

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.

FIG. 9.

Ataxin-3 binds VCP/p97. (A) Proteins bound to ataxin-3 are identified by Coomassie staining following separation by SDS-10% PAGE. Lane 1, molecular size standards (10 kDa); lane 2, NT2 cell extract (10% of input); lane 3, purified Thio-ataxin-3^Q20; lane 4, 2 mg of the NT2 cell extract applied to Thio-ataxin-3^Q20; lane 5, 2 mg of the NT2 cell extract applied to Thio-Bond resin; lane 6, 2 mg of the NT2 cell extract applied to affinity beads containing Thio; lane 7, 2 mg of the NT2 cell extract applied to affinity beads containing Thio-β-synuclein. (B) Binding of VCP by Thio-ataxin-3^Q20. Lane 1, purified Thio-ataxin-3^Q20; lanes 2 to 6, 0.5, 1, 2, 5, and 10 μg, respectively, of Thio-ataxin-3^Q20 to which 1 mg of the NT2 cell extract was applied; lane 7, mock pulldown with Thio-Bond resin. (Top panel) Thio-ataxin-3^Q20 purified on beads and visualized with Ponceau stain. (Bottom panel) VCP interacting with Thio-ataxin-3^Q20, detected with antibodies to VCP (Research Diagnostics). (C) GST-ataxin-3^Q20 and GST-ataxin-3^Q79, but not GST-S5a, bind VCP. Two milligrams of the NT2 cell extract was applied to equal amounts of immobilized GST-ataxin^Q20 (lane 2), GST-ataxin-3^Q79 (lane 3), or GST-S5a (lane 4). Lane 1, NT2 cell lysate (10% of input); lane 5, GST purified on beads; lane 6, mock pulldown with glutathione Sepharose. VCP/p97 was detected by immunoblotting with anti-VCP antibodies. (See also Fig. 5C for immunoprecipitation of VCP with ataxin-3 from 293T cells by using a polyclonal ataxin-3 antibody.) (D) VCP binds to (Ub)_2-7 chains more weakly than ataxin-3. Lane 1, 10 ng of (Ub)_2-7; lane 2, GST-VCP (1 μg); lanes 3 and 4, 1 and 2 μg, respectively, of GST-VCP plus Thio-ataxin-3^Q20; lane 5, Thio-ataxin-3^Q20 (1 μg). All lanes are from the same gel, and the immunoblot was incubated with ubiquitin-specific antibodies. Lane 5 represents a separate pulldown.

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.

FIG. 10.

Multiubiquitinated proteins can be displaced from GST-UBA1 (derived from yeast Rad23) in the presence of ataxin-3 and VCP. A protein extract was prepared from yeast cells that expressed GST-UBA1 of Rad23, and the bound ubiquitinated proteins were purified on glutathione Sepharose. Equal amounts of the GST-UBA1 beads were combined with varying amounts of VCP and ataxin-3. The proteins were incubated for several hours at 4°C, and the supernatant was subjected to 10% trichloroacetic acid precipitation, followed by an acetone wash and examination by SDS-PAGE. (Top panel) The proteins remaining on beads or displaced in the supernatant were resolved by SDS-10% PAGE, transferred to nitrocellulose membranes, and incubated with ubiquitin-specific antibodies. Lane 1, 100 μg of the yeast cell extract (3% of input); lane 2, multiubiquitinated proteins that were copurified with GST-UBA1; lane 3, supernatant (Sup) after incubation of GST-UBA1 without VCP or ataxin-3; lane 4, GST-UBA1 beads after incubation with 10 μg of VCP and 10 μg of ataxin-3; lane 5, supernatant (Sup) from lane 4 after incubation, showing partial displacement of ubiquitinated proteins; lane 6, GST-UBA1 beads after incubation with 20 μg of VCP and 20 μg of ataxin-3; lane 7, supernatant (Sup) from lane 6 after incubation; lane 8, GST-UBA1 beads after incubation with 20 μg of VCP; lane 9, supernatant from reaction in lane 8 after incubation; lane 10, GST-UBA1 beads after incubation with 20 μg of ataxin-3; lane 11, supernatant from reactions in lane 10 after incubation. Note that ubiquitinated proteins were not displaced from GST-UBA1 in the presence of VCP or ataxin-3 alone (lanes 9 and 11). (Second panel) Incubations with anti-GST antibodies showed that GST-UBA1 was not displaced from the beads during the incubation period. (Bottom panels) Ponceau-stained nitrocellulose filters show VCP, Thio-ataxin-3^Q20, and GST-UBA1 used in the experiment, and their positions are indicated on the right.

Ataxin-3 Interactions with Rad23 and Valosin-Containing Protein and Its Associations with Ubiquitin Chains and the Proteasome Are Consistent with a Role in Ubiquitin-Mediated Proteolysis

Mol Cell Biol. 2003 September;23(18):6469-6483.