Journal: The Journal of Biological Chemistry

Article Title: Distinct Modes of Ubiquitination of Peroxisome-targeting Signal Type 1 (PTS1) Receptor Pex5p Regulate PTS1 Protein Import *

doi: 10.1074/jbc.M113.527937

Figure Lengend Snippet: Lys-linked monoubiquitination of Pex5p occurs at the Lys520in vivo. A, HEK cells were transfected with plasmids encoding His-FLAG-Pex5pL and HA2-Ub as indicated at the top. Cells were cultured for 36 h and for an additional 2 h in the presence of MG132. His-FLAG-Pex5pL was immunoprecipitated with anti-FLAG IgG-agarose and analyzed by immunoblotting (WB) with the antibodies indicated on the left. Input, 10% cell lysates used for immunoprecipitation (IP). Solid and shaded arrowheads, His-FLAG-Pex5pL conjugated with endogenous ubiquitin and HA2-Ub, respectively. Open arrowhead, unmodified His-FLAG-Pex5pL. B, CHO-K1 cells were transfected with plasmids encoding the shorter and longer forms of wild-type His-Pex5p-HA (WT-S and WT-L) and the KR mutants of the L form, K-less, K520R, and 520K. After a 1-day culture, cells were solubilized and subjected to immunoprecipitation with anti-HA antibody. Immunoprecipitates and the input (5%) were analyzed by SDS-PAGE under the reducing condition and immunoblotting as indicated on the left. Solid and open arrowheads indicate ubiquitinated and unmodified forms of His-Pex5p-HA variants, respectively. Dots, mouse IgG light chains, apparently weakly cross-reactive with anti-goat IgG secondary antibody. C, top, CHO-K1 was transfected with plasmids encoding wild-type Pex5pL-HA (WT) and the KR mutants and analyzed by SDS-PAGE and immunoblotting as in B. Solid and open arrowheads, ubiquitinated and unmodified forms of Pex5pL-HA variants, respectively. Bottom, numerical diagram of the Lys-to-Arg mutations in KR variants of Pex5pL-HA used in the top. D, KR mutants of Pex5p in peroxisome-deficient CHO mutants. Plasmids encoding wild-type Pex5pL-HA (WT) and the KR mutants were separately transfected to CHO-K1, pex12 ZP109, pex2 Z65, pex14 ZP161, pex19 ZP119, and pex1 ZP107 cells. At 24 h post-transfection, cells were analyzed as in Fig. 7B. Solid and open arrowheads, Lys-ubiquitinated and unmodified forms of Pex5pL-HA mutants, respectively. The figure is shown as a composite of two separate experiments. E, the Pex10p·Pex12p complex preferentially ubiquitinates Lys449 and Lys499 in Pex5p in vitro. Top, in vitro ubiquitination assay was performed as in Fig. 5C, with MBP-fused full-length Pex10p and Pex12p, using various KR mutants of Pex5pL, as indicated at the top. A composite of two separate experiments (lanes 1–13 and 14–19) was shown. Lys-to-Arg mutations in KR variants of Pex5pL-HA are shown in the bottom panel of C. F, interaction between KR mutants of Pex5p and Pex5p-binding partners. Both S and L forms of wild-type Pex5p and KR mutants of Pex5pL were subjected to pull-down assays with GSH-Sepharose beads conjugated with GST, GST-EGFP-PTS1, GST-Pex14p, and GST-Pex13pN, respectively. Proteins in bound fractions were assessed by immunoblotting with anti-Pex5p antibody and by Coomassie Brilliant Blue (CBB) staining. Solid arrowheads indicate respective GST fusion proteins used for the assay. Dot, Pex5p variants recovered with GST-Pex14p.

Article Snippet: Purification of Recombinant Proteins MBP and GST fusion proteins were purified with amylose resin (New England Biolabs) and glutathione (GSH)-Sepharose beads (GE Healthcare), respectively, as described ( 35 ).

Techniques: Transfection, Cell Culture, Immunoprecipitation, Western Blot, SDS Page, In Vitro, Ubiquitin Assay, Binding Assay, Staining

Journal: The Journal of Biological Chemistry

Article Title: Distinct Modes of Ubiquitination of Peroxisome-targeting Signal Type 1 (PTS1) Receptor Pex5p Regulate PTS1 Protein Import *

doi: 10.1074/jbc.M113.527937

Figure Lengend Snippet: Pex5p ubiquitination is involved in matrix protein import into peroxisomes. A, complementing activity of Pex5pL KR mutants. CHO pex5 ZP139 cells were transfected with plasmids, each encoding wild type, K520R, K-less, and 520K mutants of Pex5pL-HA. After a 1-day culture, cells were immunostained with anti-catalase antibody. Bar, 10 μm. B, quantification of complementing activity of KR mutants. Cells restored for the impaired import of PTS1 (not shown) and catalase (A) in 100–150 cells were counted in three independent experiments. Complementing activities of K520R, K-less, and 520K mutants were represented as percentages relative to that of wild-type with S.E. (error bars). C, ubiquitination at Lys520 abrogates the interaction of Pex5p with the PTS1 cargo protein. Cytosolic fractions of HEK cells expressing Pex5pL-HA mutants, 520K and K-less, were incubated with GSH-Sepharose beads conjugated with GST, GST-EGFP-PTS1, and GST-Pex14pN(1–260), respectively. Proteins in the bound fractions were analyzed as in Fig. 6F by immunoblotting, as indicated on the left, and staining with Coomassie Brilliant Blue (CBB). Input, 15% cytosolic fractions (Cyto) used for the pull-down assay (lanes 1 and 2). Solid and open arrowheads, 520K mutant ubiquitinated at Lys520 and unmodified forms of the 520K and K-less mutants, respectively.

Article Snippet: Purification of Recombinant Proteins MBP and GST fusion proteins were purified with amylose resin (New England Biolabs) and glutathione (GSH)-Sepharose beads (GE Healthcare), respectively, as described ( 35 ).

Techniques: Activity Assay, Transfection, Expressing, Incubation, Western Blot, Staining, Pull Down Assay, Mutagenesis