Fig 1.
Bag-1 isoform-specific interactome analysis and classification of enriched pathways.
a. Schematic representation of TAP–Bag-1 constructs used in the interactome analysis. TAP: Tandem Affinity Purification, NLS: Nuclear localization signal, TRSEEX: Amino acid repeats, UbL: Ubiquitin-like domain, BAG: BAG domain. b. Venn diagram of candidate interaction partners enriched (>1.2 fold) in the Bag-1 isoforms’ interactomes relative to the mock interactome. c. Subset of Gene Ontology (GO) classifiers for candidate Bag-1-interactors. Percentage of proteins with the indicated GO terms within Bag-1 interactomes and the reference dataset are depicted. Blue: Bag-1S, green: Bag-1M, purple: Bag-1L and grey: Reference. d. Heatmap analysis for the relative abundances of the binding partners for each Bag-1 isoform. Enriched proteins are clustered according to their biological functions. Color code is given in log2 base.
Fig 2.
Components of isoform-specific Bag-1 complexes identified by BN PAGE/LC-MS/MS.
a. Experimental workflow. Bag-1 immunoprecipitates were run on native gel. Individual bands in each lane were cut, trypsinized and the resulting peptides were analyzed by LC-MS/MS. M: Protein marker. b-d. Seven protein complexes were characterized for Bag-1S (b), five for Bag-1M (c), and six for Bag-1L (d). Identified proteins are color-coded as following: Blue: Bag-1, green: Chaperones, yellow: ER-resident proteins, orange: Ubiquitination elements, purple: Shuttling proteins, red: Proteasome subunits.
Fig 3.
Bag-1 isoforms interact with protein quality control elements in breast cell lines and tissues.
a. Immunoblotting assays for interaction partners of purified Bag-1S, M and L proteins extracted from MCF-7 and MCF-12A. Total, unbound, and Bag-1-bound protein extracts were blotted for BiP, PDIA3, Hsp70, Hsp90, VCP/p97, Rad23B, Calnexin and Calreticulin. b. Immunocytochemistry for assessment of Bag-1’s co-localization with interaction partners. MCF-7 cells transfected with Bag-1 isoforms were stained with Bag-1 antibody and VCP/p97. c. Co-immunoprecipitation assay in tissues from breast cancer patients. Interaction of Bag-1 with VCP/p97, Rad23B, and Hsp70 was increased in tumor tissues compared to normal. n = 4 for each molecular subtype of breast cancer. T: Tumor tissue, N: Neighboring normal tissue.
Fig 4.
Structural predictions for Bag-1 complexes.
Interaction of BAG domain of Bag-1 (turquoise) with a. the ATPase domain of Hsp70 (red), and b. the monomeric VCP/p97 protein (yellow). Interaction of UbL domain of Bag-1 (purple) with c. the UbL domain (green) and UBA2 domain (orange) of Rad23B. d. Interaction of BAG domain of Bag-1 (turquoise) and UbL domain of Bag-1 (purple) with the Rpn1 protein (brown). e—j. Close up views for interaction surfaces.
Fig 5.
BAG and UbL mutants disrupt interaction of Bag-1 with protein quality control network.
Protein abundances in the interactomes of TAP–Bag-1S mutants are compared to that of wild-type TAP–Bag-1S. Fold changes are depicted in log2 base for a. R161D, b. R162E, c. R193D, and d. I103K mutants.
Fig 6.
Mutations on BAG and UbL domains affect proteasomal activity.
Proteasome activity was measured by the fluorescence of released MCA in protein extracts from MCF-7 cells after transfection with a. Bag-1 isoforms and mock vector, and b. Bag-1S mutants and wild-type Bag-1S. Proteasome activity was measured under ATP-containing (grey) and ATP-depleted (orange) conditions. Significant differences were determined by t-test.
Fig 7.
Bag-1 downregulates VCP/p97-dependent ER-associated degradation of CD147.
Bag-1 KO MCF-7 cells were transfected with mock vector, wild-type Bag-1S and mutant Bag-1S, and treated with a. glycosylation inhibitor tunicamycin, b. translation inhibitor emetine, c. VCP/p97 inhibitor CB-5083. Immunoblots of CD147 (M: Mature, CG: Core glycosylated, DeG: Deglycosylated) are shown in a-c. d. model for downregulation of CD147 degradation by Bag-1.