Table 1.
Enriched tight junction (TJ) and adherens junction (AJ) proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 2.
Enriched signaling proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 3.
Enriched kinases and phosphatases tagged by biotin ligase fused to occludin and claudin-4.
Table 4.
Enriched endosomal- and membrane-trafficking proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 5.
Enriched clathrin-dependent endocytosis and exocytosis/transcytosis trafficking proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 6.
Enriched cell adhesion proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 7.
Enriched cytoskeletal proteins tagged by biotin ligase fused to occludin and claudin-4.
Table 8.
Enriched membrane proteins tagged by biotin ligase fused to occludin and claudin-4.
Fig 1.
Ocln and Cldn4 biotin ligase fusion-proteins localize to the TJ and the lateral cell membrane.
A. Both biotin ligase fused to the N terminus (BL-Ocln, myc) and C terminus (Ocln-BL, myc) of Ocln co-localized (Merge, top right, middle right panel) with ZO-1 (top left and middle left panel), although there is also some non-junctional immunofluorescence associated with the transgene. Biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4, myc) partly co-localizes with ZO-1 (Merge, bottom right panel), and partly in the cytoplasm (Bottom middle panel). B. BL-Ocln, Ocln-BL and BL-Cldn4 (Myc signal middle panel) are distributed along the basolateral membrane. The two Ocln constructs also concentrate at the apical junction with ZO-1 (right top and middle panel). Bar, 20 microns.
Fig 2.
Streptavidin staining of biotinylated proteins reveals proteins at the TJ and lateral membranes.
Biotin was added over night to MDCKII cells expressing the various biotin ligase fusion proteins. After wash, fixation, and block fluorescent streptavidin and ZO-1 primary antibody was added. Both biotin ligase fused to the N-terminus (BL-Ocln, myc) and C-terminus (Ocln-BL, myc) of Ocln tagged proteins near ZO-1 (Merge, top right and middle right panel). Biotin ligase fused to the N-terminus of Cldn4 (BL-Cldn4) also partly tags proteins co-localized with ZO-1 but possibly to a lesser extent than Ocln (Merge, bottom right panel). Although the majority of streptavidin stained proteins are distributed along the lateral plasma membrane at cell-cell contacts, there are also cytoplasmic staining (middle panel). Streptavidin staining of cells expressing biotin ligase alone reveal a much more diffusely distributed protein staining throughout all compartments of the cell [10]. Bar, 20 microns.
Fig 3.
Coomassie-stained SDS-PAGE gels reveal that streptavidin-purified biotinylated proteins from different transgenes show differing protein patterns.
A. Shown are proteins purified from cells expressing biotin ligase alone (BL), biotin ligase fused to the N terminus of Ocln (BL-Ocln). B. Proteins purified from the N- and C- terminus of Ocln (BL-Ocln and Ocln-BL) with added biotin. C. Shown are proteins purified from cells expressing biotin ligase fused to the N terminus of Cldn4 (BL-Cldn4), with or without added biotin. The positions of the transgenes are marked with arrowheads. Triplicate samples gave very similar protein patterns.
Fig 4.
Functional analysis of top 150 enriched proteins recovered from cells expressing biotin ligase fusion proteins.
Streptavidin-purified proteins identified by mass spectrometry from cells expressing biotin ligase fused to the N terminus of Ocln (BL-Occludin, left), the C terminus of Ocln (Occludin-BL, middle) or the N terminus of Cldn4 (BL-Claudin 4, right). Functional classification revealed similar distribution for the two Ocln constructs, although individual proteins within the functional groups trafficking-, signaling-, cell adhesion etc. vary, or are more or less abundant (higher or lower average normalized-PSM/OPN). Functional groups of proteins recovered from cells expressing biotin ligase fused to the N terminus of Cldn4 shows similar functional distribution of enriched proteins as both of the Ocln fusion proteins, although there are slightly more trafficking proteins tagged by BL-Cldn4. Enriched = ≥3-fold increase compared to biotin ligase alone (as determined by the average normalized PSM/OPN).
Fig 5.
Relative abundance of proteins tagged by biotin ligase fusion proteins identified by mass spectrometry.
The y-axis is proportional to the amount of protein recovered and was calculated as follows: PSMs from each of the three isolations were normalized (PSM for each protein/total PSMs for that isolation), these normalized PSMs were averaged between the three runs and then divided by the number of theoretical observable peptide number falling in the size range detectable by MS and this value multiplied by 1000. Proteins were ordered by this value (largest to smallest); points on the x-axis indicate individual unique proteins identified using the Canis lupus familiaris Ref Seq database. Plotted are the top 150 enriched (three-fold or above biotin ligase alone levels) proteins for each fusion-protein. The top 10 most enriched proteins for each fusion protein are listed.
Fig 6.
Cellular localization of proteins identified by proteomic analysis of proteins surrounding occludin and claudin-4.
A. GFP-PLLP localizes diffusely in the cytoplasm and at cell-cell contacts (two middle panels). Co-localization with Ocln and Cldn4 appears to be at cell-cell contacts (two right panels). B. GFP-RNtre predominantly localizes to cell-cell contacts (two middle panels) where the co-localization with Ocln and Cldn4 occurs (two right panels). C. GFP-FLRT2 localizes diffusely in the cytoplasm and at cell-cell contacts (two middle panels).Co-localization with Ocln and Cldn4 appears to be at cell-cell contacts (two right panels). D. The majority of Mark3 localizes to cell-cell contacts but is also present in punctate structures in the cytoplasm (two middle panels). Ocln and Cldn4 co-localize with Mark3 at cell-cell contacts (two right panels). Bar, 20 microns (x63 oil objective).