Interaction analysis of SNX27 and retromer with MT1-MMP. (A) Lysates were prepared from MDA-MB-231 cells stably expressing GFP–MT1-MMP or GFP–MT2-MMP and allowed to bind recombinant GST-fused SNX27 and Vps35 immobilized on the glutathione sepharose beads. The protein complex was eluted and run on denatured SDS gel. Immunoblotting was performed using anti-GFP and anti-Vps26 antibodies. Amino acid sequences represent the cytoplasmic tail sequences of MT2-MMP and MT1-MMP. (B) GBP pull-down was performed with lysates from MDA-MB-231 cells expressing GFP vector, GFP-Vps29, and GFP-SNX27. Purified GST–GBP was incubated with glutathione sepharose beads and allowed to bind with the respective lysates for 1 h. Beads were washed and boiled, samples were run on SDS gel, and analysis was done by Western blotting. (C) Purified GST or GST-MT1 tails bound to glutathione sepharose beads were incubated with His-Vps26 or SNX27 in the binding buffer (see Materials and methods). Immunoblotting was done using an anti-Vps26 or anti-SNX27 antibody. (D) Calorimetric titrations of the MT1-MMP–CT peptide (400 µM) with His-tagged SNX27, Vps26, and Vps29 were performed. The upper panel shows raw ITC data for the Vps26 (40 µM) obtained from the 25 automated injections (2 µl in each injection). The lower panel shows integrated peak areas fitted using a 1:1 independent model of binding. (D′) The quality of proteins used in ITC was determined by running on SDS gel. Table 1 represents the analyzed thermodynamic parameters for the measured protein–peptide interaction. In all the performed experiments, the heat of ligand dilution was subtracted from the raw data to obtain normalized integrated heats. All ITC titrations were performed at 17°C. (E) MDA-MB-231 cells expressing the GFP–MT1-MMP or MT1-MMPΔDKV mutant were lysed and subjected to GBP pull-down as described above. Immunoblotting was performed using an antibody against Vps35, SNX27, and GFP. (F) Cells cotransfected with GFP–MT1-MMP and mCherry–MT2-MMP were fixed and analyzed by super-resolution microscopy, 3D-SIM. Images were obtained after 3D reconstruction (scale bar = 5 µm). The insets show the localization of MT1-MMP and MT2-MMP on discrete endosomal domains. WCL, whole cell lysate.
Figure 6.

Interaction analysis of SNX27 and retromer with MT1-MMP. (A) Lysates were prepared from MDA-MB-231 cells stably expressing GFP–MT1-MMP or GFP–MT2-MMP and allowed to bind recombinant GST-fused SNX27 and Vps35 immobilized on the glutathione sepharose beads. The protein complex was eluted and run on denatured SDS gel. Immunoblotting was performed using anti-GFP and anti-Vps26 antibodies. Amino acid sequences represent the cytoplasmic tail sequences of MT2-MMP and MT1-MMP. (B) GBP pull-down was performed with lysates from MDA-MB-231 cells expressing GFP vector, GFP-Vps29, and GFP-SNX27. Purified GST–GBP was incubated with glutathione sepharose beads and allowed to bind with the respective lysates for 1 h. Beads were washed and boiled, samples were run on SDS gel, and analysis was done by Western blotting. (C) Purified GST or GST-MT1 tails bound to glutathione sepharose beads were incubated with His-Vps26 or SNX27 in the binding buffer (see Materials and methods). Immunoblotting was done using an anti-Vps26 or anti-SNX27 antibody. (D) Calorimetric titrations of the MT1-MMP–CT peptide (400 µM) with His-tagged SNX27, Vps26, and Vps29 were performed. The upper panel shows raw ITC data for the Vps26 (40 µM) obtained from the 25 automated injections (2 µl in each injection). The lower panel shows integrated peak areas fitted using a 1:1 independent model of binding. (D′) The quality of proteins used in ITC was determined by running on SDS gel. Table 1 represents the analyzed thermodynamic parameters for the measured protein–peptide interaction. In all the performed experiments, the heat of ligand dilution was subtracted from the raw data to obtain normalized integrated heats. All ITC titrations were performed at 17°C. (E) MDA-MB-231 cells expressing the GFP–MT1-MMP or MT1-MMPΔDKV mutant were lysed and subjected to GBP pull-down as described above. Immunoblotting was performed using an antibody against Vps35, SNX27, and GFP. (F) Cells cotransfected with GFP–MT1-MMP and mCherry–MT2-MMP were fixed and analyzed by super-resolution microscopy, 3D-SIM. Images were obtained after 3D reconstruction (scale bar = 5 µm). The insets show the localization of MT1-MMP and MT2-MMP on discrete endosomal domains. WCL, whole cell lysate.

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