Figure 1.
Experimental strategy adopted in this work to prove the topography of VDAC.
In the figure the possible output of caspase's cleavage is shown. Right side: the C-terminus faces the cytosol, exposing the DEVD to caspase's cleavage and causing the removal of the His tag. Left side: the opposite topography will not cause any separation of the two tags.
Figure 2.
HA and His immunostaining defines the position of the C-terminal end of VDAC1 in the cell.
A) Expression and sorting of the chimera VDAC1HaDEVDHis. HeLa cells were transfected with pCMSmtDsRed-VDAC1HaDEVDHis and double-immunostained for the HA and the His tags. pCMSmtDsRed-VDAC1HaDEVDHis transfection resulted in the simultaneous expression of both mitochondrial hVDAC1-HaDEVDHis (HA green and His white) and mitochondrial mtDsRed (red) used as transfection and targeting reporter. Fluorescence distribution analysis indicates the co-localization of both the HA and the His tags with the mtDsRed in the mitochondrion. Images are representative of 50 cells analyzed for each condition in experiments performed in triplicate. The lower panel is a magnification. Scale bar, 20 µm. B) Caspase activation does not separate HA from His tag, indicating that the DEVD is not accessible. HeLa cells transfected with pCMSGFP- VDAC1HaDEVDHis were exposed to staurosporine, fixed and double immunostained for the HA and the His tags. Cells expressing the cytosolic GFP (green), positively stain for both the HA (red) and His (white) tags, even upon mild staurosporine exposure. In the upper panel cells treated with 500 nM staurosporine are shown. In the lower panel the staurosporine was used at 1 µM. Note the dramatic morphological modification induced by staurosporine. Images are representative of 50 cells analyzed for each condition in experiments performed in triplicate. Scale bar, 20 µm.
Figure 3.
DEVD is cleaved in MomDEVDGFP upon staurosporine exposure.
A) HeLa cells were transfected with the chimera MomDEVDGFP. In non apoptotic cells, the GFP localizes in the mitochondrial outer membrane facing the cytosol. When apoptosis was induced by treatment with 0.5 µM staurosporine, the GFP fluorescence become cytosolic, as a result of the DEVD cleavage. B) Co-transfection of HeLa cells with both pCMSmtDsRed-VDAC1HaDEVDHis and MomDEVDGFP. Images were obtained after fixation and immunostaining for the His tag (white). Red stains for DsRed (mitochondria), green for GFP. The upper row shows untreated cells, where all the three overexpressed proteins (mtDsRed, MomDEVDGFP and hVDAC1-HaDEVDHis) localize into the mitochondria. Growing concentrations of staurosporine (caspase activation, lower rows) results in diffusion of the GFP fluorescence form mitochondria to the cytosol. Nevertheless, in the same condition the His tag was not lost, confirming that hVDAC1-HaDEVDHis is still in mitochondria and not accessible to caspases. Images are representative of 50 cells analyzed for each condition in experiments performed in triplicate. Scale bar, 20 µm.
Figure 4.
HA and His tags of chimeric hVDAC1-HaDEVDHis are protected from proteases in the FPP assay.
A) HeLa cells transfected with pCMSGFP-VDAC1HaDEVDHis were double immunostained for the HA tag (red) and the His tag (white). B) Addition of 40 µM digitonin caused the diffusion of the GFP from the cytosol to the external medium. C) Addition of 40 µM digitonin plus proteinase K does not affect the HA and His staining. D) The same experiment, but cytochrome c (white) was stained instead of His tag. The stable localization of cytochrome c demonstrates that, in the condition here used, the intermembrane space is not accessible to proteases. The reduced cytochrome c signal in hVDAC1-HaDEVDHis transfected cells is just random. Images are representative of 50 cells analysed for each condition in experiments performed in triplicate. Scale bar, 20 µm. E) Addition of Triton X-100 damages the outer membrane and results in the entrance of proteinase K in the mitochondrial intermembrane space and in the digestion of HA and His tags. The picture represents the HA and cytochrome c staining. The Bright field is the same cell field, showing the presence of cell, even though they are quite damaged by the harsh treatment with detergent and proteases.
Figure 5.
FPP assay upon transfection with pCMSmtDsRed-VDAC1HaDEVDHis.
A) HeLa cells transfected with pCMSmtDsRed-VDAC1HaDEVDHis. Transfected cells were identified through the mitochondrial fluorescent signal of mtDsRed (red). The incubation with of 40 µM digitonin plus proteinase K does not affect the HA (green) and His (white) staining. B) The same as in A, but cytochrome c was stained instead of the His tag, showing its co-localization with VDAC (HA tag in green). The stable localization of cytochrome c demonstrates that the intermembrane space is not accessible to proteases in conditions here used. Images are representative of 50 cells analyzed for each condition in experiments performed in triplicate. Scale bar, 20 µm.
Figure 6.
Access of proteases to MomGFP, a transmembrane anchored MOM protein, in the FPP assay.
A) HeLa cells expressing the mitochondrial outer membrane protein, MomGFP were treated with digitonin and proteinase K as described. Images were taken before and after treatment with digitonin and proteinase K at the indicated time points. The GFP signal becomes weak at 210 seconds and is completely lost at 300 seconds. Scale bar, 20 µm. B) Kinetic analysis of the GFP fluorescence fading in three regions of the microscopic field described above.
Figure 7.
Relevance of VDAC topography: interaction with other proteins.
A) Topography of the yeast porin showing the position of proteases and antibodies used for the prediction of sideness. Yellow arrowheads show the position of the mAb and proteases cleavage sites as described in [13]. Purple arrowheads show the location of epitopes probably detected by the pAb anti-peptides 1-21, 195-210, 251-268, 272-283 [14]. The peptide 251-268 protrudes from both sides of the membrane. This explains its ambiguous results in [14]. The pale blue arrows show the location of FLAG epitopes inserted in yeast porin [15].
B) Topography of the mouse porin showing the position of residues and loops found important in the interaction of VDAC1 with HKI, Bcl-2 and Bax. Circled in red the residues found involved with the HK-VDAC1 binding [34]; the blue circles mark HKI binding sites as defined by SPR and peptide competition assay [35]; the yellow triangles show the position of peptides anti-Bax [36]; the purple squares mark the loop involved in the binding to Bcl 2 [37].
C) VDAC1 three-dimensional structure. Lateral view of the protein as reported in pdb: 3emn [10]. The red arrow outlines the C-terminus. Cysteines are indicated. The figure was obtained with PyMol.
D) VDAC1 structure as defined from site-directed mutagenesis experiments [11]. The red box indicates the C-terminal β-strand.