The pro-apoptotic Bcl-2 family member tBid localizes to mitochondrial contact sites
© Lutter et al; licensee BioMed Central Ltd. 2001
Received: 27 September 2001
Accepted: 8 November 2001
Published: 8 November 2001
Following cleavage by caspase 8, the C-terminus of Bid translocates from the cytosol to the mitochondria that is dependent upon structures formed by the mitochondrial-specific lipid cardiolipin. Once associated with mitochondria, truncated Bid (tBid) causes the potent release of cytochrome c, endonuclease G, and smac.
We investigated whether tBid localizes specifically to the contact sites of mitochondria purported to be rich in cardiolipin. A point mutation changing the glycine at position 94 to glutamic acid in the BH3 domain of tBid (tBidG94E) was principally used because mitochondria treated with this mutant tBid displayed better preservation of the outer membrane than those treated with wild type tBid. Additionally, tBidG94E lowers the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria. Electron microscope tomography coupled with immunogold labeling was used as a new hybrid technique to investigate the three-dimensional distributions of tBid and tBidG94E around the mitochondrial periphery. The statistics of spatial point patterns was used to analyze the association of these proteins with contact sites.
Immunoelectron tomography with statistical analysis confirmed the preferential association of tBid with mitochondrial contact sites. These findings link these sites with cardiolipin in tBid targeting and suggest a role for Bcl-2 family members in regulating the activity of contact sites in relation to apoptosis. We propose a mechanism whereby Bcl-2 proteins alter mitochondrial function by disrupting cardiolipin containing contact site membranes.
Recent work has shown that the Bcl-2 family regulates mitochondrial homeostasis during apoptosis . Pro-apoptotic members, including Bax, Bak, Bid, and Bim, promote the release of death-inducing proteins, such as cytochrome c [2, 3], smac , and endonuclease G , from mitochondria while anti-apoptotic members, such as Bcl-2 and Bcl-XL, inhibit this release. Following release into the cytosol, these death-inducing proteins promote apoptotic cell destruction through multiple pathways including caspase activation and nuclear DNA fragmentation.
In addition to controlling the release of pro-death proteins, the Bcl-2 family also alters the function of mitochondria undergoing apoptosis. Dysfunction of voltage dependent anion channel opening, ADP/ATP exchange, the electron transport chain, oxidative phosphorylation, and calcium buffering through the action of this family of proteins have been reported [6–8]. While early defects in the electron transport chain can be reversed by addition of exogenous cytochrome c, the damage eventually becomes refractory to cytochrome c addition . This finding suggests that pro-death Bcl-2 proteins can damage mitochondrial function independent of cytochrome c loss.
Recently, we demonstrated that the targeting of the pro-apoptotic protein tBid to mitochondria depends upon the presence of the mitochondria-specific lipid cardiolipin in a possibly unique structure . Cardiolipin has a defined distribution pattern within mitochondria . It is found in high concentrations throughout the inner membrane, including at contact sites where the inner membrane and outer membrane interact. Cardiolipin is present at much lower concentrations elsewhere in the outer membrane. This distinction suggests that tBid might localize to contact sites because of the arrangement of cardiolipin there. To test this hypothesis, we determined the submitochondrial localization of tBid using a new hybrid technique – immunoelectron tomography, which couples conventional immunoelectron microscopy with tomography to add the third dimension. Tomographic analysis was chosen because it enables quantitative three-dimensional examination of fine structure within the relevant mitochondrial domains in semi-thick sections and thereby facilitates accurate representation of the sometimes complex membrane topology of this organelle [11, 12]. Immunoelectron tomography is ideal for testing the independence of two types of labels, or label and structural component (as employed in this study), and their deviation from independence – colocalization or mutual inhibition – because it allows true 3-D distances to be measured. We report here an analysis of the 3-D distances of immunogold-labeled tBid from contact sites on the periphery of liver mitochondria that strongly supports the colocalization of the two.
To enhance mitochondrial membrane preservation, a point mutation changing the glycine residue at position 94 to glutamic acid in the BH3 domain of tBid was used. This mutation was previously shown to lower the cytochrome c releasing activity of tBid without affecting its targeting to mitochondria . Mitochondria treated with tBidG94E displayed better preservation of the outer membrane than those treated with WT tBid. Hence, because of better preservation of contact sites, the tBidG94E was used primarily for the tomographic analysis. For immunogold labeling, a three-myc tag was placed at residue 67 of BidG94E. Following cleavage by caspase 8, a version of tBidG94E was generated with the three-myc tag at the N-terminus of tBidG94E. Monoclonal anti-myc antibody was used for immunogold labeling to ensure high specificity of binding.
Electron tomography has several advantages over conventional two-dimensional immunogold electron microscopy that should be enumerated to understand the accuracy of the measurements made in this study. First, a more accurate localization of the 5-nm gold particles is possible because each slice of the tomographic reconstruction is about 2 nm in thickness compared with the typical 50–100 nm thickness used in conventional immuno-electron microscopy. Second, because the dimensions of contact sites (~14 nm diameter) [13, 14] are considerably smaller than 50–100 nm, they are easily missed in conventional microscopy; this problem was removed by the typically finer sampling of electron tomography (~2 nm). Third, because tomography is inherently a 3-D tool, the 3-D placement of gold-labeled tBidG94E was obtained in relation to the 3-D distribution of contact sites and gold beads at high resolution. Unlike two-dimensional electron micrographs, true Euclidean distance was measured at a resolution of 4 nm (twice the pixel size, or thickness, according to Shannon sampling theory) in tomographic reconstructions from each gold bead to the nearest contact site. These measurements were then used for a statistical analysis of association.
Distance from immunogold particles to the nearest contact site in tomographic reconstructions of liver mitochondria
Labeling type on Mitochondrion
Mean distance experimental (nm)a
Mean distance random (nm)a,b
Number of contact sites
Number of immunogold particles
WT tBid 1
53 ± 27
122 ± 56
GE tBid 1c
30 ± 14
70 ± 28
GE tBid 2
29 ± 12
129 ± 76
GE tBid 3
31 ± 6
77 ± 25
GE tBid 4
23 ± 9
104 ± 48
Mitochondria are compartmentalized into three spaces by their inner and outer membranes . The outer membrane surrounds the mitochondria and separates it from the cytosol. The inner and outer membranes unite to form mitochondrial contact sites with protein composition that is still being defined . It is important to test association of tBid with contact sites using electron microscopy because whereas purification schemes are based on assumptions about what contact sites should contain (and sometimes arrived at close to 100 proteins), these sites have always been defined structurally. As previously noted, contact sites are purported to have a unique lipid environment that is rich in cardiolipin. The presence of cardiolipin has been implicated in the function of numerous mitochondrial proteins including, sites I and III of the electron transport chain, the adenine nucleotide transporter, the protein translocation machinery, and the localization of mitochondrial creatine kinase [19–22].
tBidG94E was used predominantly for testing the hypothesis of localization of tBid to contact sites. Although this mutant translocates to mitochondria as efficiently as does the WT with diminished disruptive effect on the outer membrane, it seems conceivable that tBid harbors two distinct signals, one for translocation to mitochondria, and the other for interaction with proteins or lipids to exert its pro-apoptotic function. Disruption of this latter signal via mutation might alter tBid's submitochondrial localization, while preserving the outer membrane. We showed that WT tBid was not randomly localized with respect to contact sites. The mean experimental distance between gold particles and contact sites (53 nm; see table 1), though, was significantly higher than those values for tBidG94E (23–31 nm; table 1). This increased distance for WT tBid is likely due to two factors that affect the visualization of contact sites. First, tBid may have degraded the outer membrane, so that the nearest contact site is not always recognized as such. Second, the streaking caused by reconstructing the 10-nm gold particles used for WT tBid labeling may have obscured the nearest contact site in a few instances. Hence, whereas our analysis cannot disprove the hypothesis that by mutating tBid its submitochondrial localization was changed, it seems more likely that the greater value for WT tBid is caused by the factors mentioned above. Thus, it appears that the conclusion of tBid localization to contact sites can be applied more generally than the narrow interpretation of tBidG94E localization only.
Truncated Bid, as other BH3-only proteins, requires another protein such as Bax or Bak to exert its pro-apoptotic function [23, 24]. Interestingly, tBid was found associated with mitochondrial contact sites (Figs. 1 and 2) and was shown to destabilize lipid membranes in vitro (along with Bax) by increasing the permeabilization of artificial liposomes [25, 26]. Therefore it is possible that tBid can bind to the cardiolipin rich contact site membranes and destabilize them by inducing Bax or Bak to oligomerize. The membrane destabilization may cause a secondary effect of inhibiting the action of the proteins located there. This hypothesis would account for how tBid affects the function of many different processes without binding directly to several proteins. Indeed it was found that the targeting of tBid to mitochondria-like membranes occurs in the absence of proteins, yet depends upon cardiolipin being in a possibly unique structure as opposed to free cardiolipin . The requirement for a specific cardiolipin structure that is likely defined by the contact site is consistent with our observation that no gold particles were found in regions stripped of the outer membrane exposing the cardiolipin-rich inner membrane.
After the induction of apoptosis, mitochondria display two distinct phases of damage. Early in the process the outer membrane becomes permeable, without rupture (Although this is controversial .), to several proteins found in the mitochondrial inner membrane space including cytochrome c, in which contact sites have a probable role . During this early phase, though, the inner membrane remains intact and mitochondria still retain their protein import machinery . Later in the process there is swelling and herniation of the matrix leading to a distension of the cristae [8, 30].
Many factors could contribute to the structural changes observed in mitochondria. tBid and other pro-death proteins such as Bax or Bak could destabilize the membrane leading to the observed changes. Additionally loss of contact site and inner membrane integrity could indirectly cause alterations in ion exchange and lead to the flow of water into the matrix. The loss of cytochrome c causes a break in the electron transfer chain resulting in the production of reactive oxygen species . Reactive oxygen species are known to cause the oxidation and loss of function of many lipids including cardiolipin . Finally, caspases activated downstream of cytochrome c release have been implicated in mitochondrial damage . It appears likely that the cumulative damage of these agents could lead to the irreversible loss of mitochondrial membrane integrity and structure.
In conclusion, we have combined the techniques of immunogold microscopy and electron tomography to determine visually and by statistical analysis that tBid is preferentially concentrated at contact sites rather than being randomly distributed along the outer membrane. For the first time, immunogold electron tomography permitted a quantitative demonstration of 3-D organellar protein labeling with antibodies. The method described is envisioned to broaden biological applications of ultrastructural immunogold labeling techniques especially in investigations dealing with compartmentalization of functional elements. For example, many organelles, including the nucleus, endoplasmic reticulum, and Golgi apparatus, have a complex organization with a unique distribution of proteins throughout each. Immunoelectron tomography is starting to be a useful tool in determining how the distribution of proteins may provide clues as to their targets or scope of action .
Materials and Methods
Generation of Bid and caspase 8 proteins
Full length human Bid and caspase 8 cDNA were obtained as described (Luo et al 1998). A three myc-tag recognizing the sequence EQKLISEEDL was placed into position 67 in the cDNA of full length Bid. A point mutation changing glycine 94 to glutamic acid was made into three-myc Bid using the primers 5'-GCCCAGGTCGAGGACAGCATG-3' and 5'-CATGCTGTCCTCGACCTGGGC-3' using standard techniques. Both vectors were confirmed by DNA sequencing. Bacterially expressed proteins were made as previously described .
Preparation of mitochondria for immunogold electron microscopy
Mouse liver mitochondria were prepared fresh and treated with 1 ng myc-tagged tBidG94E/ 100 μg of mitochondrial protein. Mitochondria were then washed 2X in mitochondria isolation buffer (MIB, 250 mM mannitol, 5 mM HEPES pH 7.2, 0.5 mM EGTA, and 0.1% (w/v) bovine serum albumin) and weakly fixed with fresh 1% paraformaldehyde in MIB for 15 min on ice. Mitochondria were pelleted (in all steps at 10,000 x g for 3 min at 4°C) and resuspended in blocking buffer (1% bovine serum albumin, 1% normal goat serum, 1% cold water fish gelatin, 0.04% glycine in MIB) for 15 min on ice. They were then pelleted and resuspended in working buffer (blocking buffer diluted 10 x in MIB) containing the primary antibody, anti-myc (1:1000) for 1 hr on ice. The no-primary control omitted the previous step only. A second control consisted of replacing the primary antibody with an excess of tBidG94E and continuing as with the primary antibody. Mitochondria were washed 4 x 4 min in working buffer and incubated with the indicated anti-mouse secondary antibody conjugated to 10-nm (used for wild-type tBid) or to 5-nm (used for tBidG94E) gold beads (1:50 Amersham) for 1 hr on ice in working buffer. Subsequently, they were washed 4 x 4 min with working buffer to remove unbound secondary antibody and rinsed 1 x in cacodylate buffer (0.1 M sodium cacodylate. Electron Microscopy Sciences, Ft. Washington, PA).
Mitochondria were centrifuged and the pellet was kept intact for all remaining steps. The mitochondrial pellets were fixed in 2% glutaraldehyde in cacodylate buffer for one hour on ice and rinsed 3 x 4 min in cacodylate buffer. The mitochondrial pellets were incubated with 1% osmium tetroxide (Electron Microscopy Sciences) in cacodylate buffer for one hour on ice, followed by rinsing 3 x 4 min in ice-cold double-distilled water. The pellets were dehydrated using a successive ethanol series of 20, 50, 70, and 90% in double distilled water for 10 min each on ice. The pellets were further dehydrated at room temperature 2 x in 100% ethanol for 5 min and 2 x in 100% acetone for 5 min each.
Pellets were transferred to glass scintillation vials for resin (Durcupan ACM, EMS) infiltration. The pellets were first incubated in a sequence of 70% ethanol/30% resin, 50% ethanol/50% resin, and 30% ethanol/70% resin for 30 min each at room temperature. Pellets were then infiltrated 3 x 1 hr in 3 ml of 100% resin. After the final incubation, the resin-infiltrated pellet plus excess resin were poured into aluminum dishes and polymerized at 60°C for 36 hr.
Electron microscopy and tomographic reconstructions
Tomographic reconstructions were performed as described previously . Briefly, thin-sectioned material (~80-nm thick) was examined using a JEOL 1200FX electron microscope. Thick-sectioned material was examined with a JEOL 4000EX microscope operating at 400 keV. The 0.75 μm thick sections were tilted through 120° of rotation, and electron micrographs were taken in 2° increments. Using the SUPRIM software package, Four tilt series from different sections consisting of 61 tilt images each were then digitized, aligned, and back projected using R weighting to generate the volume information. Volume segmentation of the reconstructed mitochondria using Xvoxtrace software was used to delineate the inner boundary membrane, cristae, outer membrane, contact sites, and gold particles. This information was used to construct surface-rendered computer graphic representations using the SYNU software package. Gold particles were represented by gold spheres in the surface-rendered volume.
To determine whether immunogold-labeled tBid is spatially clustered or randomly located about contact sites, a spatial point pattern statistics was used, namely, the nearest neighbor statistic . The Euclidean distances from each immunogold particle to all of the contact sites in the reconstruction was calculated for all the particles using a similar approach to that described by Perkins and coworkers . The nearest neighbor statistic was calculated for the reconstruction using the closest observed contact site to each gold particle and then calculating the mean and standard deviation for all such measurements in a reconstructed mitochondrion. These values were compared with those derived from simulations in which the same number of gold particles determined empirically were randomly placed on the mitochondrial surface.
Mitochondrial Isolation Buffer
PAGE-Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis
- tBid – Truncated Bid:
the C-terminal portion of Bid after cleavage by caspase-8.
A point mutation changing glycine 94 to glutamic acid in three-myc Bid.
We thank the reviewers and Tom Deerinck for their helpful comments. X.W. was supported by Grant #0050769Y. Part of this work was performed at the National Center for Microscopy and Imaging Research supported by NIH Research Resource Grant # RR04050 to M. H. Ellisman and NIH grants R01 NS 14718 to MHE and P01 KD54441 to S. S. Taylor.
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