Active Bax and Bak are functional holins

Abstract

The mechanism of Bax/Bak-dependent mitochondrial outer membrane permeabilization (MOMP), a central apoptotic event primarily controlled by the Bcl-2 family proteins, remains not well understood. Here, we express active Bax/Bak in bacteria, the putative origin of mitochondria, and examine their functional similarities to the λ bacteriophage (λ) holin. As critical effectors for bacterial lysis, holin oligomers form membrane lesions, through which endolysin, a muralytic enzyme, escapes the cytoplasm to attack the cell wall at the end of the infection cycle. We found that active Bax/Bak, but not any other Bcl-2 family protein, displays holin behavior, causing bacterial lysis by releasing endolysin in an oligomerization-dependent manner. Strikingly, replacing the holin gene with active alleles of Bax/Bak results in plaque-forming phages. Furthermore, we provide evidence that active Bax produces large membrane holes, the size of which is controlled by structural elements of Bax. Notably, lysis by active Bax is inhibited by Bcl-xL, and the lysis activity of the wild-type Bax is stimulated by a BH3-only protein. Together, these results mechanistically link MOMP to holin-mediated hole formation in the bacterial plasma membrane.

Figure 1.

An active Bax mutant displays a holin-like bacterial lysis activity. (A) Diagram of Bax and miniBax. (B) Behavior of miniBax, Bax, and S105 in the pET system. BL21(DE3)pLysS E. coli transformed with the pET24b vector or pET24b constructs expressing F-miniBax, F-Bax, or S105-Flag were grown to A₅₅₀ ∼0.3. Following the addition of IPTG (0.3 mM), A₅₅₀ was measured at 5-min intervals. After 1 h, the cultures were harvested and subjected to SDS-PAGE, followed by Western blot with an α-Flag antibody.

Figure 2.

MiniBax functionally replaces bacteriophage holin (S105), causing endolysin- dependent lysis of Δ(SR) cells. (A) Diagram for the construction of the pS105-based isogenic plasmid replacing S105 with F-miniBax. The DNA segment encoding the first 92 amino acids of S105 in the S105/R lysis cassette within the plasmid pS105 was replaced by the reading frame of F-miniBax. (B) Lysis of Δ(SR) cells by the miniBax/R cassette. The plasmid pSam7 is identical to pS105 except for an Amber mutation in the S105 gene that abolished S105 expression. Upon reaching A₅₅₀ ∼0.3, the Δ(SR) cells carrying the indicated plasmids were induced by thermal induction for 15 min at 42°C, followed by aeration at 37°C. A₅₅₀ was measured for each culture at 5-min intervals. The cultures were harvested as described in the Materials and Methods and subjected to SDS-PAGE and Western blot with an α-Flag antibody. (C) Diagram of the lysis cassettes of S105 and F-miniBax with or without functional endolysin (R). (D) Lysis phenotypes of the lysis cassettes listed in C in Δ(SR) cells. Expression of the indicated proteins was detected by Western blot with an α-Flag antibody. (E) Diagram of truncation mutants of miniBax. These miniBax mutants were cloned into the pS105 plasmid by the same strategy as shown in A. (F) Lysis induced by miniBax and its truncation mutants in Δ(SR) cells was measured the same way as in B. The expression of these mutants was detected by Western blot with an α-Flag antibody. (G) Δ(SR) cells carrying either pS-F-miniBax or pS-F-miniBaxH3A were monitored by time-lapse microscopy. Still images of the cells undergoing lysis at different time points are shown. The real-time videos are provided in Supplemental Movies S1 and S2.

Figure 3.

Dependence of the bacterial lysis activity of miniBax on homo-oligomerization. (A) Lysis phenotypes of miniBax and its two BH3 mutants, miniBaxL63E and miniBaxL63P, in Δ(SR) cells. The expression of these proteins was detected by Western blot as carried out in Figure 2B. (B) Homo-oligomerization of miniBax and its mutants. BMH cross-linking was carried out as described in the Materials and Methods, followed by SDS-PAGE and Western blot analysis with an α-Flag antibody. The asterisk (*) stands for nonspecific protein.

Figure 4.

Lysis of Δ(SR) cells by active mutants of Bax or Bak, but not by any other Bcl-2 family protein. (A) Diagram for the replacement of the first 92 amino acids of S105 by Bcl-2 family proteins in the S105/R lysis cassette in pS105. (B) Lysis curves for the BH3-only proteins were measured as in Figure 2B. (C) Expression of the indicated BH3-only proteins in Δ(SR) cells. Protein samples from cells expressing F-Bid, F-BimEL, F-Bmf, and F-Bik were diluted 1:10. Protein samples from F-Bnip3 and F-Noxa were diluted 1:5. The asterisk (*) stands for nonspecific protein. (D) Lysis phenotypes of the Bax/Bak and their mutants in Δ(SR) cells. (E) Expression of Bax/Bak and their mutants. Due to low expression, the protein sample from F-BakΔH1-expressing cells was concentrated fourfold. The asterisk (*) stands for nonspecific protein. Diagrams of miniBax, miniBak, and BakΔH1 are shown at the bottom. (F) Lysis phenotypes of anti-apoptotic Bcl-2 family proteins. (G) Expression of the indicated anti-apoptotic Bcl-2 family proteins. Protein samples from cells expressing these proteins were diluted 1:20 as compared with F-miniBax. A diagram of a truncation mutant of Bcl-xL, “miniBcl-xL,” is shown at the bottom.

Figure 5.

Replacement of the holin gene by functional Bax/Bak mutants generates viable chimeric λ phages. (A) Diagrams of Bax and Bak mutants. These mutants were cloned into the pS105 plasmid as shown in Figure 2A to replace S105. (B) Lysis phenotypes of the indicated Bax/Bak mutants. Expression of the indicated Bax and Bak mutants in Δ(SR) cells was detected by Western blot with an α-Flag antibody as carried out in Figure 2B. (C) Plaque formation by Bax/Bak mutants. At 75 min, the cultures were centrifuged at 22,000g for 10 min, and the supernatant was assayed for plaque formation using the MC4100ΔtonA as indicator cells as described in the Materials and Methods.

Figure 6.

Sizing the membrane holes induced by active Bax mutants through the R-LacZ fusion proteins. (A) Diagram of the various lysis cassettes with LacZ or its truncation mutants fused to the reading frame of R. The R-LacZ fusion protein has a Myc tag on the N terminus. R-LacZt1 is the fusion between R and the first 497 amino acids of LacZ. R-LacZt2 is the fusion between R and the first 8 amino acids of LacZ. (B) Lysis curves of the different lysis cassettes listed in A in Δ(SR) cells. (C) Diagram of the lysis cassettes expressing Bax mutants in conjunction with R-LacZ. (D) Lysis curves of the indicated Bax mutants with R-LacZ cassettes in Δ(SR) cells. (E) Δ(SR) cells carrying the indicated plasmids were thermally induced. At the indicated time points, 1-mL samples of the culture were collected and pelleted by centrifugation at 22,000g at 4°C. The supernatant (S) and the pellet (P), which was resuspended in 1 mL of EBC buffer, were loaded onto SDS-PAGE followed by Western blot analysis with an α-Myc antibody.

Figure 7.

Regulation of Bax-mediated bacterial lysis by Bcl-xL and tBid. (A) Suppression of miniBaxH3A-mediated bacterial lysis by Bcl-xL. Lysis curves of F-miniBaxH3A lysogens carrying the indicated plasmids were measured at a 15-min interval. The expression of Bcl-xL and Bcl-xLmt8 at 15 min was detected using an α-Bcl-xL antibody, and the expression of miniBaxH3A was detected using an α-Flag antibody. (B) Suppression of BaxΔH6-mediated bacterial lysis by Bcl-xL. (C) Lysis phenotypes of sam7 lysogen carrying the indicated plasmids. The expression of tBid, its mutants, and Bax at 60 min was detected by Western blot with an α-Flag antibody. (D) Lysis phenotypes of F-Bax lysogen carrying indicated plasmids. (E) A diagram depicting the functional homology between MOMP and hole formation in bacterial membrane.