Basic residues in the C-segment are necessary for Bak stability and function.

<p>(<b>A</b>) C-terminal sequence of Bak variants indicating which basic residues in the C-segment that were substituted with serine. (<b>B</b>) Substitution of basic residues in the C-segment reduces Bak proapoptotic function. <i>bak^−/−bax^−/−</i> MEFs expressing Bak, BakRRS, BakRSS or BakSSS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; *p<0.05, **p<0.01. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (<b>C</b>) Substitution of basic residues in the C-segment destabilizes Bak. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakSSS, 4-fold total protein was loaded onto the gels. (<b>D</b>) Substitution of basic residues does not prevent targeting to membranes. Cells from (B) were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and for the cytosolic marker HSP70. Results are representative of two or more independent experiments.</p

The Bak C-segment is essential for stability, membrane targeting and proapoptotic function.

<p>(<b>A</b>) C-terminal sequence of Bak variants truncated at the C-terminus. The C-terminus (CT) contains a hydrophobic transmembrane (TM) domain and a hydrophilic C-segment (CS). (<b>B</b>) Truncation of the Bak C-terminus or C-segment blocks proapoptotic function. <i>bak^−/−bax^−/−</i>MEFs expressing Bak, BakΔCT and BakΔCS were left untreated, or treated with UV or etoposide for 24 h. Percentage cell death is expressed as the mean ± SEM from three independent experiments. Statistical significance for treatment when compared to Bak; ***p<0.001. Upper panel is a western blot of cell lysates immunoblotted for Bak, and for β-actin as a loading control. (<b>C</b>) Truncation of the C-segment reduces half-life. Cells from (B) were incubated with cycloheximide for up to 24 h and cell lysates immunoblotted for Bak, and for β-actin as a loading control. Note that due to low expression of BakΔCS, 4-fold total protein was loaded onto the gels. (<b>D</b>) Truncation of the Bak C-terminus prevents membrane targeting. Cells were left untreated or treated with UV, separated into cytosolic and membrane fractions, and immunoblotted for Bak and the cytosolic marker HSP70. (<b>E</b>) Bak lacking the C-terminus fails to undergo conformation change and oligomerization in response to UV. Cells treated as in (D) were exposed to oxidant (CuPhe), separated into cytosolic and membrane fractions, run on non-reducing SDS-PAGE and immunoblotted for Bak. M_X, non-activated intramolecular cross-linked monomer; M, non-crosslinked monomer; D, intermolecular crosslinked dimers. Results are representative of two or more independent experiments.</p

Bak regulation is independent of initial subcellular localization.

<p>(<b>A</b>) Schematic of Bak-mediated apoptosis initiated by Noxa-Mcl-1 signalling in MEFs. The four prosurvival proteins expressed in MEF (Mcl-1, Bcl-x_L, Bcl-w and Bcl-2) are depicted, together with their preferential binding of BH3-only proteins (Noxa, Bim and Bad) and of Bak and Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Willis1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Chen1" target="_blank">[46]</a>. The Noxa-Mcl-1-Bak pathway to apoptosis is indicated (<i>bold</i>). (<b>B</b>) Expression of Bim_S^NOXA preferentially mediates apoptosis via either Bak or Bak/BaxCS. <i>bak^−/−bax^−/−</i>MEFs expressing the indicated Bak and Bax variants were retrovirally infected with Bim_S or with Bim_S containing the Bad or Noxa BH3 domains (Bim_S^BAD and Bim_S^NOXA). Percentage cell death at 36 h (normalized to the efficiency of infection) is expressed as the mean ± SEM of three independent experiments. Statistical significance for treatment when compared to Bak; **p<0.01.</p

Bak/BaxCS translocates to mitochondria and following tBid releases cytochrome <i>c</i>.

<p>(<b>A</b>) Cytosolic and mitochondrial Bak/BaxCS both contribute to permeabilization of MEF mitochondria. Cytosol and membrane fractions from <i>bak^−/−bax^−/−</i> MEFs or from <i>bak^−/−bax^−/−</i> MEFs expressing either Bak or Bak/BaxCS were combined as shown, and incubated at 30°C in the presence of 100 nM tBid for 0, 30 or 60 mins, or without tBid for 60 mins (60-). Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. (<b>B</b>) Cytosolic Bak/BaxCS is sufficient to permeabilize mouse liver mitochondria. MEF cytosol fractions derived as in (A) were combined with mitochondria isolated from wild-type (<i>bak^+/+</i>) or <i>bak^−/−</i> mouse liver, and incubated at 37°C with or without 100 nM tBid for 60 min. Supernatant (<i>Cyt</i>) and membrane (<i>Memb</i>) fractions were immunoblotted for cytochrome <i>c</i> and for Bak. Note that Bak/BaxCS levels appear high compared to the endogenous mouse Bak, possibly due to different recognition by the anti-Bak antibody. (<b>C</b>) Mcl-1 is higher in MEF membranes than in mouse liver mitochondria. MEF membranes and mouse liver mitochondria at the concentrations used in (A) and (B) were examined for levels of Mcl-1, Bcl-x_L and VDAC1 by immunoblotting. Results are representative of two or more independent experiments.</p

A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS.

<p>(<b>A</b>) Schematic of Bax converting from a TM:groove to a TM:membrane conformation during apoptosis. Bax is shown as surface representation (<i>orange</i>) with the hydrophobic surface groove highlighted (<i>white</i>). The Bax TM domain is represented (<i>blue</i>) and the C-segment (KKMG) has the sidechains shown. Residues proposed to interact during TM:groove interaction are highlighted (S184 and D98; <i>red</i>). Images were generated in Pymol using the RCSB Protein Data Bank file 1F16 for Bax <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031510#pone.0031510-Suzuki1" target="_blank">[38]</a>. (<b>B</b>) Mutations in both the TM domain and the groove alter mitochondrial targeting. Cytosolic and membrane fractions from <i>bak^−/−bax^−/−</i> MEFs expressing the indicated Bax or Bak/BaxCS cysteine mutants were immunoblotted for Bax, Bak/BaxCS, or HSP70. (<b>C</b>) A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS. Cytosolic and membrane fractions from cells in (B) were incubated with oxidant (CuPhe) and electrophoresed under non-reducing conditions (<i>upper</i>) or reducing conditions (+2ME, <i>lower</i>) and immunoblotted for Bax or Bak/BaxCS. Intramolecular cysteine linkage (D98C:S184C in Bax; S117C:Q202C in Bak/BaxCS) results in faster migration under non-reducing conditions (<i>X-link</i>). (<b>D</b>) Etoposide treatment decreases TM:groove interaction in membrane-associated Bak/BaxCS. Cells expressing the Bak/BaxCS S117C/Q202C variant were incubated with or without etoposide for 24 h. Cytosolic and membrane fractions were incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>E</b>) tBid treatment decreases the TM:groove interaction in membrane-associated Bak/BaxCS. Cells in (D) were permeabilized and incubated with or without tBid (100 nM). Cytosolic and membrane fractions were separated and then incubated with CuPhe, electrophoresed under non-reducing conditions, and immunoblotted for Bak. (<b>F</b>) The TM domain of Bak/BaxCS inserts into membranes following tBid. Permeabilized cells were treated with or without tBid as in (E), then incubated with or without IASD. Membrane fractions were electrophoresed under reducing conditions and immunoblotted for Bak. Results are representative of two or more independent experiments.</p

<i>In vitro</i> analysis of m41 mutant viruses.

<p>(<b>A</b>) Fibroblasts were infected with WT, Rev or the indicated MCMV mutants and total cell lysates prepared 24 hr later. Immunoblot analysis was performed using antibodies specific for m41, m41.1 or IE1 as indicated. (<b>B</b>) Fibroblasts were infected with the indicated viruses (MOI = 3) and 18 h later 100 µM etoposide added. Cell viability was quantified by Trypan Blue exclusion 24 hr after the addition of etoposide (n = 6). (<b>C</b>) IC-21 macrophages were infected with the indicated viruses (MOI = 3) and cell viability assessed 48 hr later (n = 8). (<b>D</b>) Fibroblasts or (<b>E</b>) IC-21 macrophages were infected with WT MCMV (filled square), Rev (filled circle), Δm41 (open circle), Δm41.1 (cross) or Δm41/m41.1 (open diamond) (MOI = 0.05 for fibroblasts and MOI = 0.5 for IC-21) and viral replication measured at the indicated times pi (n = 6 for fibroblasts and macrophages). Dotted line indicates the limit of detection of the assay.</p

m41.1 enhances viral replication in leukocytes.

<p>(<b>A</b>) Whole blood was isolated from infected mice on day 5 pi., and red blood cells removed by isotonic lysis. The proportion of infected cells was determined by infectious centre assay. (<b>B</b>) Mice were infected with WT MCMV or the Δm41.1 mutant and spleens removed on day 4 pi. Viral burden in stromal cells (left panel), or leukocytes (right panel) were determined by plaque assay.</p

m41.1 encodes a Bak-specific inhibitor.

<p>(<b>A</b>) The predicted amino acid sequences of m41 and m41.1 in the K181-Perth MCMV strain are shown. (<b>B</b>) <i>In vitro</i> transcription/translation reactions using cDNA constructs encoding m41 or m41L were performed, the resulting protein products were separated by SDS-PAGE and detected by autoradiography. (<b>C</b>) Fibroblasts derived from WT, Bax- or Bak-deficient mice were infected with retroviruses encoding the indicated proteins. Cells were treated with 10 µM staurosporine for 24 hr and cell viability assessed. (n = 6).</p

Loss of m41.1 or m41 impairs viral replication <i>in vivo</i>.

<p>(<b>A</b>) BALB/c mice were infected with WT MCMV (filled square), Rev (filled circle) or Δm41.1 mutant (open diamond), organs were removed at the indicated times pi and viral load determined by plaque assay, mean ± S.D. of 5–6 mice per time point is plotted. ** <i>P</i><0.01, *** <i>P</i><0.0001. Dotted line indicates the limit of detection of the assay. (<b>B</b>) BALB/c mice were infected with WT MCMV (filled square), Rev (filled circle) or the Δm41 mutant (cross), organs removed at the indicated times pi and viral load determined by plaque assay. Mean ± S.D. of 5–6 mice per time point is plotted. ** <i>P</i><0.01. Dotted line indicates the limit of detection of the assay.</p

Genomic arrangement and analysis of the m41 locus.

<p>RNA isolated from MCMV infected fibroblasts at IE, E and L times post-infection was subjected to (<b>A</b>) 5′ RACE or (<b>B</b>) 3′ RACE analysis. The resulting products were separated on 1% agarose gels. (<b>C</b>) The genomic region of MCMV encompassing the annotated m41 ORF (solid arrow), the newly identified 41 exon (solid box), and adjoining ORFs (open arrows) is shown. Direction of transcription is indicated by the orientation of the arrows. Location of the common polyadenylation site used by all genes in this region is denoted by the filled oval. (<b>D</b>) Sequence of the annotated m41 ORF is shown. The annotated m41 translation start site and the location of splice acceptor and donor sites are shown above the DNA sequence. Location of mutations within the m41 ORF are shown below the DNA sequence. (<b>E</b>) The indicated m41 constructs were transiently overexpressed in Cos-7 cells, total cell lysates prepared and expression of the m41 and m41L proteins detected by immunoblot using anti-flag antibodies. (<b>F</b>) Total cell lysates were prepared from fibroblasts infected with MCMV at the indicated time pi and the expression of m41, m41L, and IE1 proteins detected by immunoblot.</p