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. 2008 Dec 26;4(12):e1000248. doi: 10.1371/journal.ppat.1000248

Figure 1. Charge substitutions of μ1 ϕ residues Lys594 and Ile595 diminish apoptosis-inducing capacity of ectopically expressed μ1 and ϕ.

Figure 1

(A) Crystal structure of a μ1 monomer (PBD∶1JMU) [36]. The μ1N, δ, and ϕ domains are indicated in red, yellow, and blue, respectively. The location of a minimal functional region of ϕ (spanning amino acids 582–611) sufficient for apoptosis induction following expression in cells [33] is indicated in dark blue. A black box indicates the position of Lys594 and Ile595. (B) 293T cells were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (C) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type or the indicated point-mutant μ1 protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for μ1 (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the PSTAIR peptide of Cdk1 (lower panel). (D) 293T cells were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Caspase-3/7 activity in cell lysates was quantified 24 h post-transfection. Results are expressed as the mean ratio of caspase-3/7 activity from transfected cell lysates to that from empty vector-transfected cells for triplicate samples. Error bars indicate SD. *, P<0.05 as determined by Student's t-test in comparison to cells infected with wild-type μ1. (E) 293T cells treated with Z-VAD-FMK were transfected with a plasmid expressing either wild-type, K594D, or I595K GFP-tagged ϕ protein. Cell lysates were prepared at 24 h post-transfection, resolved by SDS-PAGE, and immunoblotted using a MAb specific for GFP (upper panel). As a control for protein concentration, the blots also were probed with an antibody specific for the Cdk1 PSTAIR peptide (lower panel).