Mutagenesis of the Bak Membrane Anchor Figure 5. A TM:groove interaction forms in cytosolic Bax and in cytosolic Bak/BaxCS. (A) Schematic of Bax converting from a TM:groove to a TM:membrane conformation during apoptosis. Bax is shown as surface representation (orange) with the hydrophobic surface groove highlighted (white). The Bax TM domain is represented (blue) and the C-segment (KKMG) has the sidechains shown. Residues proposed to interact during TM:groove interaction are highlighted (S184 and D98; red). Images were generated in Pymol using the RCSB Protein Data Bank file 1F16 for Bax [38]. (B) Mutations in both the TM domain and the groove alter mitochondrial targeting. Cytosolic and membrane fractions from bak2/2bax2/2 MEFs expressing the indicated Bax or Bak/BaxCS cysteine mutants were immunoblotted for Bax, Bak/BaxCS, or HSP70. (C) 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 (upper) or reducing conditions (+2ME, lower) 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 (X-link). (D) 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. (E) 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. (F) 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. doi:10.1371/journal.pone.0031510.g005 PLoS ONE | www.plosone.org 7 March 2012 | Volume 7 | Issue 3 | e31510