Figure 5
The F1F0-ATP synthase is compromised in HtrA2-deficient cells. (a) ATP production was measured in live cells using a luciferin/luciferase based reporter assay. Immortalised MEFs transfected with cytosolic luciferase were perfused with luciferin (5 μM) followed by ATP (1 mM), which stimulates calcium uptake into mitochondria and therefore ATP production by the ATP synthase. Representative traces are shown for one WT and one KO coverslip. Initial luminescence is normalised to 0 and response to luciferin is normalised to 1. (b) ATP response curves were normalised to luciferin response as described, then for each pair of WT and KO coverslips the KO response was expressed as a percentage of the WT. Data are represented as mean±S.E.M. ** indicates P<0.01 compared with WT. (c) HtrA2 was tagged at the C terminus with a TAP tag consisting of a protein A sequence (for IP with IgG beads) and a calmodulin binding peptide (CBP) sequence (for IP with calmodulin resin) separated by a TEV cleavage site. (d) The F1 α-subunit (cVα) was identified in TAP eluates from HtrA2-TAP cells but not from TAP control cells. WB for HtrA2 shows endogenous processed HtrA2 (37 kDa) in TAP and HtrA2-TAP inputs, full-length and processed TAP-tagged HtrA2 (58 and 69 kDa) in the HtrA2-TAP inputs, and CBP-tagged processed HtrA2 (42 kDa) in the final eluate. * indicates a non-specific band. (e) A 2D-PAGE analysis of protein expression in WT and HtrA2-KO MEFs indicated a significant reduction in the peak corresponding to the α-subunit of the ATP synthase (60 kDa), and the appearance of a further peak identified by mass spectrometry as a truncated form of this subunit (41 kDa). The identified fragments are highlighted on the full length sequence of the α-subunit in red. In the truncated subunit good sequence coverage was obtained for the C-terminus but not the N-terminus of the protein, suggesting that truncation may remove the MTS (grey box)
