The Carboxyl-Terminal Region of the Yeast ATPase Inhibitor Is Indispensable for the Stability of the Protein in Mitochondria

Abstract

The role of the carboxyl-terminal region of the yeast mitochondrial ATPase inhibitor was investigated. Three progressive C-terminal deletion mutants of the inhibitor were constructed: (i) Ile58-->end; (ii) Ile51-->end; and (iii) Gln43-->end. The truncated inhibitor was detected in extracts of Ile58-->end mutant yeast cells. For the Ile51-->end mutant, the truncated inhibitor was only detected when the cells were grown on medium containing the membrane-permeable metal chelator, o-phenanthroline, which inhibits mitochondrial proteases. The most greatly truncated inhibitor protein, Gln43-->end, was never detected even in the cells grown in the presence of the metal chelator. The rates of ATP synthesis and hydrolysis in the mutant mitochondria containing the Ile51-->end inhibitor were similar to those in wild type control cells, while the Ile51-->end inhibitor protein was degraded in the cells unless they were incubated in the presence of the chelator. These results indicate that the carboxyl-terminal region of the ATPase inhibitor is not involved in the its inhibitory action on the F1Fo-ATPase, but is required for the stable conformation of the protein which is protected against degradation by proteases.

IEX-1 targets mitochondrial F1Fo-ATPase inhibitor for degradation

“…To test this possibility, the C terminus of IF1 was progressively truncated through a PCR-based strategy (Figure 3a). Similar to what has been described in yeast,20 removal of nine amino residues (IF1-73) at the C terminus did not affect the stability of IF1 in the presence (Figure 3b, lane 2) or absence of the protease inhibitor o-phenanthroline (data not shown). Apparently, the last nine amino residues are dispensable in IF1 stability.…”

“…In support of this, removal of the C terminus of IF1 results in its degradation in yeast 20. IF1 degradation can be inhibited by culture of the cells in a medium supplemented with a mitochondrial protease inhibitor, o-phenanthroline 20. This inhibitor also blocked IF1 degradation in the presence of IEX-1 in mammalian cells (Figure 1d), suggesting a similar protease involved in IF1 degradation between yeast and mammalian cells.…”

“…Although the C-termini of two IF1 molecules interact with each other to form a dimer in uncoupled mitochondria as revealed by the protein crystal structural study of the soluble F1-IF1 complex,8 the C terminus of IF1 may be associated with several protective proteins in the inner mitochondrial membrane in coupled mitochondria 22–24. In support of this, removal of the C terminus of IF1 results in its degradation in yeast 20. IF1 degradation can be inhibited by culture of the cells in a medium supplemented with a mitochondrial protease inhibitor, o-phenanthroline 20.…”

“…We found no alteration in the amount of IF1 mRNA expression, whether or not IEX-1 was co-transfected (data not shown), ruling out the possibility that IEX-1 regulates IF1 transcription directly. Importantly, IEX-1-mediated downregulation of IF1 was completely blocked by the divalent metal ion chelator o-phenanthroline that inhibits mitochondrial ATP-dependent proteases and prevents IF1 degradation in yeast (Figure 1d, lane 4) 20. To corroborate the involvement of an ATP-dependent mitochondrial protease, a known inhibitor of the adenine nucleotide translocator (ANT), carboxyatractyloside, was also tested.…”

“…Previous studies have shown that the C-terminal region of IF1 is not involved in its inhibitory action on the F1Fo-ATPase in yeast, but it is required for protecting the protein against degradation by mitochondrial proteases 20,22…”