The contiguous inner membrane of mitochondria consists of two morphologically distinct subdomains, namely the IBM that parallels the outer membrane and the infoldings of the inner membrane, the CM. A major conclusion from this study is that the m-AAA protease is preferentially localized in the IBM. Likewise, pCcp1, which is a substrate of the m-AAA protease, is enriched in the IBM of mitochondria lacking a functional m-AAA protease. This strongly indicates that the m-AAA protease processes pCcp1 preferentially in the inner boundary membrane.
A tempting explanation of the different protein compositions of the IBM and the CM is based on topological reasoning as the IBM is facing the outer membrane, whereas the CMs are facing each other. This appears to be a likely explanation for the enrichment of the TIM23 complex in the IBM (Wurm and Jakobs, 2006
), because the TIM23 complex interacts at least during protein translocation with the TOM complex in the outer membrane. The results of this study suggest that additional mechanisms determining the predominant localization of proteins in the IBM exist.
On import, the nuclear encoded Ccp1 is inserted into the IBM. Because the precursor form of Ccp1 is loaded with heme in wild-type cells, it could be speculated that the machinery mediating this process might retain the precursor Ccp1 in the IBM. However, the attachment of heme is not a prerequisite for proteolytic processing of Ccp1 (Esser et al., 2002
), and we found that the addition of the heme synthesis inhibitor succinyl acetone to enlarged mitochondria does not influence the localization of pCcp1-GFP (not shown). Furthermore, in wild-type cells, pCcp1 exists only in minute quantities, whereas it accumulates to substantial amounts in mitochondria lacking a functional m
-AAA protease. Hence the stoichiometric occurrence of a binding partner of pCcp1 in Δyta12
cells appears rather unlikely, suggesting that the localization of precursor Ccp1 is determined differently.
One suggestion is that the cristae junctions might restrict the movement of pCcp1 from the IBM to the CM. Also, the preferential localization of the m-AAA protease in the IBM may be explained by such a barrier function of the highly bended inner membrane at the cristae junctions because the m-AAA protease has no known direct or indirect binding partner in the outer membrane. Because mCcp1 is soluble in the IMS and iCcp is only weakly attached to the inner membrane, with a tendency to dissociate into the IMS, it is tempting to assume that mCcp1 and iCcp1 may diffuse unrestrictedly through the openings of the cristae junctions. Because the relative sizes of the CS and the IBMS are not known, a conclusion on a subcompartmentalization of the IMS based on the data presented in this study is not possible; the data are, however, in full agreement with the hypothesis that mCcp1 is uniformly distributed in the IMS of wild-type cells.
The protein distributions between CM and IBM observed in this study are not binary. Rather, we demonstrated enrichment of the m-AAA protease and of the precursor form of Ccp1 in the IBM, but we also found these proteins in the CM, albeit at lower levels. Thus, if the cristae junctions act as gate-keepers, they appear not to be strict barriers, but may allow the passage of some proteins, possibly in a regulated manner.
Most nuclear encoded integral inner membrane proteins are released laterally into the lipid phase of the membrane during the import process. For proteins that are exported from the matrix into the inner membrane, another insertion machinery exists (for reviews see Rehling et al., 2004
; Neupert and Herrmann, 2007
). The major substrates of this machinery are mitochondrial encoded hydrophobic proteins that assemble with nuclear encoded proteins into supercomplexes involved in oxidative phosphorylation. The m
-AAA protease is likely to have a safeguard function in these assembly processes. Thus the preferential localization of the m
-AAA protease in the IBM points to the intriguing idea that the insertion, assembly, and quality control of the proteins of the mitochondrial inner membrane may predominantly take place in the IBM, suggesting that the functional differences of the IBM and the CM are more significant than previously anticipated.