Mitochondrial β-barrel proteins are synthesized in the cytosol and therefore must bear targeting signals to direct them to the right organelle. Their bacterial counterparts contain an N-terminal signal sequence that mediates their translocation from the bacterial cytoplasm across the inner membrane. This signal shows some similarity to signal sequences that direct eukaryotic proteins to the ER. During evolution mitochondrial β-barrel proteins lost such an extension, and our results show that indeed bacterial PhoE with a signal sequence is assembled in reduced levels into mitochondria as compared to a construct without this extension. The presence of a signal sequence results in a protein with two competing targeting signals, one for the mitochondria (within the β-barrel domain) and one for the ER (signal sequence). Neither of these signals is dominant, resulting in a dual localization of the protein. Those molecules that reach the mitochondria integrate into the outer membrane in a stable manner. In contrast, we propose a scenario in which the signal sequence directs the other population to the SEC system in the ER, where Sig-PhoE is translocated into the lumen because there is no hydrophobic membrane-spanning segment that stops the translocation. This process is similar to the transport of the protein into the periplasm through the bacterial SEC machinery in the inner membrane (Bos et al.
). Because there is no BAM complex (or eukaryotic equivalent) in the ER, these molecules cannot get assembled into the membrane and remain in the ER lumen. Comparable accumulation of β-barrel precursors is observed in the periplasm of BamA-depleted bacterial cells (Bos et al.
). In the ER lumen, PhoE can become glycosylated and eventually destined for degradation because the yeast cell probably recognizes it as an unfolded, nonfunctional protein. Analogously, unassembled β-barrel precursors are degraded in the bacterial periplasm (Bos et al.
). Taken together, as the signal sequence appears to be counterproductive for the assembly into the mitochondrial outer membrane, these observations provide an experimental explanation for the absence of bacterial-like signal sequences in precursors of modern mitochondrial β-barrel proteins.
Rather than the presence of a linear sequence, it was suggested that the ability of a protein to adopt a membrane-embedded β-barrel-like conformation could be sufficient for its specific targeting to mitochondria (Rapaport, 2003
). Recent results supported this hypothesis by demonstrating that bacterial β-barrel proteins, like PhoE, expressed in yeast cells are targeted to mitochondria, although these proteins show no significant sequence similarity with mitochondrial β-barrel proteins (Walther et al.
). To better understand this putative structural signal, we tested if specific targeting to mitochondria requires a complete β-barrel precursor structure or whether even a fragment of such a structure would be sufficient. For this purpose, we used YadA, a member of the class of trimeric autotransporters that is found only in bacteria. These proteins are synthesized in the cytoplasm as monomers and form β-barrel-like trimers with their membrane-embedded, C-terminal domain. Recent work demonstrated that BamA, similarly to its function in the biogenesis of other β-barrel proteins, interacts directly with YadA and is essential for its membrane integration (Lehr et al.
Our data demonstrate that YadA was exclusively targeted to mitochondria where it formed native trimeric structure. Thus it appears that even fragments of a β-barrel structure are sufficient for the recognition of a β-barrel protein and its correct targeting to mitochondria. The usage of the heterologous expression system can also help to address the yet open question: In which step of the protein biogenesis is the trimeric structure formed? To investigate whether YadA monomers can form a trimeric structure already in the eukaryotic cytosol, we performed cell-free translation experiments using rabbit reticulocyte lysate. Our results suggest that a formation of cytosolic trimer is unlikely because only signals corresponding to monomeric YadA-MA were observed under these conditions (unpublished data).
The finding that YadA-MA is specifically targeted to mitochondria raised this question: Which components of the mitochondrial import machinery are used? The initial interaction between endogenous β-barrel proteins like porin or Tom40 and the general entry gate, the TOM-complex is mediated by Tom20 (Rapaport and Neupert, 1999
; Krimmer et al.
; Yamano et al
). The same appears to be true for β-barrel proteins of bacterial origin (this study and Walther et al.
), but surprisingly we found that this is not the case for YadA-MA. Similarly, Tom70 is also not required for the import of YadA, and even a slight increase in YadA-MA levels was observed in its absence. Tom70 exposes a large domain on the cytosolic surface of the outer membrane. As this receptor is part of the TOM holo complex, this bulky domain can be in the vicinity of the import pore and thus form a steric hindrance for precursor proteins that are translocated via this pore. Thus, for those proteins that are not recognized by Tom70, the absence of this receptor can even result in a slight improvement of their import efficiency. A similar observation was made by Hines et al. regarding the import of CoxIV-dihydrofolate reductase (DHFR) into mitochondria lacking Tom70 (Hines et al
Of note, Tom import receptors are not absolutely required for the translocation in vitro of bona fide mitochondrial precursor proteins. Import can still occur, albeit with low efficiency, after destroying protease-sensitive receptors (Pfaller et al
). The import via this so-called “bypass” route occurs most probably by a direct interaction of the precursor proteins with the Tom40 import pore. Alternatively, Tom22 can function as a secondary receptor and thus might be involved in the recognition of the YadA precursor. The receptor domain of Tom22 was shown recently to be required for the in vitro import of porin. Furthermore, Tom22 and Tom20 were suggested to be involved in the same step or sequential steps in similar import pathways (Yamano et al
). Hence we propose that YadA is recognized on the surface of the organelle either by Tom22 or directly by Tom40. Naturally, these two alternatives are not mutually exclusive.
The finding that the import of YadA-MA is independent of the import receptors could have evolutionary reasons. Whereas the TOB complex is most probably derived from a bacterial translocase, the TOM complex has no bacterial ancestor (Dolezal et al.
) and only three of the TOM-complex components (Tom40, Tom7, and Tom22) are commonly found in eukaryotes (Macasev et al.
). It is thought that the TOM complex developed on the way of converting the endosymbiont into an organelle. Thus, although it is not clear when trimeric autotransporters emerged, it could be hypothesized that the class of these proteins was lost in early eukaryotes before the development of the primary import receptors (Tom20 and Tom70). In such a scenario, there was never a need for the import receptors to recognize such proteins, and thus import of YadA is independent of the two receptors just mentioned. Astonishingly, the evolutionary origin of mitochondria from bacteria allows the organelle to assemble a class of proteins that are not present in modern eukaryotic organisms.
Upon leaving the TOM complex, YadA is probably exposed in its assembly pathway to the IMS as its overall import efficiency is reduced in cells lacking the small chaperones Tim8/Tim13. This reduction, however, is somewhat less significant as compared to that observed for PhoE. One possible explanation of this difference is the smaller size of hydrophobic elements in YadA as compared to those in PhoE. This proposal is supported by a previous report that larger bacterial β-barrel proteins were more dependent on the presence of all five polypeptide-transport-associated (POTRA) domains of Neisseria meningitidis
BamA as compared to small β-barrel proteins (Bos et al.
). From the IMS, precursor molecules of YadA-MA are most likely relayed to the TOB complex, and our results clearly show a strong dependence of YadA-MA assembly on the TOB subunits, Tob55 and Mas37. These findings are in accordance with our previous findings for PhoE the import of which into mitochondria is also severely affected by the deletion of Mas37 or the depletion of Tob55 (Walther et al.
). Although both PhoE and YadA-MA can be assembled by the TOB complex, they probably represent suboptimal substrates for this complex. Hence an efficient membrane integration of these proteins necessitates most likely the presence of a fully functional TOB complex. Therefore in the absence of Mas37, the Tob55-Tob38 subcomplex cannot deal efficiently with bacterial precursors, whereas it can still process mitochondrial β-barrel substrates.
Assembly of mitochondrial β-barrel proteins appears to be facilitated by the presence of a eukaryotic-specific β-signal present in the most C-terminal β-strand (Kutik et al.
). Interestingly we found that mutation of Ser-417 to glycine, a mutation that allows the last β-strand of YadA-MA to resemble the eukaryotic β-signal, led to a much higher stability of the trimer. This mutation enhances also the stability of the bacterially expressed trimeric form of full-length YadA (Lehr et al.
). Nevertheless, wild-type YadA-MA was present in higher steady-state levels than was the mutant construct. Thus it can be speculated that, although β-signal-like sequences improve the final stability of β-barrel proteins, some structural flexibility is actually an advantage in other stages in the assembly pathway of these proteins, most probably in the integration into the lipid core of the membrane.
In conclusion, our findings shed new light on the biogenesis of mitochondrial β-barrel proteins. They demonstrate that rather than a specific linear sequence, the structural information contained in four β-strands is sufficient for it to be recognized and processed by the mitochondrial import machinery.