Translocation of the hydrophilic N-tail and C-tail domains of yeast Cox2 from the mitochondrial matrix to the IMS is carried out by different mechanisms. N-tail export of pre-Cox2 is strictly dependent upon the integral membrane protein Oxa1 but does not require a potential difference across the inner membrane (21
). While export of the C-tail depends upon Oxa1, it also requires the integral membrane protein Cox18, the peripheral membrane protein Mss2 (Pnt1 assists these proteins but is dispensable), and a membrane potential (6
). While Oxa1 appears to have a role in the membrane insertion of several inner membrane proteins (24
), the interacting proteins Cox18 and Mss2 appear to be specific for Cox2 C-tail translocation (6
). Yeast mitochondria do not possess an identifiable translocation system homologous to the bacterial Sec apparatus (17
). However, the Cox2 C-tail translocation system can export a large passenger protein, Arg8m
, fused to the Cox2 C terminus (21
Oxa1 appears to have two functional domains (41
). Membrane protein insertion is carried out by the N-terminal polytypic integral membrane segment of Oxa1, which is homologous to bacterial YidC as well as yeast Cox18. The C-terminal domain of Oxa1 is exposed on the inner surface of the mitochondrial membrane and interacts with mitochondrial ribosomes, apparently to promote cotranslational targeting of nascent proteins (28
). Both Cox18, which is functionally conserved among eukaryotes (14
), and YidC lack this C-terminal domain. Fusion of this domain of Oxa1 to the C terminus of bacterial YidC generates a chimeric protein that can partially compensate for the absence of normal Oxa1 in yeast, while unmodified YidC can partially compensate for the absence of Cox18 (41
). These observations suggest that direct interaction with the ribosome presents the N terminus of pre-Cox2 to the translocase domain of Oxa1 but that other functions may be required to present the Cox2 C-terminal domain to Cox18.
Mss2 is exposed on the inner surface of the inner membrane and thus could function to recognize the Cox2 C-tail prior to translocation by Cox18 (6
). Consistent with this idea, Mss2 has a tetratricopeptide motif (6
) similar to one in the mitochondrial import receptor Tom70, which binds cytoplasmic chaperone-associated translocation substrates (9
). We observed here that newly synthesized full-length Cox2 could be coimmunoprecipitated with Mss2 from solubilized mitochondria, consistent with the idea that Mss2 recognizes the Cox2 C-tail prior to export. The coprecipitated Cox2 was processed at its N terminus, demonstrating that N-tail export can occur while Cox2 is still bound, directly or indirectly, to Mss2 on the inner surface of the inner membrane. Thus, C-tail translocation may be a posttranslational event that can occur subsequent to cotranslational N-tail export and N-terminal processing. Mss2-Cox2 coprecipitation was not observed when the entire C-tail domain was completely deleted, consistent with the possibility that Mss2 might bind to the C-tail. If so, Mss2 could hold the C-tail domain of Cox2 in an unfolded conformation, or in an intermediate folded conformation (51
), required for translocation after translation termination. (The N-tail domain of truncated Cox2 lacking the C-tail was not processed during synthesis in isolated mitochondria, making it formally possible that interaction with Mss2 depends on N-terminal processing rather than the absence of the C-tail per se.)
To explore the features of Cox2 that are required for membrane insertion and translocation, we studied the behavior of truncated Cox2 variants bearing an epitope tag at their C termini, encoded in mtDNA. The N-tail of a protein lacking the entire C-tail domain, Cox2(1-109)-HA, was efficiently translocated across the inner membrane during pulse-labeling of whole cells in a reaction that was dependent upon Oxa1 but not Cox18 or Mss2. Interestingly, N-tail export of the Cox2(1-109)-HA N-tail did not occur during pulse-labeling of isolated mitochondria, demonstrating that the isolated organelles are deficient in some feature of this process relative to intact cells.
Unlabeled Cox2(1-109)-HA accumulated (albeit in greatly diminished amounts relative to wild-type Cox2) with both its N terminus and epitope-tagged C terminus on the outside of the inner membrane. Thus, insertion of the two transmembrane domains of Cox2, and export of the C-terminal epitope, can occur in the absence of the C-tail. Both the N and C termini of Cox2(1-109)-HA accumulated in the matrix in an oxa1Δ
mutant. Since previous work indicated that Cox18 and Mss2 are required only for C-tail translocation, we expected export of the Cox2(1-109)-HA N-tail to be unaffected in cox18Δ
mutants. However, both mutations caused accumulation of detectable levels of Cox2(1-109)-HA, whose N-tail was in the matrix (cox18Δ
more so than mss2Δ
). This could indicate either a direct but minor role for Cox18 and Mss2 in N-tail export or a general inefficiency of a larger translocation complex lacking either protein, in either case leading to accumulation of species not detected during pulse-labeling. Similarly, while most of the C-terminal epitope of Cox2(1-109)-HA was exported in both mutants, demonstrating that Cox18 and Mss2 are not required for insertion of the second transmembrane domain, both mutations clearly reduced export of the C terminus relative to wild type. In this context it is important to note that the addition of the 33-residue hydrophilic epitope tag to the C terminus of the second transmembrane domain at residue 109 may reduce the efficiency of its insertion. This insertion is completely blocked by the presence of a 402-residue hydrophilic passenger protein moiety (Arg8m
) fused at the same position (21
We also examined features of the 144-residue Cox2 C-tail domain that could contribute to its recognition as a translocation substrate. A truncated variant of Cox2 lacking the 40 C-terminal residues of this domain [Cox2(1-211)-HA] accumulated in mitochondria with its N-tail exported to the IMS and its C terminus remaining in the matrix. Thus, it is clear that sequence and/or structural features of the C-tail domain itself are recognized by its translocation apparatus independently of N-tail translocation. Recognition of these features in the C-tail can also lead to translocation of a fused passenger protein (21
). Despite the fact that the truncated C-tail of Cox2(1-211)-HA is not translocated, this variant protein nevertheless did coimmunoprecipitate with Mss2. Thus, while the Cox2-Mss2 interaction is highly likely to be necessary for C-tail export, recognition of the C-tail as a translocation substrate must involve other steps as well.
Three of the four copper-ligating residues of the cytochrome c
oxidase CuA site are absent from the truncated protein Cox2(1-211)-HA. While copper insertion is thought to occur in the IMS after export (7
), the mitochondrial matrix has an accessible nonproteinaceous copper pool (10
) that could provide metal ions to the C-tail domain. However, we found that point mutations altering the copper-binding residues C225 and H229 (corresponding to C200 and H204 from the bovine Cox2 sequence) (49
) are not required for efficient C-tail translocation. Furthermore, Sco1, a mitochondrial protein required for copper delivery to Cox2 (37
), is not required for C-tail translocation (45
). Thus, a fully folded C-tail domain containing copper ions is not required for C-tail translocation.
Both the exported 22-residue N-tail and 144-residue C-tail domains of Cox2 are negatively charged, with predicted pI values of 3.8 and 4.4, respectively. Thus, these translocation systems adhere to the “positive-inside rule” (15
). However, while charge may play a role in driving the Cox2 C-tail across the membrane in a potential-dependent fashion, neither acidity nor the charged residues immediately flanking the transmembrane domains are sufficient to direct this process, since the acidic C-tail domain of the truncated variant Cox2(1-211)-HA (pI 4.0) is not translocated.
The molecular mechanism(s) of bacterial YidC has been studied extensively but remains to be precisely defined (reviewed in reference 32
). YidC is at least partially associated with the bacterial Sec translocase, although it is not required for most Sec-dependent reactions. YidC is necessary for the assembly of the Escherichia coli
respiratory chain (54
). Plasma membrane insertion of the E. coli
protein CyoA, a subunit of the bacterial cytochrome bo
oxidase that is similar to mitochondrial Cox2, also depends upon distinct mechanisms for insertion of its N- and C-terminal domains into the membrane (8
). In this case, the Oxa1/Cox18 homolog YidC is required for translocation of the N-terminal domain, while the C-terminal domain is translocated by the Sec translocase. While cells depleted for YidC do not translocate either domain of the wild-type protein, this is due to the fact that prior YidC-dependent translocation of the N-terminal sequences and insertion of the transmembrane domains are a prerequisite for Sec-dependent translocation of the C-terminal domain (8
). Interestingly, both translocation reactions are independent of membrane potential when carried out in vitro in proteoliposomes (12
). Thus, there are both similarities and clear differences between the yeast mitochondrial and bacterial systems: in both cases the early events are catalyzed by Oxa1/YidC, while in mitochondria the Cox2-specific functions of Cox18/YidC and Mss2 have taken the place of the Sec translocon.