Arrested TMDs typically lack proline residues
For several proteins of the mitochondrial inner membrane of baker's yeast, the topology is known and the intramitochondrial sorting route has been verified experimentally. The hydrophobic segments of these membrane proteins are depicted in A, and have been classified into sequences which are arrested and laterally inserted (left panel) or transferred into the matrix by the TIM23 translocase (right panel). TMDs that are arrested at the level of the inner membrane tend to be of higher hydrophobicity. When the hydrophobicity of a 17-residue wide frame within these sequences was calculated, arrested sequences showed mean lower and upper values of 0.83 and 1.78, respectively, compared with 0.62 and 1.52 for the transferred segments ( A). The same bias of arrested sequences toward an increased hydrophobicity was observed for smaller window sizes ( B). In addition, arrested sequences often were COOH-terminally followed by several charged residues ( C). However, there was a significant variability with respect to hydrophobicity and flanking charges within both groups of sequences. Some arrested TMDs, like those of Dld1 or Sco2, are less hydrophobic than many transferred sequences. This suggests that additional features of the sequences contribute as sorting determinants. Interestingly, arrested and transferred TMDs differed strikingly in their content of proline residues ( D). Proline residues were present in most of the transferred hydrophobic domains, but were almost entirely absent in arrested sequences. The only example of an arrested domain with a proline residue that we found, Yme2, showed an exceptionally high hydrophobicity score.
Figure 1. TMDs of stop transferred and conservatively sorted proteins differ in their content of proline residues. (A) Inner membrane proteins of known topogenesis are listed. The proteins were classified according to their sorting by the TIM23 translocase as sketched (more ...)
Proline residues compromise stop transfer
To assess the significance of proline residues for the intramitochondrial sorting of inner membrane proteins experimentally, we used an in vitro import assay with radiolabeled precursor proteins. We chose Cox5a as a reporter protein that follows a stop transfer route to reach its final topology in the inner membrane ( A, pathway 1). The precursor form of Cox5a ( B, pre) was imported into isolated mitochondria and converted to the mature Cox5a protein, which was inaccessible to externally added protease ( B, m). Upon hypotonic rupturing of the outer membrane (“swelling”), the imported protein was converted almost entirely to a fragment of an apparent size of ~10.5 kD ( B, lane 4). This fragment comprises the matrix and inner membrane part of Cox5a and originates from the membrane-embedded protein species (Glaser et al., 1990
). Because this fragment contains only two of the four methionine residues that are present in the mature Cox5a protein, the amount of arrested protein is underestimated by autoradiography. Quantification and correction for the different methionine content revealed that >90% of Cox5a were arrested during import at the level of the inner membrane. The small amounts of nondigested mature Cox5a protein likely are due to incomplete swelling of the mitochondria.
Figure 2. The introduction of proline residues inactivates the stop transfer function of the TMD of Cox5a. (A) Sorting pathway of wild type Cox5a (1) and of mutants that are missorted to the matrix (2). IM, inner membrane; IMS, intermembrane space. (B) (more ...)
As control for a matrix-targeted protein, we used a Cox5a mutant lacking the TMD. As expected, this protein was completely targeted to the matrix and protected from added protease after swelling ( C).
Remarkably, the introduction of a single proline residue into the TMD of Cox5a led to the mistargeting of >90% of Cox5a to the matrix ( D). From this we conclude that introduction of a single proline residue is sufficient to overrule the stop transfer signal of Cox5a.
Is the exchange of the TMD of Cox5a for that of the conservatively sorted protein Oxa1 sufficient to target Cox5a to the matrix? As shown in E, this chimeric protein (Cox5a(Oxa1)) was imported into the matrix completely. Thus, the critical sorting information for this precursor protein apparently is encoded in its TMD. However, when the proline residue present in the TMD of Oxa1 was replaced by a leucine residue (Cox5a(Oxa1P106L)), the protein was arrested partially ( F). Notably, this latter fusion protein has a significantly lower hydrophobicity score than the arrested construct Cox5aL104P ( D). Thus, the hydrophobicity of a TMD may influence the intramitochondrial sorting of proteins, but does not seem to be the major discriminating factor.
As an additional assay to monitor the intramitochondrial sorting of the various Cox5a variants, we fractionated the protease-treated mitoplasts after the import reaction by alkaline extraction ( G). Cox5a and Cox5a(Oxa1P106L) were found largely in the membrane fraction, consistent with the results of the protease-protection experiment. In contrast, Cox5aΔTM and the proline-containing construct Cox5aL104P fractionated with soluble proteins, again indicating that proline residues in the TMDs disfavor membrane integration.
Proline residues influence protein sorting in vivo
To assess the intramitochondrial sorting of the Cox5a mutants in vivo, we constructed fusion proteins of Cox5a variants with acetylornithine aminotransferase (Arg8). Arg8 is a matrix enzyme that was used to assess the sorting of protein domains in mitochondria in vivo (Steele et al., 1996
). Upon expression in a Δcox5a
strain, only the fusion protein containing the wild-type Cox5a protein allowed for growth on nonfermentable carbon sources ( A) and resulted in considerable levels of cytochrome oxidase activity ( B). To verify that this is due to missorting of the proline-containing Cox5a variant, we measured the Arg8 activities after expression in a Δarg8
strain ( C). No Arg8 activity was found with wild-type Cox5a-Arg8 fusion protein because the Arg8 domain was unstable and degraded (unpublished data). In contrast, upon expression of Cox5aΔTM-Arg8 and Cox5a(L104P)-Arg8, considerable levels of Arg8 activity were detected as Arg8 was localized to the matrix. This suggests that, in vivo like in vitro, the insertion of a proline into the TMD of Cox5a prevented the efficient arrest of the Cox5a(L104P)-Arg8 fusion protein.
Figure 3. The introduction of proline residues into the TMD of Cox5a leads to sorting into the matrix in vivo. (A) Δcox5a mutant cells expressing the indicated proteins were grown to log phase. 10-fold serial dilutions were spotted on plates containing (more ...)
Proline residues determine the sorting route
We then tested the impact of proline residues present in the TMD of the conservatively sorted protein Oxa1 into isolated mitochondria. Oxa1 is imported first into the matrix from where it inserts into the inner membrane ( A, pathway 1) (Herrmann et al., 1997
). Following its import into mitochondria, Oxa1 resided partially in the matrix and mainly was inserted into the inner membrane. The latter species could be converted into specific COOH-terminal fragments of ~27 kD by protease treatment of mitoplasts ( B, f27
Figure 4. The exchange of one hydrophobic domain leads to the arrest of the normally conservatively sorted protein Oxa1. (A) Conservative sorting of Oxa1, a protein containing five TMDs (1). Correct insertion of Oxa1 allows proteolytic digestion to a COOH-terminal (more ...)
To test whether this protein can be redirected to the stop transfer pathway, we replaced the second TMD of Oxa1 by the TMD of Cox5a. We used the second TMD because mature Oxa1 does not contain methionine residues NH2-terminal of its first TMD, so that NH2-terminal fragments of arrested Oxa1 proteins would not be detected by autoradiography. The Oxa1(Cox5a) preprotein was imported efficiently into mitochondria. Upon swelling and protease treatment, a fragment of 19 kD appeared ( C, black arrowhead). The size corresponded well to the calculated 18.6 kD that was expected for an arrested mature Oxa1(Cox5a) species (compare A, f19). This fragment contains 4 out of the 10 methionine residues that are present in mature Oxa1. Quantification revealed that >70% of the imported Oxa1(Cox5a) precursor was arrested at the level of the inner membrane. The insertion of proline residues, but not of glutamine residues, into this chimeric fusion protein restored the conservative sorting of the protein. The insertion of two proline residues reduced the amount of arrested protein from 72.1% to 27.1%, whereas upon insertion of two glutamine residues, 70.1% were arrested at the level of the inner membrane (). Thus, the presence or absence of proline residues determined the intramitochondrial sorting of this membrane protein.
The bacterial homologue of Oxa1, YidC, was shown to tolerate significant changes in its primary sequence without affecting its function. Even mutants in which complete TMDs were replaced remained functional (Jiang et al., 2003
). This inspired us to test whether the different Oxa1 mutants can complement an oxa1
deletion strain. The replacement of the second TMD of Oxa1 by that of Cox5a did not lead to a functional protein. However, the introduction of one proline residue into the hydrophobic segment restored the function of Oxa1 partially, and that of two proline residues led to almost wild-type growth ( G). This suggests that the addition of proline residues also redirects Oxa1(Cox5a) onto a conservative sorting pathway in vivo, and thereby, reestablishes the respiration-competent phenotype ( G). In contrast, the insertion of alanine or glutamine at the same position did not restore respiratory activity. The lack of growth of the Oxa1(Cox5a)-expressing mutant most likely is due to a sorting deficiency of the protein; this is indicated by the lack of a 27-kD proteolytic fragment that is characteristic for the properly inserted protein. However, this fragment was regained by introduction of the proline residues into the TMD of Oxa1(Cox5a) ( H).
Our findings suggest that the presence of proline residues in TMDs is necessary and sufficient to disfavor lateral insertion of proteins into the inner membrane. The presence of proline residues together with the hydrophobicity of the TMD and the presence of following charges serve as crucial sorting information that is deciphered by the TIM23 translocase. The identification of these signals should help to improve predictions on the intramitochondrial sorting routes of individual proteins (see Supplemental material). Consistent with our results, the insertion of proline residues into the hydrophobic sorting sequence of cytochrome b2
compromised its translocation arrest and caused its transfer into the matrix (Beasley et al., 1993
). Interestingly, in an extensive screen in which the impact of individual amino acid residues on the insertion of TMDs into the ER membrane was studied in a systematic way, proline residues counteracted membrane insertion significantly more than it was expected from their hydrophobicity (Hessa et al., 2005
). Thus, the impact of proline residues on the transfer of hydrophobic stretches does not seem to be limited to mitochondria. However, mitochondria apparently use the effect of proline residues in a physiologic reaction to direct inner membrane proteins to two different sorting pathways.
The presence of a proline residue in the first TMD of Oxa1 was critical for its transfer across the inner membrane into the matrix. Interestingly, this proline residue is well-conserved among animal and fungal members of the Oxa1 family (see Table SI). On the contrary, no proline residues are found in most of the first TMDs of bacterial homologues of Oxa1. Possibly, these proline residues appeared during evolution of mitochondria and have been maintained since that time. Proline residues also are present frequently in the first, and always present in the second, TMDs of mitochondrial members of the Yta10/Yta12 family (see Table SII). The strict conservation of the proline residue in the second TMD of Yta10/Yta12 proteins in fungi, animals, and plants indicates its general relevance. However, it is unclear whether this is due to the impact of proline residues on the intramitochondrial sorting, to functional constraints, or both.
The recently resolved three-dimensional structure of the Sec translocase provided exciting insights into the mechanisms by which a protein translocase can catalyze the membrane insertion of hydrophobic segments (Van den Berg et al., 2004
). Further studies will elucidate how the TIM23 translocase deciphers the sorting signals in hydrophobic segments, and manages to function as a channel allowing passage of TMDs and as an insertase that mediates the transfer of such segments into the lipid phase of the inner membrane of mitochondria.