Proline residues of transmembrane domains determine the sorting of inner membrane proteins in mitochondria

Abstract

Most inner membrane proteins of mitochondria are synthesized in the cytosol and reach the inner membrane using one of two alternative sorting pathways. On the stop transfer route, proteins are arrested during import at the level of the inner membrane. The conservative sorting pathway involves translocation through the inner membrane and insertion from the matrix. It is unclear how the translocase of the inner membrane 23 protein translocation machinery differentiates between the two classes of proteins. Here we show that proline residues in hydrophobic stretches strongly disfavor the translocation arrest of transmembrane domains (TMDs) and favor the transfer of preproteins to the matrix. We propose that proline residues, together with the hydrophobicity of the TMD and the presence of charged residues COOH-terminally flanking the TMD, are determinants of the intramitochondrial sorting of inner membrane proteins.

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 on the top. Monotopic proteins, which are sorted by the stop transfer pathway, are shown on the left. Import along the conservative sorting pathway was verified experimentally for the proteins shown on the right. Sequences depict the putative TMDs of the proteins. Proline residues are highlighted by black boxes. The minimal and maximal hydrophobicity scores of the segments were calculated as described in the Materials and methods section and are indicated beside the sequences. IM, inner membrane; IMS, intermembrane space. (B) The mean maximal hydrophobicity scores of arrested and transferred TMDs were calculated using window sizes from 5 to 17 residues. The standard deviations are indicated. (C) The numbers of charged residues in a sequence of 5 to 15 residues COOH-terminal of the TMDs were counted. The mean values and standard deviations are depicted. (D) The graph depicts the ratio of the frequencies of specific amino acid residues in the two groups of proteins. For the calculation, the frequency of a given residue (i.e., the number of a given residue divided by the number of all residues) in the transferred TMDs was divided by the frequency of the same residue in arrested domains.

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) Radiolabeled Cox5a precursor protein (pre) was incubated for 5 min at 25°C with isolated mitochondria. The mitochondria were exposed to proteinase K (PK) without or after hypotonic swelling (sw) as indicated. The radioactive bands of the mature form (m) and the protease fragment (frag) generated are depicted by white and black arrowheads, respectively. Radioactive signals were quantified by densitometry and corrected for their specific methionine content. The percentage of arrested (i.e., membrane-inserted) protein was calculated from the ratio of protease-accessible/total imported protein. For comparison, lane 1 shows 10% of the precursor protein used per import reaction. Efficiency of swelling was controlled by Western blotting using antibodies against a protein of the intermembrane space (cytochrome b₂, Cyt b₂) and of the matrix (Mge1). (C–F) Import of the Cox5a derivatives Cox5aΔTM, Cox5a^L104P, Cox5a(Oxa1), and Cox5a(Oxa1^P106L) were performed as described for B. The sequences and minimal and maximal hydrophobicity scores of the hydrophobic domains of the preproteins are shown on top of the panels. (G) The fusion proteins used for B to F were imported into mitochondria. Following swelling and protease treatment, mitoplasts were fractionated by carbonate treatment into a membrane pellet (P) and a soluble fraction (S). The numbers represent the fraction of imported protein found in the membrane pellet.

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 glucose or glycerol and incubated at 30°C for 2 and 3 d, respectively. (B) Δcox5a cells expressing the indicated fusion proteins were grown on galactose medium. Mitochondria were isolated and the enzymatic activities of cytochrome oxidase (COX) and malate dehydrogenase (MDH) were measured. (C) The indicated fusion proteins were expressed in Δarg8 cells on lactate medium. Mitochondria were purified and the activities of Arg8 and MDH were measured.

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 fragment of 27 kD (f₂₇). Translocation arrest at the second TMD and lateral insertion into the membrane is expected to lead to a fragment of ∼19 kD (f₁₉, 2). IM, inner membrane; IMS, intermembrane space. (B–F) Radiolabeled Oxa1, Oxa1(Cox5a), Oxa1(Cox5a^L206P), Oxa1(Cox5a^{L206P L211P}), and Oxa1(Cox5a^{L206Q L211Q}) were imported for 10 min at 25°C into mitochondria and further processed as described for Fig. 2. White arrowheads depict the two protein species that represent products of the conservative sorting pathway. An f₁₉ fragment derived from arrested preprotein is indicated by black arrowheads. (G) Oxa1-deficient yeast mutants were transformed with plasmids expressing the indicated Oxa1 variants. Growth of the cells on fermentable and nonfermentable carbon source was assessed as described for Fig. 3A. (H) Mitochondria were isolated from Δoxa1 mutants expressing the proteins indicated. The outer membrane of the mitochondria was opened by hypotonic swelling, and the resulting mitoplasts were incubated with 100 μg proteinase K on ice. The full-length protein and COOH-terminal fragments (white arrowhead) of Oxa1 were detected by Western blotting of the resulting samples.