Association of Tim50 with the TIM23 Complex
Previous work suggested Tim23 to be the binding partner of Tim50 in the TIM23 complex (Geissler et al., 2002
; Yamamoto et al., 2002
; Mokranjac et al., 2003a
; Alder et al., 2008b
). It remained unclear, however, whether Tim23 is the only interaction partner of Tim50 within the complex. To gain more insight into the association of Tim50 with the TIM23 complex, we performed coimmunoprecipitation experiments using wild-type yeast mitochondria. Mitochondria were lysed with digitonin and subjected to immunoprecipitation with affinity-purified antibodies to Tim17, Tim23, and Tim50. Tim50 was equally well precipitated with antibodies to Tim17 and to Tim23 (A). In addition, the efficiencies of precipitation of Tim17 and Tim23 with antibodies to Tim50 were essentially the same. This suggested that Tim17 may be an additional binding partner of Tim50 in the TIM23 complex. To analyze whether there is a subcomplex between Tim17 and Tim50 in the absence of Tim23, we isolated mitochondria from yeast cells depleted of Tim23. Depletion of Tim23 did not influence the levels of Tim17 and Tim50; however, a stable subcomplex between these two proteins was not observed upon coimmunoprecipitation from digitonin-solubilized mitochondria (B). In contrast, the subcomplex consisting of Tim23 and Tim50 was previously found to be stable in the absence of Tim17 (Mokranjac et al., 2003b
). Also, depletion of Tim44, Tim14, or Tim16 had no effect on the association of Tim50 with Tim17-Tim23 (Mokranjac et al., 2003a
) nor did deletions of Pam17 and Tim21 (Popov-Čeleketić et al., 2008
). This suggests that the only stable interaction of Tim50 in the TIM23 complex is to Tim23, at least when analyzed by this method.
Figure 1. Association of Tim50 with the TIM23 complex. Wild type (A) or mitochondria depleted of Tim23 (B) were solubilized with digitonin and incubated with affinity-purified antibodies to Tim17, Tim23, or Tim50 or antibodies from preimmune serum (PI) bound to (more ...)
We further studied the assembly of the TIM23 complex in the absence of Tim50. In lysates of wild-type mitochondria antibodies to Tim16, Tim17, Tim23, and Tim50 precipitate all known components of the complex, however, with different efficiencies due to the reported instability of the complex upon solubilization (C). When the same experiment was performed with mitochondria from cells depleted of Tim50, all subunits of the complex analyzed were precipitated with antibodies to Tim16, Tim17, and Tim23 with virtually same the efficiency as in wild type. As expected, only residual amounts of Tim50 and none of the other TIM23 subunits were precipitated with antibodies to Tim50, confirming the specificity of precipitation. Thus, the assembly of the TIM23 complex appears to be largely unaffected in the absence of Tim50.
In summary, Tim23 appears to be the major component that recruits Tim50 to the TIM23 complex. The lack of Tim50 does not greatly affect the assembly of the rest of the translocase.
Domain Analysis of Tim50
To analyze the contributions of the various segments and domains of Tim50 to its function, we generated several truncation mutants (A). The first mutant, denoted Tim50Δmatrix, lacked the matrix localized N-terminal segment in front of the transmembrane domain. In the second mutant, DLDTim50, we exchanged the N-terminal and the transmembrane segments of Tim50 with the corresponding segments of yeast d-lactate dehydrogenase. The third mutant, b2Tim50, consisted of first 167 residues of yeast cytochrome b2 fused to the IMS domain of Tim50 (residues 132–476). In this mutant, the b2-sorting signal was expected to be cleaved off at the IMS side of the inner membrane by Imp1. Thereby, only the IMS domain of Tim50 would be present soluble in the IMS. When expressed in yeast cells all truncation mutants were able to support growth of yeast cells in the absence of the wild-type copy of Tim50 (B). Yeast cells carrying the truncation mutants were essentially indistinguishable from wild type when analyzed for growth on fermentable and nonfermentable carbon sources at 24, 30, and 37°C (Supplementary Figure S1). Likewise endogenous levels of various mitochondrial proteins and ability to import proteins into mitochondria in vitro were similar to those in wild type. Therefore, the only essential part of Tim50 appears to be its IMS domain. Mitochondria harboring wild-type or truncation mutants of Tim50 were subjected to carbonate extraction to differentiate between soluble and integral membrane proteins. Full-length Tim50, Tim50Δmatrix, and DLDTim50 were found in the pellet fraction as was the integral membrane protein Tim17 (C). In contrast, b2Tim50 was recovered in the soluble fraction like Mge1. This result proves that the b2-sorting signal was properly processed and that b2Tim50 was not anchored in the membrane. We conclude that the soluble IMS domain of Tim50 is sufficient to support the function of the full-length protein.
Figure 2. The soluble IMS domain of Tim50 is sufficient to support growth of yeast cells. (A) Schematic representation of Tim50 domain structure and of truncation mutants. (B) A haploid deletion strain of TIM50 carrying a wild-type copy of TIM50 on URA plasmid (more ...)
Tim50 Recognizes All Types of TIM23 Substrates
We next asked which types of substrates of the TIM23 complex interact with Tim50. Various TIM23 substrates were added to de-energized wild-type yeast mitochondria. Under these conditions precursors are only partially translocated across the outer membrane. To analyze which types of TIM23 substrates interacted with Tim50, samples were incubated with the cross-linking reagent DFDNB and subsequently subjected to immunoprecipitation with antibodies to Tim50 or preimmune serum as a control. We first analyzed the interaction of Tim50 with precursors of soluble matrix proteins. A construct consisting of the first 55 residues of cytochrome b2, which contain its matrix targeting signal, fused to mouse DHFR was efficiently cross-linked to Tim50 (A). The same was observed with the precursor of the matrix protein Jac1 (B). Likewise, precursors of inner membrane proteins that are conservatively sorted, such as Oxa1 and Rieske FeS protein, were cross-linked to Tim50 (, C and D). On reestablishment of the membrane potential all of these precursors were efficiently chased into mitochondria with the concomitant loss of cross-linking adducts to Tim50, demonstrating that Tim50-bound species are productive import intermediates (Supplementary Figure S2, A–D). We conclude that apparently all matrix-targeted precursors are recognized by Tim50.
Figure 3. Tim50 recognizes matrix-targeted proteins. Precursors of soluble, matrix-destined proteins pb2(1–55)DHFR (A) and pJac1 (B) and of inner membrane proteins conservatively sorted through the matrix pOxa1 (C) and pRieske (D) were synthesized in the (more ...)
Is Tim50 also involved in recognition of precursors that are laterally sorted by the TIM23 complex? We first used a precursor consisting of the first 72 residues of DLD fused to mouse DHFR which is laterally sorted and remains anchored in the inner membrane. A second precursor, which consisted of the first 167 residues of cytochrome b2 fused to DHFR, is laterally sorted, cleaved by Imp1 and released into the IMS. These precursors were incubated with de-energized mitochondria so that they only partially crossed the outer membrane, as described above. Cross-linking followed by immunoprecipitation showed them to be recognized by Tim50 (, A and B).
Figure 4. Tim50 also serves as a receptor for substrates laterally sorted by TIM23 and for TIM23 substrates with internal targeting signals. (A and B) Same as except that precursors of laterally sorted proteins pDLD(1–72)DHFR (A) and pb2(1–167)DHFR (more ...)
Are these species productive intermediates on the way to the inner membrane? We used a precursor consisting of the first 220 residues of cytochrome b2
fused to DHFR and bound it to de-energized mitochondria. On reestablishment of membrane potential one aliquot was analyzed for cross-linking to Tim50 and another one for further transport into mitochondria. Precursor initially bound to mitochondria was efficiently chased to its final destination as observed by processing first by MPP and then by Imp1 (C, bottom panel). At the same time, all three adducts to Tim50 observed with precursor bound to de-energized mitochondria disappeared in a time-dependent manner (C, top panel). Interestingly, the adduct with the lowest mobility disappeared first followed by the middle one. In contrast, the adduct with the highest mobility first increased in its intensity and then disappeared like the other ones. Similar observations were made for the other two laterally sorted precursors used in this study (Supplementary Figure S2, E and F). Thus, interaction of laterally sorted precursors with Tim50 represents the initial step of their transport via the TIM23 complex. The presence of multiple cross-linking adducts suggests that transfer to and release from Tim50 occur in distinct steps in which precursors exist in different conformations. It should be noted that multiple cross-linking adducts were also observed for precursors cross-linked to Tom40 (Esaki et al., 2004
We also analyzed whether Tim50 serves as a receptor for TIM23 substrates which are targeted to mitochondria by a signal that consists of a transmembrane domain followed by a presequence-like segment. The precursor of Tim14, as a representative of this class of substrates, was cross-linked to Tim50 (D) on its way to the mitochondrial inner membrane (Supplementary Figure S2G). In contrast, precursors independent of the TIM23 complex, like ATP/ADP carrier (AAC) and Tim23, were not cross-linked to Tim50 (E), demonstrating the specificity of interactions.
In summary, Tim50 recognizes all known types of TIM23 substrates showing that it serves as a general receptor of the TIM23 complex and that it most likely binds to the presequence or presequence-like elements.
Receptor Function of Tim50 Requires Its Association with the TIM23 Complex
Does the receptor function of Tim50 depend on its association with the TIM23 complex or can Tim50 function in a manner independent of TIM23? Cross-linking of precursors to Tim50 in mitochondria depleted of Tim23 yielded virtually no cross-linking adducts to Tim50 (A). In contrast, cross-linking of precursors to Tim50 was indistinguishable from wild type in control mitochondria depleted of Mia40, an essential mitochondrial protein whose function is unrelated to the TIM23 import pathway (B). This demonstrates specificity of the removal of Tim23 and argues against secondary effects due to mere depletion of an essential mitochondrial protein. To obtain additional support for this finding, we used mitochondria carrying a temperature-sensitive mutant of Tim23 in which specifically the interaction between Tim50 and Tim23 is affected (Gevorkyan-Airapetov et al., 2008
). Cross-linked adducts of Tim50 to precursors partially translocated across the outer membrane were virtually absent (B). In conclusion, the receptor function of Tim50 in mitochondria appears to depend on its association with TIM23.
Figure 5. Receptor function of Tim50 depends on its association with TIM23. (A) 35S-labeled matrix destined precursor pb2(1–167)Δ19DHFR was incubated with wild type (WT) and mitochondria depleted of Tim23 (Tim23↓) in the absence of membrane (more ...)
Tim50 Interacts with Precursors at a Very Early Stage of Translocation
When are the presequences seen by Tim50? The precursor consisting of the first 55 residues of cytochrome b2 and full-length DHFR was incubated with de-energized mitochondria at 25°C in the presence of methotrexate to prevent unfolding of the DHFR domain. The efficiency of cross-linking to Tim50 was reduced to ca. 30% of the cross-linking efficiency observed in the absence of methotrexate (A). This demonstrates that the DHFR domain had to be at least partially unfolded to allow movement of the targeting signal sufficiently far in to be seen by Tim50. A similar, though less pronounced effect, was seen when the incubation was performed in the absence of methotrexate but at a temperature of 4°C, at which unfolding of DHFR is greatly reduced because of the reduced thermal fluctuations. There are five lysine residues in the 35-resides long b2 presequence that could participate in cross-linking: Four of them are among the first 12 residues (residues 3, 5, 9, and 12). Therefore, a stretch of at least 55 residues can be trapped within the TOM complex before becoming accessible to the TIM23 complex. This result predicts that extending the cytochrome b2 segment in front of DHFR to a certain length should abolish these effects. Indeed, the precursor pb2(1–167)ΔDHFR, which has 148 residues in front of DHFR (first 167 residues of cytochrome b2 with the 19 residues of the sorting signal deleted) was cross-linked to Tim50 with essentially the same efficiency irrespective of whether it was preincubated with methotrexate or not (111 vs. 100%). Likewise, no difference in cross-linking was observed when the incubation with mitochondria was performed at 25 or at 4°C (B).
Figure 6. Translocation across the TOM complex is coupled to recognition by Tim50. (A–C) Presequence-containing precursors with cytochrome b2 segments of different lengths in front of the DHFR domain were incubated with de-energized wild-type mitochondria (more ...)
How many residues of the polypeptide chain can be bound to the TOM complex before they are seen by Tim50? We extended the b2
segment from 55 to 68 residues in the pb2
(1–87)Δ19DHFR. The efficiencies of cross-linking to Tim50 in the presence and absence of methotrexate were essentially the same (96 compared with 100%; C). Therefore, 55 residues can be accommodated in the TOM complex and are not accessible to Tim50, in contrast to 68 residues that cannot be contained within the TOM complex and are already exposed to the translocation machinery of the inner membrane. Apparently, at this stage of translocation, matrix targeted precursors are not yet in a fully extended state because ca. 60 residues are sufficient to span both TOM and TIM23 complexes when the import motor of the TIM23 complex has engaged in driving vectorial movement into the matrix (Ungermann et al., 1994