Model of matrix translocation by the TIM23 complex

Based on these results an updated model of protein translocation across the TIM23 com-plex can be proposed. While the precursor is inserted into the channel, Hsp70 undergoes multiple rounds of binding and release to drive translocation into the matrix by a ratchet mechanism. The regulatory and scaffolding PAM subunits Pam18 and Tim44 leave and integrate into the translocase during this process. It remains to be elucidated whether both subunits have the same oscillation frequency at the translocase as Hsp70.

One can imagine a mechanism where the Pam16-Pam18 complex is positioned at the translocase exit site and a conformational change of the J-complex is required to stimulate the ATPase activity of Hsp70 (Mokranjac et al., 2006). After activation of Hsp70, the interaction of Pam16-Pam18 is destabilized, resulting in a loss of Pam18. Tim21 could preferentially assemble to this complex, displacing the remaining PAM subunits and form-ing TIM23^SORT. Pam16 and Tim44 might also be lost from TIM23 directly after Pam18 after the Hsp70 stimulation. Tim21 would subsequently be recruited to TIM23^CORE. This process is most likely regulated by Mgr2 as it is the coupling partner for Tim21 and affects Pam18 assembly. The process could serve precursor probing in order to prevent complete matrix translocation of an inner membrane protein. The displacement of the import mo-tor could be a prerequisite for lateral sorting in case the positioning of the PAM subunits at TIM23 blocks a potential lateral gate.

An alternative to the Tim21 recruitment to TIM23^MOTOR that just lost Pam18, is the assembly of a new Pam18 subunit. This would result in a “recharged” TIM23^MOTOR that can stimulate a new round of Hsp70 activity (Figure 41). The same would hold true for Tim44, except that its recruitment seems to be Mgr2 independent. Aspam17Δ mitochondria showed phenotypes similar tomgr2Δ(all subunits at TIM23, strong import defect), similar investigations in this mutant could shed additional light on the regulation of the import motor.

Clearly, many of the steps of this proposed mechanism await to be tested by rigorous experiments. These would strongly benefit from a structure of the membrane embedded part of the presequence translocase.

DISCUSSION

matrix IMS

Δψ – – – + + +

dissociation recharging Hsp70 recruitment

ADP +

ADP ADP

+ ATP

+ ADP

+ ATP hydrolysis

ADP

+ TIM23^MOTOR substrate

PAM Tim44

Mgr2 Tim23/17 Tim50

Mge1 Hsp70

16 18

ATP hydrolysis

Fig. 41: Model of subunit oscillation during import motor function- During translo-cation of a polypeptide chain into the mitochondrial matrix ATP hydrolysis by Hsp70 is stimulated by Pam18. This leads to a destabilization of the regulatory PAM sub-units (Pam16, Pam18 and Tim44) at the translocase and their subsequent dissociation from the exit site. For a new round of Hsp70 recruitment the translocase has to be recharged with these PAM subunits in order to support continuous Hsp70 stimulation and hence vectorial movement of the substrate.

4 Summary and Conclusion

Within the eukaryotic cell mitochondria play an important role in metabolism and reg-ulation of apoptosis. Due to their endosymbiotic origin and the transfer of most of its genetic information to the nucleus, the organelle largely relies on protein import for its biogenesis. Proteins carrying an N-terminal targeting signal (presequence) constitute the largest fraction among the imported proteins. Recognition of the presequence by the re-ceptors Tom20 and Tom22 of the translocase of the outer membrane (TOM) initiates the transport process that will guide the proteins into the mitochondrial matrix or allow them to be laterally sorted into the inner membrane by the presequence translocase (TIM23).

In this thesis the mechanism of precursor recognition by the TIM23 complex as well as the regulation of the presequence translocase-associated import motor (PAM) are de-scribed. Using purified p-benzophenylalanine (BPA)-containing presequence peptides we identified Tim50 as the primary presequence receptor of the TIM23 complex. In its in-termembrane space domain it contains a C-terminal presequence binding domain (PBD) as well as a second binding site in close proximity to a negatively charged groove. While both sites are sufficient to bind the targeting signal in vitro, the PBD is essential for cell viability due to its role in precursor recognition during protein transport across the inner membrane. We found that after initial presequence binding by Tim50, the targeting signal is then handed over to the channel-forming Tim23 via a trimeric intermediate. The role of the second binding site in Tim50 as well as the interplay of both binding sites with the receptor domain of Tim23 will be an attractive topic for future studies.

Once the precursor is transfered to the Tim23 channel, the membrane potential across the inner membrane drives transport of the positively charged presequence. However, full translocation into the mitochondrial matrix relies on the ATP driven import motor which consists of the mtHsp70, a tethering factor (Tim44), a J-protein (Pam18) as well as a regulatory J-like protein (Pam16). The activity of this motor is required to stabilize translocation intermediates that span the TOM and TIM23 complexes. Based on this, we developed an assay which revealed that Pam18 and Tim44 are able to integrate into

SUMMARY AND CONCLUSION

the active TIM23 translocase. This integration is required for efficient protein import, as a TIM23 mutant (mgr2Δ), defective in the recharging of the translocase with Pam18, shows drastic reductions in matrix import.

Due to the tight interaction of Pam18 with Pam16, conformational changes in Pam18 have long been speculated to be required for the stimulation of mtHsp70’s ATPase activ-ity. Does this lead to a subsequent destabilization of the interaction of Pam18 with the translocase? If so, one would expect that the exchange of regulatory subunits is coupled to the ATPase and recruitment cycle of mtHsp70. In the future it will be interesting to study potential conformational changes of the Pam18-Pam16 complex as well as possible synchronization of the regulatory PAM subunits with mtHsp70.

What would be the benefit of continuous recharging of the regulatory PAM subunits during import? This question might be answered by the present finding that the TIM23 subunit Tim21 also integrates into the active translocase. It has been previously shown that Tim21 association with TIM23 transforms the complex into a form that functions in lateral sorting of inner membrane proteins. Interestingly, Mgr2 which is required for efficient Pam18 assembly, is also important for Tim21 association. Hence, the labile association of regulatory PAM subunits and Tim21 could allow an Mgr2 mediated in-terconversion between the motor-associated and the sorting form of the TIM23 complex.

Such a mechanism would be required to support biogenesis of proteins that contain a long segment between the presequence and the sorting signal and are therefore dependent on the import motor before they are released into the inner membrane.

5 Materials and Methods