Different views have been discussed on the mechanism of protein sorting at the mitochondrial inner membrane, ranging from a single-entity translocase, where TIM23 complex and PAM are permanently associated, to the existence of two TIM23 forms (TIM23-PAM and TIM23-Tim21) that function separately for protein translocation into the matrix rather than protein sorting into the inner membrane. We accumulated b2-DHFR model preproteins at distinct stages of mitochondrial import and monitored the formation of active, preprotein-carrying translocase complexes that were used as snapshots to define the composition of translocases in active mitochondria. Our results indicate that neither a single-entity presequence translocase nor two permanently separated translocase forms are adequate to describe the mechanism of inner membrane protein translocation but suggest that two different modular forms of the presequence translocase are in a dynamic exchange during preprotein translocation and sorting.
The hypothesis of a single-entity presequence translocase predicts that TIM23, Tim21, and PAM are permanently present in one complex and thus would exclude the existence of two forms, TIM23-Tim21 and TIM23-PAM (27
). However, several independent findings provide evidence for the existence of two distinct forms of an active TIM23 complex. (i) Using the accumulated b2
-DHFR proteins, we demonstrate that mitochondria contain both forms of the TIM23 complex in an active, preprotein-carrying state: TIM23-PAM and TIM23-Tim21. The sorting signal and the import stage of a preprotein are critical for the in organello accumulation in the distinct complexes. (ii) To exclude possible concerns on the functionality of protein A-tagged Tim21 (33
), we also used a Pam18His
strain. Use of both strains, Tim21ProtA
, yielded the identical conclusion that the vast majority of TIM23 complexes did not contain PAM and Tim21 together. Moreover, the TIM23-Tim21 complex purified from a Tim21ProtA
strain inserted sorted preproteins into the membrane in a proteoliposome assay in the absence of motor components (46
). Mitochondria containing Tim21ProtA
were competent in accumulating a matrix-targeted preprotein in the inner membrane, like mitochondria containing Tim23ProtA
. (iii) In agreement with these findings, the results of Popov-Celeketic et al. (33
) showed that only small, substoichiometric amounts of Tim21 were copurified with Pam16, and Mokranjac et al. (28
) reported that they were not able to detect Tim21 together with precipitated Pam16. Similarly, only small, substoichiometric amounts of PAM subunits (Tim44 and Pam18) were copurified with Tim21His
). (iv) It may be argued that PAM or Tim21 was lost during coprecipitation because the complexes may be detergent labile. The following observations exclude this possibility. PAM and Tim21 were efficiently copurified with TIM23CORE
separately, whereas the copurification of both together showed only a very low yield. Since the same detergent (digitonin) was used in the different coprecipitation experiments, the TIM23-PAM and TIM23-Tim21 interactions were stable in the detergent. Moreover, both forms of the TIM23 complex carried the accumulated preprotein and were found in association with the TOM complex under these detergent conditions. We conclude that PAM and Tim21 are not simply lost from TIM23CORE
during the purification procedure but are present in two different forms of the translocase. (v) The results obtained with yeast mutant or overexpression strains strongly support the view of a dynamic, competitive situation for association of PAM and Tim21 with TIM23CORE
. The partial inactivation of Pam16 in a yeast mutant increased the yield of copurification of Tim21 with TIM23CORE
), whereas overexpression of Pam17 reduced the amount of Tim21 associated with TIM23CORE
). We show here that overexpression of Tim21 decreased the copurification of PAM subunits with TIM23CORE
. Taken together, we conclude that the components of the mitochondrial presequence translocase and import motor do not function as a permanently associated single entity but consist of several modules that cooperate in a dynamic manner.
We found that both TIM23-PAM and TIM23SORT are involved in the import of matrix-targeted preproteins and inner membrane-sorted preproteins. Tim21 not only is found in association with inner membrane-sorted preproteins but also is a component of the TOM-TIM23 supercomplex that carries a matrix-targeted preprotein yet lacks stoichiometric amounts of PAM. A second form of the TOM-TIM23 supercomplex contains PAM but lacks stoichiometric amounts of Tim21. We observed that preproteins can shift between the TIM23SORT complex and the TIM23-PAM complex. Thus, the two forms of the presequence translocase are not separated throughout the entire import process but are in dynamic exchange with each other. At an early stage of import, matrix-targeted preproteins are preferentially associated with TIM23SORT, whereas in a later stage of import, the proteins are found mainly in TIM23-PAM.
For inner membrane-sorted preproteins with a hydrophobic signal behind the matrix-targeting sequence, the reported studies gave different views. TIM23SORT
(containing Tim21) was reported to promote membrane insertion (46
), while in organello studies indicated that PAM also was involved when the preprotein contained folded domains (4
). Moreover, Popov-Celeketic et al. (33
) reported that inner membrane-sorted preproteins were not coisolated with the TIM23 complex. To resolve this puzzling situation, we established an in organello pulse-chase assay to dissect the import of inner membrane-sorted proteins into two stages, accumulation in translocation contact sites and subsequent lateral sorting. The accumulated intermediate form was clearly copurified with TIM23SORT
. With tim17
mutants that differentially affected PAM binding and lateral sorting, we show that the PAM activity promotes the accumulation of the preprotein in the import sites but delays the subsequent sorting process. The dynamic switch between PAM form and Tim21 form provides an explanation for a surprising observation. Merlin et al. (25
) reported that a b2
-DHFR preprotein with a partially inactivated sorting signal was missorted into the matrix; however, upon partial inactivation of PAM, the preprotein was laterally sorted, i.e., the impairment of the sorting signal was relieved by an impairment of the import motor. Thus, both PAM form and Tim21 form of the presequence translocase are involved in the import process of the sorted preprotein and the balance of switching between their activities is important for an efficient sorting process.