Import of Presequence-Containing Precursor Proteins into Mitochondria

The presequence pathway is utilized by over 70% of all cytosolically translated proteins destined to the mitochondria, emphasizing the significance imparted by presequence import on mitochondria translocation. Presequence docking at the main mitochondrial entry gate, the translocase of the outer mitochondrial membrane (TOM), has been the target of a vast number of publications throughout the past two and a half decades. However, the dynamic docking events leading up to the engagement of the presequence with the TIM23 complex (also known as the presequence translocase) is still poorly understood. This study presents Tom40 as a presequence-active TOM complex subunit, and maps its presequence-interacting sites employing presequence probes in a joint photo- crosslinking/mass spectrometry based approach. Moreover, the interacting regions of Tom40 were placed within a recently published, biochemically grounded Tom40 homology model, where two presequence-active regions were identified. A posttranslationally active residue was identified within one of the defined presequence- interacting surfaces. Phospho-mimetic and phospho-block Tom40 point mutants were shown to selectively augment local presequence import kinetics at the TOM complex, resulting in alteration of overall import kinetics. Finally, the established intermembrane space (IMS) domain of Tom22 was functionally linked to Tom40 presequence interaction. Examining the following stages of presequence import, this study presents a series of dynamic subunit exchange events leading to presequence presentation at the channel of the presequence translocase. A novel presequence translocase interaction between Tim50 and Tim21 was characterized and shown in vitro to be coordinated by the IMS domain of Tim23 via a Tim50 interaction mechanism. Moreover, the interaction of Tim50 and Tim21 was shown in organello to be signal sequence sensitive, as Tim21 is demonstrated to dissociate from Tim50 within the presequence translocase in a presequence-“priming” dependent manner. Finally, the aforementioned presequence translocase priming event is linked to matrix translocation as the early presequence associated motor (PAM) subunit Pam17 was shown to be recruited to the TIM23 complex concomitantly with Tim21 dissociation during TIM23 priming. Taken together, these data shine new light on the presently elusive transfer mechanism of presequence-containing substrates from the outer membrane resident TOM channel to the inner membrane bound TIM23 complex