Conserved regions of budding yeast Tim22 have a role in structural organization of the carrier translocase

Abstract

Mitochondrial biogenesis requires efficient sorting of various proteins into different mitochondrial sub-compartments, mediated by dedicated protein machinery present in the outer and inner membrane. Among them, the TIM22 complex enables the integration of complex membrane proteins with internal targeting signals into the inner membrane. Although the Tim22 protein forms the core of the complex, the dynamic recruitment of subunits to the channel is still enigmatic. In this study, we highlight that the intermembrane space (IMS) and transmembrane 4 (TM4) regions of Tim22 are critically required for interactions with the membrane-embedded subunits, including Tim54, Tim18, and Sdh3, and thereby maintain the functional architecture of the TIM22 translocase. Furthermore, we find that the TM1 and TM2 regions of Tim22 are important for association with Tim18, whereas TM3 is exclusively required for the interaction with Sdh3. Moreover, impairment of TIM22 complex assembly influences its translocase activity, the mitochondrial network, and the viability of cells lacking mitochondrial DNA. Overall, our findings provide compelling evidence highlighting the significance of conserved regions of Tim22 that are important for the maintenance of the TIM22 complex and mitochondrial integrity.

Functional crosstalk between the carrier translocase machinery and YME1 complex maintains mitochondrial proteostasis and integrity

“…The Tim18 protein expression in tim18 Δ yme1 Δ strain was confirmed by immunoblotting (Figure 1E). Furthermore, we utilized tim22 conditional mutants to validate the genetic link between these complexes (Kumar et al, 2020). Interestingly, the deletion of Yme1 in a tim22 mutant background ( tim22 K127A ) exhibited partial rescue in the growth defects of yme1 Δ at 37°C in YPG media (Figure 1F) (Kumar et al, 2020).…”

“…Furthermore, we utilized tim22 conditional mutants to validate the genetic link between these complexes (Kumar et al, 2020). Interestingly, the deletion of Yme1 in a tim22 mutant background ( tim22 K127A ) exhibited partial rescue in the growth defects of yme1 Δ at 37°C in YPG media (Figure 1F) (Kumar et al, 2020). Taken together, these findings provide genetic evidence for a novel functional link between the TIM22 and YME1 complexes.…”

“…The R149W mutation in YME1L1 was introduced by PCR-based site-directed mutagenesis using the Pfu Turbo DNA polymerase (Stratagene). The Tim22 mutant and other overexpression constructs used in this study are described in detail in an earlier report (Kumar et al, 2020).…”

“…Antisera specific for PGK1 (1:7000 dilution used) was gifted by Prof. P. N. Rangarajan (Indian Institute of Science, Bangalore). Antibodies against Tim18 (1:2500 dilution used) and Tim23 (1: 5000 dilution used) were raised in rabbits as reported previously (Pareek et al, 2013, Kumar et al, 2020). FLAG tag antibody (1:1000 dilution used) raised in mouse (F1804) was obtained from Sigma-Aldrich and Porin antibody (1:5000 dilution used) raised in mouse (16G9E6BC4) was purchased from Invitrogen.…”

Defining the architecture of the human TIM22 complex by chemical crosslinking

“…2B), suggesting an interaction interface between TIM22 and TIM29 within the matrix. Interestingly, one of the crosslinks with the matrix domain of TIM29 involves a conserved residue of TIM22 (K127) which, in yeast, has been shown to be essential for the interaction of Tim22 with Tim18 [54]. This suggests that this is a functional interaction region and it could be that TIM29 in metazoa has structurally replaced the yeast Tim18.…”