TIM29 is a subunit of the human carrier translocase required for protein transport

Abstract

Hydrophobic inner mitochondrial membrane proteins with internal targeting signals, such as the metabolite carriers, use the carrier translocase (TIM22 complex) for transport into the inner membrane. Defects in this transport pathway have been associated with neurodegenerative disorders. While the TIM22 complex is well studied in baker's yeast, very little is known about the mammalian TIM22 complex. Using immunoprecipitation, we purified the human carrier translocase and identified a mitochondrial inner membrane protein TIM29 as a novel component, specific to metazoa. We show that TIM29 is a constituent of the 440 kDa TIM22 complex and interacts with oxidized TIM22. Our analyses demonstrate that TIM29 is required for the structural integrity of the TIM22 complex and for import of substrate proteins by the carrier translocase.

The TIM22 complex regulates mitochondrial one-carbon metabolism by mediating the import of Sideroflexins

“…Based on our analysis showing reduced import and assembly of SFXN proteins in mitochondria lacking AGK and Tim9, we suggest that that this family of proteins represent novel substrates of the TIM22 complex. Indeed, independent mass spectrometric analysis of AGKKO HEK293 cells shows a striking downregulation of SFXN proteins and identified SFXN2 as a high confidence client protein of AGK (Vukotic et al, 2017), while SFXN1 has also been enriched following Tim22 immunoprecipitation (Callegari et al, 2016). The identification of SFXNs as TIM22 complex substrates prompts a re-think of the mechanisms of TIM22 complex membrane insertion.…”

“…Interestingly, MTHFD2 was one of the most significantly downregulated proteins in the Tim22 KD (Figure 2E -F, lane 2), again suggesting that TIM22 complex dysfunction leads to remodelling of the mitochondrial 1C metabolism pathway. To the contrary, depletion of Tim29 led to reduced levels of Tim23 and Tim17b (Figure 2E), TIM22 substrates that are known to require Tim29 for efficient import (Callegari et al, 2016;Kang et al, 2016). Tim29 knock-down had no effect on the abundance of SFXN proteins (Figure 2E-G), as is the case for mitochondrial carrier proteins, which also use the TIM22 complex for import.…”

“…The TIM22 complex has been extensively studied in yeast, however recent analyses in human cells has revealed substantial divergence of the complex in higher eukaryotes. The human TIM22 complex consists of: (i) Tim22, the core pore-forming subunit; (ii) the intermembrane space chaperones Tim9, Tim10, and Tim10b; and (iii) Tim29 (Callegari et al, 2016;Kang et al, 2016) and AGK (Kang et al, 2017;Vukotic et al, 2017), which are metazoan-specific subunits of the complex.…”

Mutations in TIMM50 compromise cell survival in OxPhos‐dependent metabolic conditions

“…A multimeric chaperone complex encompassing TIMM9 and TIMM10, or less frequently TIMM8 and TIMM13, shuttles these proteins from the TOM to the TIM22 complex. Human TIM22 complex consists of the channel‐forming subunit TIMM22 and two mammalian specific constituents: the lipid kinase AGK and TIMM29 (Callegari et al , ; Kang et al , ; Vukotic et al , ).…”

Dissecting the Roles of Mitochondrial Complex I Intermediate Assembly (MCIA) Complex Factors in the Biogenesis of Complex I

“…4I, right panel; Supplementary Table 4) whereas complex I, III and IV subunits were not strongly enriched. The enrichment of TMEM186 with the TIM22 machinery (TIM22, TIM29, AGK, small TIMs) may be due to TMEM186 using the TIM22 import pathway for membrane integration (Callegari et al, 2016;Kang et al, 2016;Kang et al, 2018;Kang et al, 2017;Vukotic et al, 2017) as well as overexpression of the Flag tagged protein. Mitochondria from cells expressing TMEM186 Flag were subjected to alkali extraction (Na2CO3) followed by SDS-PAGE and western blot analysis.…”