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Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability

View Article: PubMed Central - PubMed

ABSTRACT

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

Doi:: http://dx.doi.org/10.7554/eLife.17463.001

No MeSH data available.


Related in: MedlinePlus

TIM23 complex substrates are reduced in cells depleted of Tim29.(A) Mitochondria were isolated from control cells (scrambled siRNA) or cells transfected with Tim29 siRNA target for 72 hr. Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. The levels of the TIM23 complex substrates, COXIV, NDUFV2 and NDUFV1 was quantified relative to protein levels in control mitochondria and normalised against the loading control Mfn2. Data are shown as mean ± SD (n = 3). (B and C) [35S]-labelled NDUFV1 and NDUFV3 were imported into mitochondria isolated from control and Tim29 knockdown cells for the indicated times. Re-isolated mitochondria were treated with protease and separated by SDS-PAGE and analysed by autoradiography. For quantification of imported NDUFV1 and NDUFV3, the longest incubation time (45 min) was set to 100%. Data are shown as mean ± SD (n = 2, NDUFV1; n = 3 NDUFV3). p, precursor and m, mature.DOI:http://dx.doi.org/10.7554/eLife.17463.012
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fig4s2: TIM23 complex substrates are reduced in cells depleted of Tim29.(A) Mitochondria were isolated from control cells (scrambled siRNA) or cells transfected with Tim29 siRNA target for 72 hr. Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. The levels of the TIM23 complex substrates, COXIV, NDUFV2 and NDUFV1 was quantified relative to protein levels in control mitochondria and normalised against the loading control Mfn2. Data are shown as mean ± SD (n = 3). (B and C) [35S]-labelled NDUFV1 and NDUFV3 were imported into mitochondria isolated from control and Tim29 knockdown cells for the indicated times. Re-isolated mitochondria were treated with protease and separated by SDS-PAGE and analysed by autoradiography. For quantification of imported NDUFV1 and NDUFV3, the longest incubation time (45 min) was set to 100%. Data are shown as mean ± SD (n = 2, NDUFV1; n = 3 NDUFV3). p, precursor and m, mature.DOI:http://dx.doi.org/10.7554/eLife.17463.012

Mentions: Next we assessed the impact of KD of Tim29 on TIM22 complex substrates at the steady state protein level. As can be seen, Tim29 (Figure 4A) and hTim22 (Figure 4B) were depleted from mitochondria while the control protein Mfn2 remained unaffected (Figure 4A and B, bottom panels). Specific depletion of TIM22 complex substrates, including hTim23, ANT3 and the glutamate carrier was observed in both Tim29 and hTim22 depleted mitochondria (quantifications shown in Figure 4A and B, lower panels). The depletion of Tim29 also led to a significant reduction in the levels of hTim22 (Figure 4A), while the lack of hTim22 had no obvious effect on the levels of Tim29, indicating Tim29 is imported in a TIM22-independent manner (Figure 4B). BN-PAGE analysis also revealed that depletion of Tim29 caused a reduction in the assembly of ANT3 and hTIM23 complexes (Figure 4C), with the latter causing a reduction in the steady state level of TIM23 complex substrates (COXIV, NDUFV2 and NDUFV1) (Figure 4—figure supplement 2A) and import of [35S]-NDUFV1 and [35S]-NDUFV2 (Figure 4—figure supplement 2B and C).10.7554/eLife.17463.010Figure 4.Knockdown of Tim29 reduces the steady state protein levels of TIM22 substrates.


Tim29 is a novel subunit of the human TIM22 translocase and is involved in complex assembly and stability
TIM23 complex substrates are reduced in cells depleted of Tim29.(A) Mitochondria were isolated from control cells (scrambled siRNA) or cells transfected with Tim29 siRNA target for 72 hr. Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. The levels of the TIM23 complex substrates, COXIV, NDUFV2 and NDUFV1 was quantified relative to protein levels in control mitochondria and normalised against the loading control Mfn2. Data are shown as mean ± SD (n = 3). (B and C) [35S]-labelled NDUFV1 and NDUFV3 were imported into mitochondria isolated from control and Tim29 knockdown cells for the indicated times. Re-isolated mitochondria were treated with protease and separated by SDS-PAGE and analysed by autoradiography. For quantification of imported NDUFV1 and NDUFV3, the longest incubation time (45 min) was set to 100%. Data are shown as mean ± SD (n = 2, NDUFV1; n = 3 NDUFV3). p, precursor and m, mature.DOI:http://dx.doi.org/10.7554/eLife.17463.012
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fig4s2: TIM23 complex substrates are reduced in cells depleted of Tim29.(A) Mitochondria were isolated from control cells (scrambled siRNA) or cells transfected with Tim29 siRNA target for 72 hr. Mitochondrial proteins were subjected to SDS-PAGE and immunoblotting using the indicated antibodies. The levels of the TIM23 complex substrates, COXIV, NDUFV2 and NDUFV1 was quantified relative to protein levels in control mitochondria and normalised against the loading control Mfn2. Data are shown as mean ± SD (n = 3). (B and C) [35S]-labelled NDUFV1 and NDUFV3 were imported into mitochondria isolated from control and Tim29 knockdown cells for the indicated times. Re-isolated mitochondria were treated with protease and separated by SDS-PAGE and analysed by autoradiography. For quantification of imported NDUFV1 and NDUFV3, the longest incubation time (45 min) was set to 100%. Data are shown as mean ± SD (n = 2, NDUFV1; n = 3 NDUFV3). p, precursor and m, mature.DOI:http://dx.doi.org/10.7554/eLife.17463.012
Mentions: Next we assessed the impact of KD of Tim29 on TIM22 complex substrates at the steady state protein level. As can be seen, Tim29 (Figure 4A) and hTim22 (Figure 4B) were depleted from mitochondria while the control protein Mfn2 remained unaffected (Figure 4A and B, bottom panels). Specific depletion of TIM22 complex substrates, including hTim23, ANT3 and the glutamate carrier was observed in both Tim29 and hTim22 depleted mitochondria (quantifications shown in Figure 4A and B, lower panels). The depletion of Tim29 also led to a significant reduction in the levels of hTim22 (Figure 4A), while the lack of hTim22 had no obvious effect on the levels of Tim29, indicating Tim29 is imported in a TIM22-independent manner (Figure 4B). BN-PAGE analysis also revealed that depletion of Tim29 caused a reduction in the assembly of ANT3 and hTIM23 complexes (Figure 4C), with the latter causing a reduction in the steady state level of TIM23 complex substrates (COXIV, NDUFV2 and NDUFV1) (Figure 4—figure supplement 2A) and import of [35S]-NDUFV1 and [35S]-NDUFV2 (Figure 4—figure supplement 2B and C).10.7554/eLife.17463.010Figure 4.Knockdown of Tim29 reduces the steady state protein levels of TIM22 substrates.

View Article: PubMed Central - PubMed

ABSTRACT

The TIM22 complex mediates the import of hydrophobic carrier proteins into the mitochondrial inner membrane. While the TIM22 machinery has been well characterised in yeast, the human complex remains poorly characterised. Here, we identify Tim29 (C19orf52) as a novel, metazoan-specific subunit of the human TIM22 complex. The protein is integrated into the mitochondrial inner membrane with it’s C-terminus exposed to the intermembrane space. Tim29 is required for the stability of the TIM22 complex and functions in the assembly of hTim22. Furthermore, Tim29 contacts the Translocase of the Outer Mitochondrial Membrane, TOM complex, enabling a mechanism for transport of hydrophobic carrier substrates across the aqueous intermembrane space. Identification of Tim29 highlights the significance of analysing mitochondrial import systems across phylogenetic boundaries, which can reveal novel components and mechanisms in higher organisms.

Doi:: http://dx.doi.org/10.7554/eLife.17463.001

No MeSH data available.


Related in: MedlinePlus