Biology subjects

Sharpe, A. J.

Publications and source records attributed to Sharpe, A. J..

5 recordsLinked to original sources

bioRxiv2026

Proximity labelling of the BAK macropore uncovers a new role for SLC35A4-MP in mitochondrial dynamics

Mitochondrial permeabilization by the apoptotic executioners BAK and BAX represents a critical stage of mitochondrial apoptosis and facilitates the release of pro-inflammatory mitochondrial DNA via herniation of the inner mitochondrial membrane. This study utilises TurboID proximity labelling to investigate the temporal changes of the BAK proximal proteome during mitochondrial herniation and apoptosis. In doing so, we detail a comprehensive BAK proximal proteome, both at steady state and during apoptosis and observe the loss of MICOS complex stability and proximity to the BAK pore as apoptosis proceeds. In addition, we identify the mitochondrial microprotein SLC35A4-MP proximal to the BAK pore and reveal a SLC35A4-MP dependent modulation of OPA1 processing. Furthermore, loss of SLC35A4-MP delays mitochondrial fragmentation in response to a variety of stressors, uncovering a previously unrecognised role for SLC35A4-MP in fine-tuning mitochondrial rearrangement during apoptotic stress.

cell biology↗

bioRxiv2026

Characterization of human Metaxin proteins reveals functional diversification of SAM37 homologs MTX1 and MTX3

The biogenesis of outer mitochondrial membrane {beta}-barrel proteins relies on the mitochondrial Sorting and Assembly Machinery (SAM) complex. In humans, the SAM complex contains SAM50 along with Metaxin (MTX) accessory subunits. MTX1 and MTX3 are homologous yet their functional similarities and differences have scarcely been investigated. Homozygous null mutations in the MTX2 gene are linked to a rare progeroid syndrome that causes severe depletion of MTX1. Here, we uncover unique phenotypes associated with the loss of MTX1 or MTX3 in human cells. Loss of MTX1 confers a deficiency in mitochondrial volume and causes network-wide mitochondrial morphology abnormalities. MTX3 loss resulted in negligible consequences for the biogenesis of {beta}-barrel proteins but resulted in increased mitochondrial mass. We also find that both MTX1 and MTX3 stability are dependent on the presence of MTX2, with MTX1 deficiency causing defective import and assembly. Collectively, our findings support the notion that MTX1 and MTX3 are functionally diverse homologs and are unlikely to be functionally redundant.

biochemistry↗

bioRxiv2021

Mitochondrial COA7 is a heme-binding protein involved in the early stages of complex IV assembly.

Cytochrome c oxidase assembly factor 7 (COA7) is a metazoan-specific assembly factor, critical for the biogenesis of mitochondrial complex IV (cytochrome c oxidase). Although mutations in COA7 have been linked in patients to complex IV assembly defects and neurological conditions such as peripheral neuropathy, ataxia and leukoencephalopathy, the precise role COA7 plays in the biogenesis of complex IV is not known. Here we show that the absence of COA7 leads to arrest of the complex IV assembly pathway at the initial step where the COX1 module is built, which requires incorporation of copper and heme cofactors. In solution, purified COA7 binds heme with micromolar affinity, through axial ligation to the central iron atom by histidine and methionine residues. Surprisingly, the crystal structure of COA7, determined to 2.4 [A] resolution, reveals a banana-shaped molecule composed of five helix-turn-helix (/) repeats, tethered by disulfide bonds, with a structure entirely distinct from proteins with characterized heme binding activities. We therefore propose a role for COA7 in heme binding/chaperoning in the mitochondrial intermembrane space, this activity being crucial for and providing a missing link in complex IV biogenesis. Significance StatementAssembly factors play key roles in the biogenesis of many mitochondrial protein complexes regulating their stability, activity and incorporation of essential cofactors. COA7 is a metazoan-specific assembly factor, the absence or mutation of which in humans accompanies complex IV assembly defects and neurological conditions. Here we report the crystal structure of COA7 to 2.4 [A] resolution, revealing a banana-shaped molecule composed of five helix-turn-helix (/) repeats, tethered by disulfide bonds. Characterization of pathogenic variants reveals significantly lower stabilities, correlating with the associated disease outcomes. Fascinatingly, COA7 binds heme with micromolar affinity, despite the fact that the protein structure does not resemble previously characterized heme-binding proteins. This provides a possible missing link for heme handling in the mitochondrial intermembrane space.

biochemistry↗

bioRxiv2020

Optic Atrophy-associated TMEM126A is an assembly factor for the ND4-module of Mitochondrial Complex I

Mitochondrial disease is a debilitating condition with a diverse genetic aetiology. Here, we report that TMEM126A, a protein that is mutated in patients with autosomal recessive optic atrophy, participates directly in the assembly of mitochondrial complex I. Using a combination of genome editing, interaction studies and quantitative proteomics, we find that loss of TMEM126A results in an isolated complex I deficiency and that TMEM126A interacts with a number of complex I subunits and assembly factors. Pulse-labelling interaction studies reveal that TMEM126A associates with the newly synthesised mtDNA-encoded ND4 subunit of complex I. Our findings indicate that TMEM126A is involved in the assembly of the ND4 distal membrane module of complex I. Importantly, we clarify that the function of TMEM126A is distinct from its paralogue TMEM126B, which acts in assembly of the ND2-module of complex I, helping to explain the differences in disease aetiology observed between these two genes.

biochemistry↗

bioRxiv2019

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

Mitochondrial Complex I harbors 7 mitochondrial and 38 nuclear-encoded subunits. Its biogenesis requires the assembly and integration of distinct intermediate modules, mediated by numerous assembly factors. The Mitochondrial Complex I Intermediate Assembly (MCIA) complex, containing assembly factors NDUFAF1, ECSIT, ACAD9, and TMEM126B, is required for building the intermediate ND2-module. The role of the MCIA complex and the involvement of other proteins in the biogenesis of this module is unclear. Cell knockout studies reveal that while each MCIA component is critical for complex I assembly, a hierarchy of stability exists centred on ACAD9. We also identify TMEM186 and COA1 as bona fide components of the MCIA complex with loss of either resulting in in MCIA complex defects and reduced complex I assembly. TMEM186 enriches with newly translated ND3, while COA1 enriches with ND2. Our findings provide new functional insights into the essential nature of the MCIA complex in complex I assembly.

biochemistry↗