Biogenesis of the mitochondrial DNA inheritance machinery in the mitochondrial outer membrane of Trypanosoma brucei

The eukaryotic porin superfamily consists of two families, voltage-dependent anion channel (VDAC) and Tom40, which are both located in the mitochondrial outer membrane. In Trypanosoma brucei , only a single member of the VDAC family has been described. We report the detection of two additional eukaryotic porin-like sequences in T. brucei . By bioinformatic means, we classify both as putative VDAC isoforms.

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MITOL-dependent ubiquitylation negatively regulates the entry of PolγA into mitochondria

PLoS Biology ◽

10.1371/journal.pbio.3001139 ◽

2021 ◽

Vol 19 (3) ◽

pp. e3001139

Author(s):

Mansoor Hussain ◽

Aftab Mohammed ◽

Shabnam Saifi ◽

Aamir Khan ◽

Ekjot Kaur ◽

...

Keyword(s):

Mitochondrial Dna ◽

Outer Membrane ◽

E3 Ligase ◽

Insoluble Fraction ◽

Progressive External Ophthalmoplegia ◽

Brdu Incorporation ◽

Mitochondrial Outer Membrane ◽

Wild Type ◽

Mtdna Replication ◽

Enhanced Degradation

Mutations in mitochondrial replicative polymerase PolγA lead to progressive external ophthalmoplegia (PEO). While PolγA is the known central player in mitochondrial DNA (mtDNA) replication, it is unknown whether a regulatory process exists on the mitochondrial outer membrane which controlled its entry into the mitochondria. We now demonstrate that PolγA is ubiquitylated by mitochondrial E3 ligase, MITOL (or MARCH5, RNF153). Ubiquitylation in wild-type (WT) PolγA occurs at Lysine 1060 residue via K6 linkage. Ubiquitylation of PolγA negatively regulates its binding to Tom20 and thereby its mitochondrial entry. While screening different PEO patients for mitochondrial entry, we found that a subset of the PolγA mutants is hyperubiquitylated by MITOL and interact less with Tom20. These PolγA variants cannot enter into mitochondria, instead becomes enriched in the insoluble fraction and undergo enhanced degradation. Hence, mtDNA replication, as observed via BrdU incorporation into the mtDNA, was compromised in these PEO mutants. However, by manipulating their ubiquitylation status by 2 independent techniques, these PEO mutants were reactivated, which allowed the incorporation of BrdU into mtDNA. Thus, regulated entry of non-ubiquitylated PolγA may have beneficial consequences for certain PEO patients.

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Biogenesis of a Mitochondrial Outer Membrane Protein in Trypanosoma brucei

Journal of Biological Chemistry ◽

10.1074/jbc.m116.755983 ◽

2017 ◽

Vol 292 (8) ◽

pp. 3400-3410 ◽

Cited By ~ 8

Author(s):

Julia Bruggisser ◽

Sandro Käser ◽

Jan Mani ◽

André Schneider

Keyword(s):

Outer Membrane ◽

Trypanosoma Brucei ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Targeting ◽

Targeting Signal ◽

Transgenic Cells ◽

Membrane Spanning ◽

Necessary And Sufficient ◽

Inducible Rnai ◽

Positively Charged

The mitochondrial outer membrane (OM) contains single and multiple membrane-spanning proteins that need to contain signals that ensure correct targeting and insertion into the OM. The biogenesis of such proteins has so far essentially only been studied in yeast and related organisms. Here we show that POMP10, an OM protein of the early diverging protozoan Trypanosoma brucei, is signal-anchored. Transgenic cells expressing variants of POMP10 fused to GFP demonstrate that the N-terminal membrane-spanning domain flanked by a few positively charged or neutral residues is both necessary and sufficient for mitochondrial targeting. Carbonate extraction experiments indicate that although the presence of neutral instead of positively charged residues did not interfere with POMP10 localization, it weakened its interaction with the OM. Expression of GFP-tagged POMP10 in inducible RNAi cell lines shows that its mitochondrial localization depends on pATOM36 but does not require Sam50 or ATOM40, the trypanosomal analogue of the Tom40 import pore. pATOM36 is a kinetoplastid-specific OM protein that has previously been implicated in the assembly of OM proteins and in mitochondrial DNA inheritance. In summary, our results show that although the features of the targeting signal in signal-anchored proteins are widely conserved, the protein machinery that mediates their biogenesis is not.

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Mmm2p, a mitochondrial outer membrane protein required for yeast mitochondrial shape and maintenance of mtDNA nucleoids

The Journal of Cell Biology ◽

10.1083/jcb.200308012 ◽

2004 ◽

Vol 164 (5) ◽

pp. 677-688 ◽

Cited By ~ 113

Author(s):

Matthew J. Youngman ◽

Alyson E. Aiken Hobbs ◽

Shawn M. Burgess ◽

Maithreyan Srinivasan ◽

Robert E. Jensen

Keyword(s):

Mitochondrial Dna ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Mitochondrial Outer Membrane ◽

Dynamic Interactions ◽

Cell Biol

The mitochondrial outer membrane protein, Mmm1p, is required for normal mitochondrial shape in yeast. To identify new morphology proteins, we isolated mutations incompatible with the mmm1-1 mutant. One of these mutants, mmm2-1, is defective in a novel outer membrane protein. Lack of Mmm2p causes a defect in mitochondrial shape and loss of mitochondrial DNA (mtDNA) nucleoids. Like the Mmm1 protein (Aiken Hobbs, A.E., M. Srinivasan, J.M. McCaffery, and R.E. Jensen. 2001. J. Cell Biol. 152:401–410.), Mmm2p is located in dot-like particles on the mitochondrial surface, many of which are adjacent to mtDNA nucleoids. While some of the Mmm2p-containing spots colocalize with those containing Mmm1p, at least some of Mmm2p is separate from Mmm1p. Moreover, while Mmm2p and Mmm1p both appear to be part of large complexes, we find that Mmm2p and Mmm1p do not stably interact and appear to be members of two different structures. We speculate that Mmm2p and Mmm1p are components of independent machinery, whose dynamic interactions are required to maintain mitochondrial shape and mtDNA structure.

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Mmm1p, a Mitochondrial Outer Membrane Protein, Is Connected to Mitochondrial DNA (Mtdna) Nucleoids and Required for Mtdna Stability

The Journal of Cell Biology ◽

10.1083/jcb.152.2.401 ◽

2001 ◽

Vol 152 (2) ◽

pp. 401-410 ◽

Cited By ~ 157

Author(s):

Alyson E. Aiken Hobbs ◽

Maithreyan Srinivasan ◽

J. Michael McCaffery ◽

Robert E. Jensen

Keyword(s):

Mitochondrial Dna ◽

Outer Membrane ◽

Fluorescent Protein ◽

Mitochondrial Outer Membrane ◽

Temperature Sensitive ◽

Restrictive Temperature ◽

Yeast Saccharomyces Cerevisiae ◽

Saccharomyces Cerevisiae Mitochondria ◽

The Matrix ◽

Green Fluorescent

In the yeast Saccharomyces cerevisiae, mitochondria form a branched, tubular reticulum in the periphery of the cell. Mmm1p is required to maintain normal mitochondrial shape and in mmm1 mutants mitochondria form large, spherical organelles. To further explore Mmm1p function, we examined the localization of a Mmm1p–green fluorescent protein (GFP) fusion in living cells. We found that Mmm1p-GFP is located in small, punctate structures on the mitochondrial outer membrane, adjacent to a subset of matrix-localized mitochondrial DNA nucleoids. We also found that the temperature-sensitive mmm1-1 mutant was defective in transmission of mitochondrial DNA to daughter cells immediately after the shift to restrictive temperature. Normal mitochondrial nucleoid structure also collapsed at the nonpermissive temperature with similar kinetics. Moreover, we found that mitochondrial inner membrane structure is dramatically disorganized in mmm1 disruption strains. We propose that Mmm1p is part of a connection between the mitochondrial outer and inner membranes, anchoring mitochondrial DNA nucleoids in the matrix.

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Characterization of mitochondrial outer membrane proteins involved in mitochondrial protein import in Trypanosoma brucei

The FASEB Journal ◽

10.1096/fasebj.22.1_supplement.813.2 ◽

2008 ◽

Vol 22 (S1) ◽

Author(s):

Shvetank Sharma ◽

Ujjal K Singha ◽

Minu Chaudhuri

Keyword(s):

Membrane Proteins ◽

Outer Membrane ◽

Trypanosoma Brucei ◽

Protein Import ◽

Outer Membrane Proteins ◽

Mitochondrial Protein ◽

Mitochondrial Outer Membrane ◽

Mitochondrial Protein Import

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Cryo-Electron Microscopy and Correlation Averaging of Frozen-Hydrated, Polymorphic 2D Crystals of the Mitochondrial Outer Membrane Channel

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100179257 ◽

1990 ◽

Vol 48 (1) ◽

pp. 100-101

Author(s):

Xiao-Wei Guo

Keyword(s):

Outer Membrane ◽

Hexagonal Lattice ◽

Mitochondrial Outer Membrane ◽

Rectangular Lattice ◽

Electron Microscopic ◽

Cryo Electron Microscopy ◽

Voltage Dependent ◽

Outer Membrane Channel ◽

2D Crystals ◽

Vdac Channel

Voltage-dependent, anion-selective channels (VDAC) are formed in the mitochondrial outer membrane (mitOM) by a 30-kDa polypeptide. These channels form ordered 2D arrays when mitOMs from Neurospora crassa are treated with soluble phospholipase A2. We obtain low-dose electron microscopic images of unstained specimens of VDAC crystals preserved in vitreous ice, using a Philips EM420 equipped with a Gatan cryo-transfer stage. We then use correlation analysis to compute average projections of the channel crystals. The procedure involves Fourier-filtration of a region within a crystal field to obtain a preliminary average that is subsequently cross-correlated with the entire crystal. Subregions are windowed from the crystal image at coordinates of peaks in the cross-correlation function (CCF, see Figures 1 and 2) and summed to form averages (Figure 3).The VDAC channel forms several different types of crystalline arrays in mitOMs. The polymorph first observed during phospholipase treatment is a parallelogram array (a=13 run, b=11.5 run, θ==109°) containing 6 water-filled pores per unit cell. Figure 1 shows the CCF of a sub-field of such an “oblique” array used to compute the correlation average of Figure 3A. With increased phospholipase treatment, other polymorphs are observed, often co-existing within the same crystal. For example, two distinct (but closely related) types of lattices occur in the field corresponding to the CCF of Figure 2: a “contracted” version of the parallelogram lattice (a=13 run, b=10 run, θ=99°), and a near-rectangular lattice (a=8.5 run, b=5 nm). The pattern of maxima in this CCF suggests that a third, near-hexagonal lattice (a=4.5 nm) may also be present. The correlation averages of Figures 3B-D were computed from polycrystalline fields, using peak coordinates in regions of CCFs corresponding to each of the three lattice types.

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