21 research outputs found
A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion
A monoclonal antibody raised against bacterially expressed MPV17 sequences shows peroxisomal, endosomal and lysosomal localisation in U2OS cells
Recessive mutations in the MPV17 gene cause mitochondrial DNA depletion syndrome, a fatal infantile genetic liver disease in humans. Loss of function in mice leads to glomerulosclerosis and sensineural deafness accompanied with mitochondrial DNA depletion. Mutations in the yeast homolog Sym1, and in the zebra fish homolog tra cause interesting, but not obviously related phenotypes, although the human gene can complement the yeast Sym1 mutation. The MPV17 protein is a hydrophobic membrane protein of 176 amino acids and unknown function. Initially localised in murine peroxisomes, it was later reported to be a mitochondrial inner membrane protein in humans and in yeast. To resolve this contradiction we tested two new mouse monoclonal antibodies directed against the human MPV17 protein in Western blots and immunohistochemistry on human U2OS cells. One of these monoclonal antibodies showed specific reactivity to a protein of 20 kD absent in MPV17 negative mouse cells. Immunofluorescence studies revealed colocalisation with peroxisomal, endosomal and lysosomal markers, but not with mitochondria. This data reveal a novel connection between a possible peroxisomal/endosomal/lysosomal function and mitochondrial DNA depletion
Channel-Forming Activities in the Glycosomal Fraction from the Bloodstream Form of Trypanosoma brucei
Background: Glycosomes are a specialized form of peroxisomes (microbodies) present in unicellular eukaryotes that belong to the Kinetoplastea order, such as Trypanosoma and Leishmania species, parasitic protists causing severe diseases of livestock and humans in subtropical and tropical countries. The organelles harbour most enzymes of the glycolytic pathway that is responsible for substrate-level ATP production in the cell. Glycolysis is essential for bloodstream-form Trypanosoma brucei and enzymes comprising this pathway have been validated as drug targets. Glycosomes are surrounded by a single membrane. How glycolytic metabolites are transported across the glycosomal membrane is unclear. Methods/Principal Findings: We hypothesized that glycosomal membrane, similarly to membranes of yeast and mammalian peroxisomes, contains channel-forming proteins involved in the selective transfer of metabolites. To verify this prediction, we isolated a glycosomal fraction from bloodstream-form T.brucei and reconstituted solubilized membrane proteins into planar lipid bilayers. The electrophysiological characteristics of the channels were studied using multiple channel recording and single channel analysis. Three main channel-forming activities were detected with current amplitudes 70–80 pA, 20–25 pA, and 8–11 pA, respectively (holding potential +10 mV and 3.0 M KCl as an electrolyte). All channels were in fully open state in a range of voltages 6150 mV and showed no sub-conductance transitions. The channel with current amplitude 20–25 pA is anion-selective (P K+/P Cl2,0.31), while the other two types of channels are slightl
The human mitochondrial DNA depletion syndrome gene MPV17 encodes a non-selective channel that modulates membrane potential
Abstract
The human MPV17-related mitochondrial DNA depletion syndrome is an inherited autosomal recessive disease caused by mutations in the inner mitochondrial membrane protein MPV17. Although more than 30 MPV17 gene mutations were shown to be associated with mitochondrial DNA depletion syndrome, the function of MPV17 is still unknown. Mice deficient in Mpv17 show signs of premature aging. In the present study, we used electrophysiological measurements with recombinant MPV17 to reveal that this protein forms a non-selective channel with a pore diameter of 1.8 nm and located the channel’s selectivity filter. The channel was weakly cation-selective and showed several subconductance states. Voltage-dependent gating of the channel was regulated by redox conditions and pH and was affected also in mutants mimicking a phosphorylated state. Likewise, the mitochondrial membrane potential (Δψm) and the cellular production of reactive oxygen species were higher in embryonic fibroblasts from Mpv17−/− mice. However, despite the elevated Δψm, the Mpv17-deficient mitochondria showed signs of accelerated fission. Together, these observations uncover the role of MPV17 as a Δψm-modulating channel that apparently contributes to mitochondrial homeostasis under different conditions
Electron microscopy of cellular organelles separated by Optiprep gradient centrifugation.
<p>Fractions enriched in glycosomes (fractions 2–5, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>), fragments of flagella (fractions 8–11) or mitochondria and other organelles (fractions 15–18) were combined and processed for EM examination (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). (<b>A</b> and <b>B</b>) Isolated glycosomes shown at lower (<b>A</b>) and higher (<b>B</b>) magnifications. The fraction consists mostly of glycosomes. Some contamination by fragments of flagella is also visible. Importantly, fragments of flagella (paraflagellar rods and axonemes) show no sign of attachment to the flagellar membrane. Note the presence of intact glycosomes as electron-dense vesicles surrounded by a single membrane (marked by arrows in panel <b>B</b>). (<b>C</b> and <b>D</b>) Fractions enriched in flagella at low (<b>C</b>) and high (<b>D</b>) magnifications. One can see many paraflagellar rods in longitudinal section (<b>C</b>) and recognize flagellar axonemes (marked by arrows in panel <b>D</b>). Some glycosomes are also visible in panel <b>C</b>. (<b>E</b> and <b>F</b>) Composition of the fraction from the top of the Optiprep gradient that is enriched with mitochondria. Several types of organelles – mitochondria, lysosomes, lipid droplets, clathrin-coated vesicles, and components from the flagellar apparatus – can be observed. Note the shrinking of the mitochondrial inner membrane (see panel <b>F</b>) apparently due to osmotic misbalance. Scale bars: 2 µm (<b>C</b> and <b>E</b>); 1 µm (<b>A</b>); 0.5 µm (<b>D</b> and <b>F</b>), and 0.1 µm (<b>B</b>).</p
Detection of channel-forming activities in subcellular fractions.
<p>Fractions 2–4 (glycosomes), 8–11 (fragments of flagella), and 15–18 (mitochondria) from Optiprep density gradients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g001" target="_blank">Figure 1A</a>) were combined and treated with Genapol X-080 to solubilize membrane proteins (see the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#s4" target="_blank">Materials and methods</a> section). After sedimentation of insoluble material, aliquots of the resulting supernatants were used for MCR (<b>A</b>–<b>C</b>) or SCA (<b>D</b>). (<b>A</b>) Traces of the current monitoring in the presence of glycosomal (upper panel) or mitochondrial (lower panel) preparations. The middle trace represents a timescale-expanded current recording of the upper trace. The bath solution contained 3 M KCl and the applied voltage was +10 mV. (<b>B</b>) Histograms of insertion events registered in subcellular fractions (see panel <b>A</b>). Bin size is 4.0 pA. The total number of insertion events (I.e.) is indicated. Here and in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3</a> C (upper panel) all insertion events with current increments over 180 pA (for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figure 3C</a>, lower panel −90 pA) are combined in one bin (180 pA or 90 pA, respectively). Note that the amount of insertion events in the flagella fraction (see <b>B</b>, middle panel) is lower than that observed in other fractions. This is mainly due to low channel-forming activity (per protein content) in the preparations of this fraction. For the sake of compatibility we used the same amounts of protein for measurements in different fractions. (<b>C</b>) Histograms of insertion events detected for glycosomal preparations using NH<sub>4</sub>Cl as the electrolyte. Bin size: 4 pA (upper panel) or 2 pA (lower panel). See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g003" target="_blank">Figures 3A and 3B</a> for other details. (<b>D</b>) Trace of the current monitoring using the glycosomal fraction (initial holding potential +10 mV) indicating the insertion (marked by one asterisk) of a large-conductance channel that spontaneously closed (marked by two asterisks) after stepwise (each step is +10 mV) increase in the holding potential up to 50 mV.</p
SCA of a very-low-conductance channel.
<p>(<b>A</b>) Current recording of a single very-low-conductance channel. The bath solution (panels <b>A</b>, <b>B</b>, and <b>C</b>) contained 3 M KCl. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>A</b></a> for other details. (<b>B</b>) Current trace of the channel in response to the shown voltage-ramp protocol. Dotted line indicates the current level at zero holding potential. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Current traces of a single channel in response to the indicated voltage-step protocol. (<b>D</b>) Ion-selectivity of the channel. See legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0034530#pone-0034530-g004" target="_blank">Figure 4<b>D</b></a> for details.</p
SCA of a high-conductance channel.
<p>(<b>A</b>) Current trace of a single high-conductance channel. The insertion event (marked by an asterisk) was registered at +10 mV and the applied voltage was then switched to −10 mV. The dashed line indicates the current level (zero) before insertion of the channel. The data in panels <b>A</b>, <b>B</b>, and <b>C</b> were collected using 3 M KCl as the electrolyte. (<b>B</b>) Current trace of the channel in response to the indicated voltage-ramp protocol. Note the near linear dependence of the current on the applied voltage. (<b>C</b>) Single channel currents in response to the indicated voltage-step protocol. (<b>D</b>) Dependence of the single channel conductance on the KCl concentration. After detection of a single channel insertion using 3 M KCl as bath solution (holding potential +10 mV), the electrolyte was diluted and registration of the current amplitudes of the same channel was conducted at 2.0 M and 1.0 M KCl, respectively. Data points are mean±SD for at least 4 independent measurements. (<b>E</b>) Current traces of a single channel in response to a low-speed linear increase (upper trace) or decrease (lower trace) of the holding potential. The bath solution contained 1.0 M NH<sub>4</sub>Cl, 20 mM Tris-Cl, pH 7.8, and 2 mM DTT at both sides of the membrane. Note that the channel was still open even at hyperpolarizing holding potentials of ±150 mV. (<b>F</b>) Current-voltage relationship of the high-conductance channel under asymmetric salt conditions: 3.0 M KCl <i>trans</i>/1.5 M KCl <i>cis</i> compartment. The insertion of a single channel was detected at 3 M KCl at both sides of the membrane and at a voltage of +10 mV, then the electrolyte concentration in the <i>cis</i> compartment was decreased by dilution and an initial current recording was conducted at zero potential followed by stepwise (±10 mV) change of the applied voltage. Data points are mean±SD, n = 4–5. Bars in some cases are smaller than symbols.</p