rim2 protein  (Cytomatrix Pty Ltd)

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) A sectoring screen was performed to find mutations that conferred synthetic lethality when combined with Δmrs3/Δmrs4 as described previously [20]. The synthetic lethal mutant J116 (tsa1-116 Δmrs3Δmrs4 [pTSV31A-MRS4-URA3]) was complemented by low-(pRS415-RIM2) and high-copy (YEp351-RIM2)-containing plasmids carrying the RIM2 open reading frame and flanking regions. The transformants were streaked on 5-fluoroorotic acid plates to remove the covering copy of MRS4, and only the RIM-containing transformants were viable (left-hand side). Similarly, the synthetic lethal mutant J386 (rad50-386 Δmrs3/Δmrs4 [pTS-V31A-MRS4-URA3]) was analysed and only the RIM2-containing transformants were viable (right-hand side). These mutants were also complemented by TSA1 for J116 and RAD50 for J386 respectively. (B) RIM2 overexpression improves the iron-dependent growth defect of the Δmrs3/Δmrs4 mutant. The Δmrs3/Δmrs4 strain [MATa ura3-52 lys2-801(amber) trp1-Δ1 ade2-101(ochre) his3-Δ200 cyh2 Δmrs4::KAN Δmrs3::URA3] was transformed with a high-copy-number plasmid carrying the genomic fragment of RIM2 or empty plasmid. Serial dilutions of the transformants were spotted on to defined medium plates with different available iron concentrations. All plates contained standard defined medium, 1 mM ferrozine and 50 mM Mes buffer (pH 6.5). In addition, 0, 25, 100 and 350 μM ferrous ammonium sulfate (from top to bottom) were added back.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Mutagenesis, Over Expression, Transformation Assay, Plasmid Preparation

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) The triple mutant Δmrs3Δmrs4Δyfh1 was maintained anaerobically and transformed with empty plasmid YEp351 or YEp351-RIM2. The WT control strain YPH499 was handled similarly. The transformants were then streaked on to standard defined medium in air. The triple mutant transformed with RIM2 showed enhanced growth compared with empty plasmid (left-hand plate, left side). (B) Rim2 overexpression restores the rate of iron insertion into porphyrin in Δmrs3Δmrs4Δyfh1 mutant mitochondria. Mitochondria (0.5 mg) were treated with 5 mM NADH for 2 min. PPO (2 μM) was added and protoporphyrin IX fluorescence was measured over time (excitation at 410 nm, emission at 632 nm). After 100 s, 1 μM ferrous ammonium sulfate was added and tracing was continued. At the end of the 5 min time trace, fluorescent emissions were scanned from 550 nm to 700 nm (bottom panel). Cells were cultured under low oxygen in argon-bubbled flasks in defined glucose medium and then shifted to air for 4 h in defined raffinose medium. Mitochondria were isolated. white circle, YPH499; black circle, Δmrs3Δmrs4Δyfh1[YEp351-RIM2]; grey circle, Δmrs3Δmrs4Δyfh1. (C) Immunoblot analysis of mitochondrial proteins from: WT YPH499 (lane 1); ΔΔΔ, Δmrs3Δmrs4Δyfh1 (lane 2); ΔΔΔ/Rim2, triple mutant transformed with YEp351-Rim2 (lane 3). Mitochondria (100 μg equivalent) were separated by SDS/PAGE (13% gel) and transferred on to a Protran nitrocellulose membrane for blotting with antibodies as indicated. Aco1, aconitase; Rim2, Yfh1 (yeast frataxin homologue); Cyc1, cytochrome c.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Mutagenesis, Transformation Assay, Plasmid Preparation, Control, Over Expression, Fluorescence, Cell Culture, Isolation, Western Blot, SDS Page, Membrane

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) Immunoblots of mitochondrial proteins. Mitochondria were isolated from WT grown in raffinose medium (YPH499, lane 1), or from Gal-Rim2 Δmrs3Δmrs4 grown in raffinose medium (Gal-Rim2↓Δ3/4, lane 2) or galactose medium (Gal-Rim2↑Δ3/4, lane 3). A second set of cultures was supplemented with 100 μM ferrous ammonium sulfate (lanes 4–6). Mitochondria were analysed by immunoblotting with antibodies against mitochondrial proteins. (B) Cytochromes in whole cells. Cells from WT (middle trace, YPH499), Rim2-induced (top trace, Gal-Rim2↑Δ3/4) or Rim2-repressed (bottom trace, Gal-Rim2↓Δ3/4) were evaluated for their low temperature (– 191°C) UV–visible spectra [25]. Cells were grown for 48 h in defined medium with raffinose (WT and repressed cells) or raffinose-galactose (induced cells) as the carbon source. Cytochrome peaks (c, c1, b, a + a3) and the zinc protoporphyrin peak (Zn-PPIX) are indicated. (C) A fluorescence assay for haem synthesis from added porphyrin and iron in isolated mitochondria. Rim2-induced (black trace, Gal-Rim2↑Δ3/4) or Rim2-repressed (grey trace, Gal-Rim2↓Δ3/4) mitochondria were evaluated as described in the legend to Figure 2(B). Time traces are shown in the top panel and emission scans are shown in bottom panel. (D) 55Fe incorporation into haem. Mitochondria were isolated from YPH499, Gal-Rim2↓Δ3/4, Gal-Rim2↑Δ3/4 or the same grown in standard defined or iron-supplemented media as described in (A). Haem synthesis was measured as follows: mitochondria (0.2 mg) were incubated with 5 mM NADH and 2 μM PPO for 3 min at room temperature (25°C), followed by the addition of 5 μM ferrous [55Fe]ascorbate. After a 5 min incubation, the reaction was stopped by the addition of hydrochloric acid and haem-associated 55Fe was determined by organic extraction with methyl ethyl ketone and scintillation counting of the radionuclide. Results shown are from three assays, and are means ± S.D.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Western Blot, Isolation, Fluorescence, Incubation, Extraction

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) Mitochondria from Gal-Rim2↓ or Gal-Rim2↑ in a WT context were subjected to immunoblotting for Rim2, aconitase (Aco1), cytochrome c (Cyc1), ferrochelatase (Hem15), yeast frataxin homologue (Yfh1) and phosphate carrier protein (Mir1). (B) Cytochromes in whole cells. Cells from the Rim2-induced (Gal-Rim2↑) or Rim2-repressed (Gal-Rim2↓) cultures were evaluated for their low temperature UV–visible spectra [25]. In a separate culture, the repressed Gal-Rim2 strain was grown in raffinose medium supplemented with 100 μM iron as ferric ammonium sulfate (Gal-Rim2↓ + Fe). Low temperature UV–visible spectra were collected [25]. Cytochrome peaks (c, c1, b, a + a3) and the zinc protoporphyrin peak (Zn-PPIX) are indicated. (C) Fluorescent haem synthesis assay. Mitochondria were incubated with PPO for 100 s followed by iron addition (1 μM ferrous ammonium sulfate) and protoporphyrin IX fluorescence was monitored. Open circle, WT or YPH499; black circle, Gal-Rim2↑; grey circle, Gal-Rim2↓). Assays were performed as described in the legend for Figure 2.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Western Blot, Incubation, Fluorescence

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: Mitochondria were isolated from WT, Gal-Rim2↓ or Gal-Rim2↑ cultures. In some cases, mitochondria were ruptured by sonication in hypotonic buffer [50 mM Tris/HCl (pH 7.5)]. Top panel: haem synthesis was measured following the addition of 2 μM PPO and 5 μM [55Fe]ferrous ascorbate to intact mitochondria or mitochondrial lysate. After a 5 min incubation at room temperature (25°C), radiolabelled haem was extracted and counted in a scintillation counter. Bottom panel: haem synthesis was measured as described above, except that the iron concentration for the assay was 1 μM [55Fe]ferrous ascorbate.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Isolation, Sonication, Incubation, Concentration Assay

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) Enzyme activities for aconitase (left-hand panel), succinate dehydrogenase (middle panel) and malate dehydrogenase (right-hand panel) were measured for mitochondria from Gal-Rim2↓ or Gal-Rim2↑. Results shown are arbitrary units from three assays, and are means ± S.D. (B) Fe–S cluster synthesis. Mitochondria from Gal-Rim2↓ (lanes 1–3 and 7–9) or Gal-Rim2↑ (lanes 4–6 and 10–12), were examined for Fe–S cluster synthesis activity by labelling with [35S]cysteine and separation of the matrix fraction on a native gel. In some samples (lanes 7–12), 10μM ferrous ascorbate was added during the labelling reaction. Labelling reactions were performed for 15, 30 or 60 min in each case. The labelled band, Aco1[Fe-35S], represents newly formed Fe–S clusters on aconitase.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Activity Assay

Journal: The Biochemical journal

Article Title: Rim2, a pyrimidine nucleotide exchanger, is needed for iron utilization in mitochondria

doi: 10.1042/BJ20111036

Figure Lengend Snippet: (A) TTP uptake. Mitochondria energized with NADH were incubated with 0.4 μCi (6 nM final) of [3H]TTP for 5 min at room temperature (25°C). After washes in buffer [50 mM Tris/HCl (pH 7.5) and 0.6 M sorbitol], the radioactivity of the mitochondrial pellet was measured in a scintillation counter. White bars, Gal-Rim2↓Δ3/4; grey bars, Gal-Rim2↑Δ3/4; and black bars, YPH499. (B) [3H]TTP exchange. Gal-Rim2↑Δ3/4 or Gal-Rim2↓3/4 mitochondria were labelled with [3H]TTP and unincorporated radioactivity was removed. Mitochondria were resuspended in 100 μl of buffer [50 mM Tris/HCl (pH 7.5) and 0.6 M sorbitol] with no nucleotides, or with 1 mM TTP or 1 mM TMP. After 10 min incubation at room temperature, samples were centrifuged to separate mitochondria and the supernatant, and the radioactivity was determined by scintillation counting. Top panel, Gal-Rim2↑Δ3/4 mitochondria, white boxes indicate the supernatant, black boxes indicate the mitochondrial pellet. Bottom panel, Gal-Rim2↓Δ3/4 mitochondria, white boxes indicate supernatant, grey boxes indicate the mitochondrial pellet. TTP- or TMP-dependent export of [3H]TTP radionuclide occurs only in the setting of Rim2 expression. (C) [3H]TTP uptake and 55Fe incorporation into haem in parallel experiments. Top panel, [3H]TTP uptake. Gal-Rim2↑ or Gal-Rim2↓ mitochondria were tested for radionuclide TTP uptake in HS buffer [50 mM Hepes/KOH and 0.6 M sorbitol] or the same buffer with 50 mM NaCl added (HS + NaCl). Uptake was allowed to proceed for 5 min at room temperature. Mitochondrial-associated [3H]TTP was determined in triplicate assays. Bottom panel, 55Fe incorporation into haem. Iron (5 μM [55Fe]ascorbate) and porphyrin (2 μM PPO) were added to mitochondria in HS buffer or HS + NaCl. Uptake was allowed to proceed for 5 min and haem was extracted. (D) Porphyrin stimulation of Rim2-dependent TTP exchange in mitochondria. Gal-Rim2↑Δ3/4 or Gal-Rim2↓Δ3/4 mitochondria were incubated with [3H]TTP to load them and unincorporated radioactivity was removed. The mitochondria were resuspended in buffer [50 mM Tris/HCl (pH 7.5) and 0.6 M sorbitol] with or without 1 μM PPO. Exchanges was allowed to proceed for 10 min, and supernatant and mitochondria were separated and subjected to scintillation counting. White boxes, supernatants; black boxes, mitochondrial pellets.

Article Snippet: An antibody against Rim2 protein was made by injecting rabbits with the unique Rim2 peptide (SIEKFGYQAEGTKSTSEKVKEWC) conjugated to KLH (keyhole-limpet haemocyanin) followed by affinity-purification of the immune serum (Covance).

Techniques: Incubation, Radioactivity, Expressing

Journal: PLoS ONE

Article Title: Calcium Channel-Dependent Molecular Maturation of Photoreceptor Synapses

doi: 10.1371/journal.pone.0063853

Figure Lengend Snippet: Antibodies.

Article Snippet: RIM2 , Mouse , 1∶100 , Recombinant protein (aa 461–987) of rat RIM2. , , Synaptic Systems.

Techniques: Transduction, Recombinant, Purification, Sequencing

Journal: PLoS ONE

Article Title: Calcium Channel-Dependent Molecular Maturation of Photoreceptor Synapses

doi: 10.1371/journal.pone.0063853

Figure Lengend Snippet: Several protein–protein interactions are affected such as the association of Ribeye, Piccolo and Bassoon. The investigated proteins in the model are depicted with a red outline. The proteins with an abnormal expression and/or distribution are shown in grey. Synaptic ribbons form a complex with several other proteins such as CAST, RIM2, Rab3a, and Munc13. In the Ca V 1.4 (α1F) -KO, RIM2 expression is compromised. The vesicle related machinery (VAMP2, synaptophysin and complexin 3 and 4) is not affected in the Ca V 1.4 (α1F) -KO. Other presynaptic proteins such PMCA, PSD-95 and Veli3 are also affected in the Ca V 1.4 (α1F) -KO. Furthermore, β-Dystroglycan, a protein necessary for the invagination of bipolar cells is absent from the photoreceptor synapses in the Ca V 1.4 (α1F) -KO.

Article Snippet: RIM2 , Mouse , 1∶100 , Recombinant protein (aa 461–987) of rat RIM2. , , Synaptic Systems.

Techniques: Protein-Protein interactions, Expressing