Mitochondrial metabolism

Tim44 is an essential component of the translocase of the inner mitochondrial membrane (TIM) complex that mediates transport of nuclear encoded mitochondrial precursors across the inner membrane. Here, we have investigated the topology of Tim44 by probing mitochondria with membrane impermeable 3-(N-maleimidopropionyl)biocytin (MPB) followed by the specific immunoprecipitation of modified proteins. Our data indicate that a single cysteine residue, Cys-369, located in the C-terminal domain of the yeast Tim44 is exposed to the mitochondrial intermembrane space [1].

MPB/3-(N-maleimidopropionyl)biocytin labelling. [1]
Mitochondria protein (800 microgram) were resuspended in 1 ml of buffer containing 0.6 M sorbitol, 20 mM Hepes–KOH, pH 7.4 for yeast mitochondria and 0.23 M mannitol, 70 mM sucrose, 20 mM Hepes–KOH, pH 7.2 for rat liver mitochondria. MPB (0.5 mM) was added and the mitochondrial suspension was divided into two aliquots. One sample was incubated with MPB/3-(N-maleimidopropionyl)biocytin for 30 min, 4 °C. The reaction was stopped by incubation with 2 mM DTT for 10 min, 4 °C. Mitochondria were solubilised in 0.5% Triton X-100 and centrifuged at 14 000 rpm, 10 min. Supernatants were subjected to immunoprecipitation for 3–4 h, 4 °C using antisera against mitochondrial proteins followed by incubation with ProteinA-Sepharose. Pellets were washed four times with TBS-Tween buffer, loaded with Laemmli solubilisation buffer and boiled 5 min. The second aliquot of the mitochondrial suspension (order 2) was treated as the previous sample except that mitochondria were solubilised with Triton X-100 at the beginning of the MPB treatment.

[1].Probing the membrane topology of a subunit of the mitochondrial protein translocase, Tim44, with biotin maleimide. Biochem Biophys Res Commun. 2002 Apr 26;293(1):321-6.

3-(n-maleimidopropionyl)biocytin is a peptide.

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Fig. 1 shows the results of labelling yeast mitochondria with MPB/3-(N-maleimidopropionyl)biocytin in the absence and in the presence of Triton X-100. After labelling, proteins were subjected to immunoprecipitation with respective antibodies (left column) followed by Western blotting using avidin-peroxidase (right column). Proteins of the outer mitochondrial membrane, Tom40 and Tom70, were readily labelled with MPB in intact mitochondria. The degree of labelling of Tom70 was greater upon solubilisation of mitochondrial membranes with detergent. It has to be mentioned that amounts of immunoprecipitated proteins were equal in both samples as verified by staining of blots with respective antibodies (not shown). Imp1 was labelled in intact as well as in solubilised mitochondria which was in agreement with topology of this protein described previously. Tim44 was another inner membrane protein that could be labelled with MPB in intact mitochondria. The degree of Tim44 labelling by MPB/3-(N-maleimidopropionyl)biocytin was similar in intact and solubilised mitochondria suggesting that the only present Cys-369 was free and exposed to the intermembrane space. Two other inner membrane proteins, Yta10 and Yta12, as well as matrix located α-MPP were biotinylated only upon solubilisation of mitochondria with Triton X-100 suggesting that the inner membrane was indeed impermeable for MPB. It has to be stressed that at high concentrations (⩾ 200 uM) MPB was reported to penetrate plasma membrane of mammalian cells [18]. In our experiments with intact mitochondria we did not observe any significant modification (⩽5%) of cysteine residues located in the matrix proteins even at MPB concentration of 500 uM. At lower concentrations of MPB efficiency of protein modification decreased but the pattern of protein labelling in different intra-mitochondrial compartments was similar.

Rat Tim44 contains three cysteine residues and one of these residues is located close to the Tim44 C-terminus. We also probed the Tim44 topology in isolated rat liver mitochondria. Under the conditions used rat Tim44 could not be modified with MPB/3-(N-maleimidopropionyl)biocytin even in solubilised mitochondria indicating that all cysteines were inaccessible to MPB. Rat mHsp70 was strongly labelled with MPB after solubilisation of the mitochondrial membranes, moreover, labelled mHsp70 was coimmunoprecipitated with anti-Tim44 antibodies (data not shown). Previously, we have observed that the in vitro synthesised precursor of Nicotiana plumbaginifolia ATP-synthase F1β subunit could be modified with MPB only when the precursor was unfolded on the mitochondrial surface. Cysteines hidden in the folded protein were not available for modification due to the hydrophilic nature and bulkiness of MPB. This might be the case for rat Tim44. Alternatively, cysteine groups can form inter or intra-molecular disulfide bridges also preventing their modification. Sequence alignment of yeast and rat Tim44 showed no conservation in the position of cysteines at their C-terminal domain (Fig. 2A). Yeast mitochondrial protein import shows no sensitivity to treatment with sulfhydryl-specific reagents in contrast to plant and mammalian mitochondrial protein import systems. Taken together, these data suggest different characteristics of cysteine residues in Tim44 from yeast and other species.

In conclusion, we have shown that in the intact yeast mitochondria a single cysteine, Cys-369 located in the C-terminal domain of the Tim44 was labelled with membrane-impermeable maleimide, MPB/3-(N-maleimidopropionyl)biocytin. The C-terminal portion of Tim44 from a variety of species has been predicted to have positionally conserved stretch of hydrophobic amino acids. In yeast Tim44, this segment contains Cys-369 accessible from the outside of the inner membrane. Taken together these results suggest that the C-terminal part of Tim44 is exposed to the mitochondrial intermembrane space.[1]

C23H33N5O7S

523.60

523.21

102849-12-7

127195

White to off-white solid powder

1.3±0.1 g/cm3

962.5±65.0 °C at 760 mmHg

535.9±34.3 °C

0.0±0.6 mmHg at 25°C

1.569

-1.5

5

8

15

36

893

4

C1[C@H]2[C@@H]([C@@H](S1)CCCCC(=O)NCCCC[C@@H](C(=O)O)NC(=O)CCN3C(=O)C=CC3=O)NC(=O)N2

KWNGAZCDAJSVLC-OSAWLIQMSA-N

InChI=1S/C23H33N5O7S/c29-17(7-2-1-6-16-21-15(13-36-16)26-23(35)27-21)24-11-4-3-5-14(22(33)34)25-18(30)10-12-28-19(31)8-9-20(28)32/h8-9,14-16,21H,1-7,10-13H2,(H,24,29)(H,25,30)(H,33,34)(H2,26,27,35)/t14-,15-,16-,21-/m0/s1

(2S)-6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoic acid

3-(N-Maleimidopropionyl)biocytin; 102849-12-7; N-(3-Maleimidopropionyl)biocytin; 3-MPB; 3-(N-Maleimidylpropionyl)biocytin; (2S)-6-[5-[(3aS,4S,6aR)-2-oxo-1,3,3a,4,6,6a-hexahydrothieno[3,4-d]imidazol-4-yl]pentanoylamino]-2-[3-(2,5-dioxopyrrol-1-yl)propanoylamino]hexanoic acid; Nalpha-(3-Maleimidylpropionyl)Biocytin; 3-(N-Maleimido-propionyl)biocytin;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Typically soluble in DMSO (e.g. 10 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)