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-maleimide propionyl)biotin is a peptide. Figure 1 shows the results of labeling yeast mitochondria with MPB/3-(N-maleimide propionyl)biotin in the presence and absence of Triton X-100. After labeling, the proteins were immunoprecipitated with the corresponding antibodies (left column), followed by Western blot analysis using avidin-peroxidase (right column). In intact mitochondria, the outer membrane proteins Tom40 and Tom70 were readily labeled with MPB. Tom70 showed a higher degree of labeling after dissolving the mitochondrial membrane with detergent. It should be noted that the amount of immunoprecipitated protein was equal in both samples, as verified by staining the blots with the corresponding antibodies (not shown). Imp1 was labeled in both intact and dissolved mitochondria, consistent with the previously described topology of this protein. Tim44 is another inner membrane protein that can be labeled with MPB in intact mitochondria. The degree to which Tim44 was labeled with MPB/3-(N-maleimide propionyl)biotin was similar in both intact and dissolved mitochondria, suggesting that the only present Cys-369 was free and exposed in the intermembrane space. The other two inner membrane proteins, Yta10 and Yta12, as well as α-MPP located in the mitochondrial matrix, were only biotinylated after mitochondria were dissolved with Triton X-100, indicating that the inner membrane is indeed impermeable to MPB. It is worth noting that high concentrations (≥200 μM) of MPB have been reported to penetrate the plasma membrane of mammalian cells [18]. In our experiments using intact mitochondria, we did not observe any significant modification (≤5%) of cysteine residues in the matrix proteins, even at MPB concentrations as high as 500 μM. At lower concentrations of MPB, protein modification efficiency decreased, but protein labeling patterns were similar across different mitochondrial compartments. Rat Tim44 contains three cysteine residues, one of which is located near the C-terminus of Tim44. We also investigated the topological structure of Tim44 in isolated rat liver mitochondria. Under the conditions used, even in dissolved mitochondria, rat Tim44 could not be modified by MPB/3-(N-maleimide propionyl)biotin, indicating that all cysteine residues could not be modified by MPB. After rat mitochondrial membrane dissolution, mHsp70 was strongly labeled with MPB; furthermore, labeled mHsp70 could undergo co-immunoprecipitation with anti-Tim44 antibody (data not shown). Previously, we observed that the in vitro synthesized tobacco ATP synthase F1β subunit precursor could only be modified by MPB when it unfolded on the mitochondrial surface. Due to the hydrophilicity and large size of MPB, cysteine residues hidden in folded proteins cannot be modified. The same may be the case with rat Tim44. In addition, cysteine residues can also form intermolecular or intramolecular disulfide bonds, thereby preventing their modification. Sequence alignment of yeast and rat Tim44 showed that the position of cysteine residues in their C-terminal domains is not conserved (Figure 2A). Unlike the mitochondrial protein importation systems of plants and mammals, yeast mitochondrial protein importation is insensitive to treatment with thiol-specific reagents. In summary, these data suggest that cysteine residues in Tim44 of yeast and other species have different characteristics. In conclusion, we have demonstrated that in intact yeast mitochondria, a single cysteine Cys-369 located in the C-terminal domain of Tim44 is labeled with the membrane-impermeable maleimide MPB/3-(N-maleimide propionyl)biotin. The C-terminal portion of Tim44 is predicted to have a positionally conserved hydrophobic amino acid sequence in multiple species. In yeast Tim44, this fragment contains Cys-369 and is accessible from the outer side of the inner membrane. These results combined suggest that the C-terminal portion 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.)