Isolation of the Tim9·Tim10 complex

For the purification of the Tim9·Tim10 complex, we inserted a sequence encoding the Tim10 protein tagged with nine histidine residues under the control of its endogenous promotor into the pCB1179 plasmid. The expression construct was then transformed into wild type N. crassa strain 74A. This procedure yielded heterokaryotic transformants, all expressing a nanohistidinyl-tagged version of the Tim10 protein, in addition to the wild-type protein. These two versions of the Tim10 protein were present in different ratios in different strains, and hence, transformants were examined for expression levels of the tagged vs. the untagged protein. Two heterokaryonic strains (TA2-1 and TA2-14) with the favourable expression of the Tim10his9 protein were chosen among a large number of transformants. These two strains were further subjected to a microconidiation procedure and from all the homokaryonic strains originating from these microconidia the TA2-14-3 strain was selected. This strain is a homokaryon expressing roughly equal amounts of the Tim10 and Tim10his9 proteins. It grew comparably to the parental wild type strain (data not shown). Mitochondria isolated from the mycelium of this strain were used to purify the Tim9·Tim10 complex, combining metal- affinity and ion-exchange chromatography technics.

To optimize the Ni-NTA purification procedure, following parameters were tested: sodium phosphate, Tris, potassium acetate, HEPES and MOPS buffers, various pH values, different imidazole, zinc and salt concentrations in washing buffers, and different reducing agents. Protein yields and the purity of the Tim9·Tim10 complex released from mitochondria by solubilization with Triton X-100 to those obtained upon disruption of the mitochondrial membrane integrity through sonication were also compared. Although both methods produced complexes of comparable purity, the yield was significantly higher when TX-100 was used. The optimal conditions, established and used from then on, are noted in the “Material and methods” section.

A large scale purification procedure was also established. Different amounts of the loaded protein with respect to column volume were evaluated and subsequently set at 5 g of total protein per 1 ml of the Ni-NTA column matrix. To minimize the possibility of protein degradation during purification steps, flow rate was increased to 5.5 ml/min. This reduced the time required for the loading of the solubilized material but still allowed for complete protein binding as determined by analysis of the flow-through fractions Because of a higher column pressure resulting from the increased flow rate, it was necessary to use the Superflow matrix,

which endures pressures up to 1 MPa, instead of the conventional Ni-NTA Agarose (both from Qiagen).

Figure 14. Purification of the Tim9·Tim10 complex. (A) Tim9·Tim10 complex was isolated from TA2- 14-3 N.crassa mitochondria lysed in sodium phosphate buffer containing 1% (v/v) Triton X-100. The solubilized material was subjected to metal-affinity chromatography; fractions 5-8 from the Ni-NTA column were loaded onto a Resource Q ion-exchange chromatography column. Samples of the entire purification procedure were analysed by high Tris urea SDS-PAGE, and the gel was stained with Coomassie Blue. (lmw, low molecular weight marker; mmw, mini molecular weight marker; MW molecular weight). (B) Tim9·Tim10 complex isolated over a Ni-NTA column in MOPS buffer, was loaded onto a Q Sepharose ion exchanger; the respective fractions were analysed by high Tris urea PAGE, and the gel was stained with silver nitrate. (C) Proteins of fraction three of the Resource Q ion-exchanger were analysed by SDS-PAGE and immunodecoration with

The Tim9·Tim10 complex eluted from the Ni-NTA column contained several contaminating proteins (Figure 14A). Therefore, we went on to investigate possibilities for segregating the complex from these protein contaminants. The size exclusion chromatography turned out to be an undependable tool. The loss in protein amount due to weak, yet undeniably present, unspecific interactions of the Tim9·Tim10 complex with the gel filtration column’s matrices, overshaddowed the overall modest improvements gained in purity-level. The ion exchange chromatography was tested next. Fractions eluted from a Ni-NTA column were pooled together and subjected to either cation- or anion-exchange chromatographies (Figure 14A). The cation-exchange chromatography was found to be inadequate, since the Tim9·Tim10 complex could not be eluted with high salt buffers at all pH values tested (pH 6- 10, at half-unit increments). The anion-exchangers, on the contrary, showed excellent potential for improving the Tim9·Tim10 complex purification procedure. When using the pH values from 6.0 till 8.0, the complex was in the flow-through of the anion-exchange column, while the contaminants remained bound. The only exception was pH 6.0, where some contaminants were also found in the flow-through. At pH values equal to and higher than 9.0, Tim9·Tim10 complex was bound to the ion-exchanger. However, for all subsequent purifications more physiological pH values were selected, closer to that of the mitochondrial intermembrane space (pH 7.5-8.0). Replacing the Resource Q with Q Sepharose anion exchanger enabled me to substantially decrease both protein amount losses, caused by unspecific binding to the ion-exchanger matrix, and the amount of contaminating proteins (compare A to B in Figure 14).

The protein bands of the purified complex were identified by mass spectroscopy (data not shown) and immunodecoration (Figure 14C).

To essay whether there are some residual TX-100 traces in the samples containing the Tim9·Tim10 complex, I have performed a thin-layer chromatography (TLC) of these probes. Every step of the purification procedure was monitored via this method, which can detect TX- 100 concentrations as low as 0.005% (v/v). Minor amounts of TX-100 were found in the washing fractions of the Ni-NTA column. In the Q Sepharose flow-through fractions, dialysed against buffers without TX-100, the detergent was completely eliminated (data not shown). To syllogise, N. crassa Tim9 and Tim10 proteins are the sole constituents of the purified complex.