Ni-chelate-affinity purification and crystallization of the yeast mitochondrial F1-ATPase

Department of Biochemistry and Molecular Biology, The Chicago Medical School, USA.
Protein Expression and Purification (Impact Factor: 1.7). 11/2004; 37(2):479-85. DOI: 10.1016/j.pep.2004.06.035
Source: PubMed


The yeast mitochondrial ATPase has been genetically modified to include a His(6) Ni-affinity tag on the amino end of the mature beta-subunit. The modified beta-subunit is imported into the mitochondrion, properly processed to the mature form, and assembled into a mature and fully active ATP synthase. The F(1)-ATPase has been purified from submitochondrial particles after release from the membrane with chloroform, followed by Ni-chelate-affinity and gel filtration chromatography. The final enzyme is a homogeneous preparation with full activity and no apparent degradation products. This enzyme preparation has been used to obtain crystals that diffract to better than 2.8 A resolution.

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    • "Furthermore, subunits of the abundant F 1 F o -ATP synthase, were not recovered on the beads with the His Aac2 protein (monitored by F 1 ␣), further demonstrating the specificity of the association of the Aac2 protein with the cytochrome bc 1 -COX and TIM23 complexes. We also performed in parallel affinity purification of the abundant F 1 F 0 -ATP synthase complex using His-tagged F 1 ␤ or Su e proteins, components of the F 1 -and F 0 -ATP synthase sectors, respectively (Brunner et al., 2002; Mueller et al., 2004). Recovery of the F 1 F 0 -ATP synthase was successfully achieved using these tagged proteins, as demonstrated by the recovery of F 1 ␣ on the Ni-NTA beads. "
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    ABSTRACT: The ADP/ATP carrier (AAC) proteins play a central role in cellular metabolism as they facilitate the exchange of ADP and ATP across the mitochondrial inner membrane. We present evidence here that in yeast (Saccharomyces cerevisiae) mitochondria the abundant Aac2 isoform exists in physical association with the cytochrome c reductase (cytochrome bc(1))-cytochrome c oxidase (COX) supercomplex and its associated TIM23 machinery. Using a His-tagged Aac2 derivative and affinity purification studies, we also demonstrate here that the Aac2 isoform can be affinity-purified with other AAC proteins. Copurification of the Aac2 protein with the TIM23 machinery can occur independently of its association with the fully assembled cytochrome bc(1)-COX supercomplex. In the absence of the Aac2 protein, the assembly of the cytochrome bc(1)-COX supercomplex is perturbed, whereby a decrease in the III(2)-IV(2) assembly state relative to the III(2)-IV form is observed. We propose that the association of the Aac2 protein with the cytochrome bc(1)-COX supercomplex is important for the function of the OXPHOS complexes and for the assembly of the COX complex. The physiological implications of the association of AAC with the cytochrome bc(1)-COX-TIM23 supercomplex are also discussed.
    Molecular biology of the cell 08/2008; 19(9):3934-43. DOI:10.1091/mbc.E08-04-0402 · 4.47 Impact Factor
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    • "Analysis of the electrophoretic profile of the subunits, which copurified with Oxa1 His , was similar to that of the ATP synthase complex purified in parallel under the same Triton X-100 lysis conditions by using the His-tagged version of the ␤-subunit of the F 1 -sector (Mueller et al., 2004; Figure 5D), suggesting that an intact or very late assembly intermediate of the ATP synthase complex was associated with Oxa1. "
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    ABSTRACT: The yeast Oxa1 protein is involved in the biogenesis of the mitochondrial oxidative phosphorylation (OXPHOS) machinery. The involvement of Oxa1 in the assembly of the cytochrome oxidase (COX) complex, where it facilitates the cotranslational membrane insertion of mitochondrially encoded COX subunits, is well documented. In this study we have addressed the role of Oxa1, and its sequence-related protein Cox18/Oxa2, in the biogenesis of the F(1)F(o)-ATP synthase complex. We demonstrate that Oxa1, but not Cox18/Oxa2, directly supports the assembly of the membrane embedded F(o)-sector of the ATP synthase. Oxa1 was found to physically interact with newly synthesized mitochondrially encoded Atp9 protein in a posttranslational manner and in a manner that is not dependent on the C-terminal, matrix-localized region of Oxa1. The stable manner of the Atp9-Oxa1 interaction is in contrast to the cotranslational and transient interaction previously observed for the mitochondrially encoded COX subunits with Oxa1. In the absence of Oxa1, Atp9 was observed to assemble into an oligomeric complex containing F(1)-subunits, but its further assembly with subunit 6 (Atp6) of the F(o)-sector was perturbed. We propose that by directly interacting with newly synthesized Atp9 in a posttranslational manner, Oxa1 is required to maintain the assembly competence of the Atp9-F(1)-subcomplex for its association with Atp6.
    Molecular Biology of the Cell 06/2007; 18(5):1897-908. DOI:10.1091/mbc.E06-10-0925 · 4.47 Impact Factor
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    ABSTRACT: The crystal structure of yeast mitochondrial F(1) ATPase contains three independent copies of the complex, two of which have similar conformations while the third differs in the position of the central stalk relative to the alpha(3)beta(3) sub-assembly. All three copies display very similar asymmetric features to those observed for the bovine enzyme, but the yeast F(1) ATPase structures provide novel information. In particular, the active site that binds ADP in bovine F(1) ATPase has an ATP analog bound and therefore this structure does not represent the ADP-inhibited form. In addition, one of the complexes binds phosphate in the nucleotide-free catalytic site, and comparison with other structures provides a picture of the movement of the phosphate group during initial binding and subsequent catalysis. The shifts in position of the central stalk between two of the three copies of yeast F(1) ATPase and when these structures are compared to those of the bovine enzyme give new insight into the conformational changes that take place during rotational catalysis.
    The EMBO Journal 12/2006; 25(22):5433-42. DOI:10.1038/sj.emboj.7601410 · 10.43 Impact Factor
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