<?xml version="1.0" encoding="utf-8"?><!DOCTYPE article PUBLIC "-//ES//DTD journal article DTD version 5.1.0//EN//XML" "art510.dtd" [<!ENTITY gr1 SYSTEM "gr1" NDATA IMAGE>]><article version="5.1" docsubtype="chp"><item-info><jid>BS:ABCD</jid><aid>5</aid><ce:pii>B978-0-000-00000-0.00000-X</ce:pii><ce:doi>10.1016/B978-0-000-00000-0.00000-X</ce:doi><ce:copyright type="full-transfer" year="2010">Elsevier Inc.</ce:copyright></item-info><ce:floats><ce:figure id="fig1"><ce:label>Figure 1</ce:label><ce:caption><ce:simple-para>Cross-linking of a mitochondrial preprotein to Tim17. [<ce:sup>35</ce:sup>S]-radiolabeled Su9(1–112)-DHFR was arrested in the mitochondrial import channel prior to incubation in the absence (lane 1) or presence of the cross-linking reagent DSS (lanes 2–4). Mitochondria were lysed and either loaded directly on the gel (lanes 1 and 2), or the resulting extract was used for immunoprecipitation with serum against Tim17 (αTim17, lane 3) or with preimmune serum (p.i., lane 4). Samples loaded in lanes 1 and 2 (total) correspond to 20% of the material used for immunoprecipitation. The cartoon shows a schematic representation of immunoprecipitated Tim17 cross-linked to radiolabeled precursor protein that has been processed by the mitochondrial processing peptidase.</ce:simple-para></ce:caption><ce:link locator="gr1"/></ce:figure></ce:floats><head><ce:title>Cross-Linking of Radiolabeled Precursor Proteins to Subunits of the TIM23 Complex</ce:title><ce:author-group><ce:author><ce:given-name>Johannes M.</ce:given-name><ce:surname>Herrmann</ce:surname><ce:cross-ref refid="aff1"><ce:sup>⁎</ce:sup></ce:cross-ref></ce:author><ce:author><ce:given-name>Benedikt</ce:given-name><ce:surname>Westermann</ce:surname><ce:cross-ref refid="aff2"><ce:sup>†</ce:sup></ce:cross-ref><ce:e-address type="email">benedikt.westermann@uni-bayreuth.de</ce:e-address></ce:author><ce:affiliation id="aff1"><ce:label>⁎</ce:label><ce:textfn>Institut für Zellbiologie, Universität Kaiserslautern, 67663 Kaiserslautern, Germany</ce:textfn></ce:affiliation><ce:affiliation id="aff2"><ce:label>†</ce:label><ce:textfn>Institut für Zellbiologie, Universität Bayreuth, 95440 Bayreuth, Germany</ce:textfn></ce:affiliation></ce:author-group><ce:abstract><ce:section-title>Purpose</ce:section-title><ce:abstract-sec><ce:simple-para>The analysis of protein–protein interactions is of particular importance for the study of the biogenesis and function of mitochondrial proteins. A number of techniques have been used successfully to analyze protein–protein interactions within cells and organelles. Among these, coimmunoprecipitation, copurification and chemical cross-linking have proven to be particularly useful to study various aspects of mitochondrial biogenesis. These techniques are powerful methods for the analysis of the sequential interactions of precursor proteins with the various components of the translocation machineries in the mitochondrial membranes. Moreover, they are useful for the investigation of protein–protein interactions during mitochondrial protein synthesis, folding and degradation, and for the determination of the molecular composition of oligomeric protein complexes, as for example the respiratory chain complexes or protein translocation machineries. Here, we outline the rationale behind the analysis of protein–protein interactions in mitochondria. Relevant methods are described and illustrated with specific examples (Herrmann et al., 2001). The methods described here have been developed for yeast as a model organism; however, they can be readily applied to other organisms.</ce:simple-para></ce:abstract-sec></ce:abstract></head><body><ce:sections><ce:section id="sec1"><ce:section-title>Theory</ce:section-title><ce:para>Several points have to be considered before starting an analysis of protein–protein interactions.<ce:list><ce:list-item><ce:label>1.</ce:label><ce:para>Protein–protein interactions are often weak and unstable <ce:italic>in vitro</ce:italic>. For example, in the case of membrane protein complexes, solubilization with detergents may destabilize oligomers, thereby preventing their analysis in extracts.</ce:para></ce:list-item><ce:list-item><ce:label>2.</ce:label><ce:para>Interactions can be transient. This may prevent coisolation of the interaction partners, since their physical interaction might not last during the isolation procedure.</ce:para></ce:list-item><ce:list-item><ce:label>3.</ce:label><ce:para>Proteins often interact with several different binding partners. For example, during import into mitochondria a precursor protein contacts several proteins of the translocation machinery in a sequential manner.</ce:para></ce:list-item><ce:list-item><ce:label>4.</ce:label><ce:para>Many proteins tend to bind to other proteins upon extraction. For example, membrane proteins and nonassembled subunits of complexes often expose hydrophobic surfaces, causing them to stick to other proteins. These nonnative interactions can be surprisingly specific and strong, and it is often difficult to distinguish them from authentic endogenous interactions.</ce:para></ce:list-item></ce:list> For these reasons, it is crucial that experiments addressing protein–protein interactions are well controlled and backed up by the use of different approaches that should consistently reveal the same interactions. Various methods have been employed to monitor interactions of mitochondrial proteins. Copurification procedures are the standard assays to detect protein–protein interactions, since they typically reflect in vivo interactions reliably and allow for the isolation of previously unknown binding partners.</ce:para><ce:para>Chemical cross-linkers introduce covalent bonds within proteins or between proteins that are in close proximity to each other. This makes cross-linking a powerful method for the detection of transient or weak interactions that might not be detected by coimmunoprecipitation. Cross-linking has been used extensively to assess interactions between mitochondrial proteins. A limitation of cross-linking is the dependence on specific amino acid side chains in the proteinaceous interaction partners. Obviously, these side chains need to be in a certain distance and orientation to allow cross-linking. Cross-linking is usually not very efficient: in the case of stable interactions cross-linking yields might exceed 10%; however, in the case of transient interactions, they are typically less than 1%. Therefore, a sensitive method is required to detect the cross-linked products. For stable interactions of abundant binding partners (e.g., subunits of the respiratory chain complexes), cross-linked products can be detected by Western blotting. Transient interactions are usually assessed using radiolabeled proteins. Synchronization of the interactions is often required to increase the yield of specific cross-links. Chemical cross-linkers can stabilize weak or short-lived interactions and therefore have been used extensively to monitor transient interactions. However, cross-linking often is rather inefficient, and the isolation and identification of yet unknown interaction partners is therefore difficult. In the case of experiments addressing protein translocation across mitochondrial membranes, radiolabeled precursor proteins containing a C-terminal fusion to DHFR can be used. DHFR can be folded stably by addition of the substrate analog methotrexate, which arrests the protein as a translocation intermediate of defined length. This has been used in many cases to cross-link precursor proteins to components of the mitochondrial protein import machineries (<ce:cross-refs refid="bib3 bib25">Berthold et al., 1995; Vestweber et al., 1989a</ce:cross-refs>). In this protocol the fusion protein, pSu9(1-112)-DHFR, is arrested as a translocation intermediate in an <ce:italic>in vitro</ce:italic> mitochondrial import assay and then cross-linked. Following lysis of the mitochondria, samples are immunoprecipitated using an anti-Tim17 antibody, identifying it as a cross-linked adduct during the import reaction (<ce:cross-ref refid="fig1">Fig. 1</ce:cross-ref><ce:float-anchor refid="fig1"/>).</ce:para><ce:para>There are two different approaches using cross-linking reagents:<ce:list><ce:list-item><ce:label>1.</ce:label><ce:para>(Photoreactive) cross-linkers can be incorporated into proteins, either co- or posttranslationally. Then, these proteins are allowed to interact with other proteins and the cross-linking reaction is triggered by a light flash (<ce:cross-refs refid="bib6 bib7">Brunner, 1993, 1996</ce:cross-refs>). This type of cross-linking led to the identification of Tom40 as a component of the translocation machinery in the mitochondrial outer membrane (<ce:cross-refs refid="bib25 bib26">Vestweber et al., 1989a,b</ce:cross-refs>).</ce:para></ce:list-item><ce:list-item><ce:label>2.</ce:label><ce:para>The second type uses bifunctional cross-linking reagents to connect preexisting complexes. This method is versatile and can be used to investigate many different processes. For example, cross-linking of subunits of the respiratory chain complexes has been used extensively to identify nearest neighbors within these multisubunit oligomers (<ce:cross-refs refid="bib4 bib20 bib22">Briggs and Capaldi, 1977; Smith et al., 1978; Todd and Douglas, 1981</ce:cross-refs>). It has also been used in several cases to screen for components in the proximity of a precursor protein during mitochondrial import or folding (<ce:cross-refs refid="bib9 bib16 bib17">Curran et al., 2002; Mesecke et al., 2005; Mokranjac et al., 2003</ce:cross-refs>). A large variety of bifunctional cross-linkers are commercially available (e.g., from Pierce, Rockford IL or Molecular Probes, Eugene OR).</ce:para></ce:list-item></ce:list></ce:para><ce:para>Cross-linking reagents differ in several properties:<ce:list><ce:list-item><ce:label>1.</ce:label><ce:para>Depending on their reactive groups cross-linkers differ in their specificity. Targets can be primary amines (the amino terminus and lysine residues), sulfhydryls (cysteine residues), carboxy groups (aspartate and glutamate residues and the carboxy terminus), or carbohydrates. Some photoreactive reagents, like phenylazides, are more or less nonselective. Both homo- and heterobifunctional cross-linkers are available.</ce:para></ce:list-item><ce:list-item><ce:label>2.</ce:label><ce:para>Some cross-linkers have to be activated by a light flash which allows the trapping of protein–protein interactions at a specific time point.</ce:para></ce:list-item><ce:list-item><ce:label>3.</ce:label><ce:para>Cross-linking reagents differ in the length of the spacer arm, which is usually in the range of 0.4–1.2 nm.</ce:para></ce:list-item><ce:list-item><ce:label>4.</ce:label><ce:para>Some cross-linkers allow the cleavage of the cross-bridge to release the adduct from the bait.</ce:para></ce:list-item><ce:list-item><ce:label>5.</ce:label><ce:para>Some cross-linkers can be radiolabeled. This can be used, for example, to specifically iodinate binding partners to enable their identification after cleavage of the cross-bridge.</ce:para></ce:list-item><ce:list-item><ce:label>6.</ce:label><ce:para>Several cross-linkers are available in membrane-permeable and membrane-impermeable versions.</ce:para></ce:list-item></ce:list></ce:para><ce:para>Since the yields of cross-links between interacting proteins are not predictable, several cross-linkers (e.g., differing in the length of the spacer arm) have to be titrated and the conditions for each interaction have to be optimized. Crosslinking reagents that have been used recently to detect interactions of mitochondrial proteins include m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), 1,5-difluoro-2,4-dinitrobenzene (DFDNB), dithiobis (succinimidyl propionate) (DSP), disuccinimidyl glutarate (DSG), disuccinimidyl suberate (DSS), and 1,4-di (30-20-pyridyldithio-propionamido)butane (DPDPB). DSS belongs to N -hydroxysuccinmide esters (NH S esters) which are the most commonly used cross- linking reagents. They react with unprotonated primary amines which excludes the use of Tris- or glycine-containing buffers during crosslinking. Instead, Tris or glycine (pH 7.5) are often used as quenching reagents following cross-linking</ce:para><ce:para>Besides chemical cross-linking, several other methods are commonly employed to assess protein–protein interactions. These include blue native gel electrophoresis and compositional analysis of mitochondrial protein complexes using size exclusion chromatography or gradient centrifugation (refs). While both techniques give information on the sizes of native protein complexes, they typically do not allow for the identification of interaction partners of given proteins. Several biophysical methods can be used to study interactions of purified proteins. For example, plasmon resonance was used to measure the binding affinities of preproteins and receptors of the mitochondrial outer membrane (<ce:cross-refs refid="bib15 bib18 bib24">Iwata and Nakai, 1998; Okamoto et al., 2002; Vergnolle et al., 2005</ce:cross-refs>). Interactions of proteins can also be tested by two-hybrid analysis in the yeast Saccharomyces cerevisiae (<ce:cross-ref refid="bib10">Fields, 1993</ce:cross-ref>). This method has been used in several cases to track down interacting domains of mitochondrial proteins (<ce:cross-refs refid="bib2 bib5 bib8 bib27">Armstrong et al., 1999; Brown et al., 1994; Cerveny and Jensen, 2003; Yano et al., 2003</ce:cross-refs>). Large-scale analyses have begun to systematically test each of the ca. 6300 yeast genes for their two-hybrid interactions in arrays covering the entire yeast genome (<ce:cross-ref refid="bib14">Ito et al., 2001</ce:cross-ref>; Phizicky et al., 2003; Uetz et al., 2000). It is not difficult to predict that these ca. 40 million combinations will reveal a number of new protein–protein interactions of mitochondrial proteins. However, as protein–protein interactions in the yeast two-hybrid system occur in the nucleus, they always have to be confirmed by assaying the interaction partners in their natural environment. Advantages and limitations of different approaches for the analysis of protein–protein interactions have been discussed in a number of excellent reviews (<ce:cross-refs refid="bib11 bib19 bib23">Gietz, 2006; Phizicky and Fields, 1995; Uetz, 2002</ce:cross-refs>).</ce:para></ce:section><ce:section id="sec2"><ce:section-title>Equipment</ce:section-title><ce:para><ce:list><ce:list-item><ce:para>Centrifuge</ce:para></ce:list-item><ce:list-item><ce:para>Boiling water bath</ce:para></ce:list-item><ce:list-item><ce:para>Platform rotator/mixer</ce:para></ce:list-item><ce:list-item><ce:para>Aspirator</ce:para></ce:list-item><ce:list-item><ce:para>SDS-PAGE gel rig</ce:para></ce:list-item><ce:list-item><ce:para>Power supply</ce:para></ce:list-item><ce:list-item><ce:para>Gel dryer</ce:para></ce:list-item><ce:list-item><ce:para>Film developer</ce:para></ce:list-item><ce:list-item><ce:para>Micro pipetors</ce:para></ce:list-item><ce:list-item><ce:para>Pipetor tips</ce:para></ce:list-item><ce:list-item><ce:para>1.5 ml polypropylene tubes</ce:para></ce:list-item><ce:list-item><ce:para>Autoradiography film</ce:para></ce:list-item></ce:list></ce:para></ce:section><ce:section id="sec3"><ce:section-title>Materials</ce:section-title><ce:para>Bovine serum albumin <ce:list><ce:list-item><ce:para>KCl</ce:para></ce:list-item><ce:list-item><ce:para>MOPS</ce:para></ce:list-item><ce:list-item><ce:para>Sucrose</ce:para></ce:list-item><ce:list-item><ce:para>KH<ce:inf>2</ce:inf>PO<ce:inf>4</ce:inf></ce:para></ce:list-item><ce:list-item><ce:para>K<ce:inf>2</ce:inf>HPO<ce:inf>4</ce:inf></ce:para></ce:list-item><ce:list-item><ce:para>KOH</ce:para></ce:list-item><ce:list-item><ce:para>MgOAc</ce:para></ce:list-item><ce:list-item><ce:para>MnCl<ce:inf>2</ce:inf></ce:para></ce:list-item><ce:list-item><ce:para>Methotrexate</ce:para></ce:list-item><ce:list-item><ce:para>[<ce:sup>35</ce:sup>S]-radiolabeled pSu9(1–112)-DHFR<ce:list><ce:list-item><ce:para>(via protocol: <ce:underline>import of proteins into mitochondria,</ce:underline> Stojanovski et al., 2007)</ce:para></ce:list-item></ce:list></ce:para></ce:list-item><ce:list-item><ce:para>DMSO</ce:para></ce:list-item><ce:list-item><ce:para>DSS</ce:para></ce:list-item><ce:list-item><ce:para>Tris base</ce:para></ce:list-item><ce:list-item><ce:para>Sorbitol</ce:para></ce:list-item><ce:list-item><ce:para>HEPES</ce:para></ce:list-item><ce:list-item><ce:para>SDS</ce:para></ce:list-item><ce:list-item><ce:para>Triton X-100</ce:para></ce:list-item><ce:list-item><ce:para>Digitonin, recrystallized</ce:para></ce:list-item><ce:list-item><ce:para>NaCl</ce:para></ce:list-item><ce:list-item><ce:para>EDTA</ce:para></ce:list-item><ce:list-item><ce:para>Phenylmethylsulfonyl fluoride (PMSF)</ce:para></ce:list-item><ce:list-item><ce:para>Protein A-Sepharose CL-4 B</ce:para></ce:list-item><ce:list-item><ce:para>Anti-Tim17 antibody</ce:para></ce:list-item><ce:list-item><ce:para>Preimmune serum</ce:para></ce:list-item></ce:list></ce:para><ce:section id="sec4"><ce:section id="sec5"><ce:section-title>Solutions & buffers</ce:section-title><ce:section id="sec6"><ce:section-title>Step 1</ce:section-title><ce:para>Potassium phosphate buffer, pH 7.2 (100 mM)<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="3"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Stock</entry><entry colname="col3" align="left">Amount/liter</entry></row><row valign="top"><entry colname="col1" align="left">KH<ce:inf>2</ce:inf>PO<ce:inf>4</ce:inf></entry><entry colname="col2" align="left">0.2 M</entry><entry colname="col3" align="left">140 ml</entry></row><row valign="top"><entry colname="col1" align="left">K<ce:inf>2</ce:inf>HPO<ce:inf>4</ce:inf></entry><entry colname="col2" align="left">0.2 M</entry><entry colname="col3" align="left">360 ml</entry></row><row valign="top"><entry namest="col1" nameend="col3" align="left">Add water to 1 liter</entry></row></tbody></tgroup></ce:table></ce:display></ce:para><ce:para>Import buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/liter</entry></row><row valign="top"><entry colname="col1" align="left">Sucrose</entry><entry colname="col2" align="left">220 mM</entry><entry colname="col3" align="left">500 mM</entry><entry colname="col4" align="left">440 ml</entry></row><row valign="top"><entry colname="col1" align="left">KCl</entry><entry colname="col2" align="left">80 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">80 ml</entry></row><row valign="top"><entry colname="col1" align="left">MOPS-KOH, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">100 mM</entry><entry colname="col4" align="left">100 ml</entry></row><row valign="top"><entry colname="col1" align="left">BSA</entry><entry colname="col2" align="left">3%</entry><entry colname="col3"></entry><entry colname="col4" align="left">30 g</entry></row><row valign="top"><entry colname="col1" align="left">Potassium phosphate</entry><entry colname="col2" align="left">25 mM</entry><entry colname="col3" align="left">100 mM</entry><entry colname="col4" align="left">250 ml</entry></row><row valign="top"><entry colname="col1" align="left">MgOAc</entry><entry colname="col2" align="left">5 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">5 ml</entry></row><row valign="top"><entry colname="col1" align="left">MnCl<ce:inf>2</ce:inf></entry><entry colname="col2" align="left">1 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">1 ml</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 1 liter</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec7"><ce:section-title>Step 2</ce:section-title><ce:para>DSS 30mM stock in DMSO</ce:para><ce:para>Dissolve 5.5 mg DSS in 500 μl DMSO</ce:para></ce:section><ce:section id="sec8"><ce:section-title>Step 3</ce:section-title><ce:para>Sorbitol buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/liter</entry></row><row valign="top"><entry colname="col1" align="left">Sorbitol</entry><entry colname="col2" align="left">600 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">600 ml</entry></row><row valign="top"><entry colname="col1" align="left">KCl</entry><entry colname="col2" align="left">250 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">250 ml</entry></row><row valign="top"><entry colname="col1" align="left">Hepes, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">Tris-HCl, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 1 liter</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display></ce:para><ce:para>SDS lysis buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/500 ml</entry></row><row valign="top"><entry colname="col1" align="left">Tris-HCl, pH 7.4</entry><entry colname="col2" align="left">100 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">50 ml</entry></row><row valign="top"><entry colname="col1" align="left">SDS</entry><entry colname="col2" align="left">1%</entry><entry colname="col3" align="left">20%</entry><entry colname="col4" align="left">25 ml</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 500 ml</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display></ce:para><ce:para>Lysis buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/500 ml</entry></row><row valign="top"><entry colname="col1" align="left">Triton X-100</entry><entry colname="col2" align="left">1%</entry><entry colname="col3"></entry><entry colname="col4" align="left">5 ml</entry></row><row valign="top"><entry colname="col1" align="left">NaCl</entry><entry colname="col2" align="left">300 mM</entry><entry colname="col3" align="left">5 M</entry><entry colname="col4" align="left">30 ml</entry></row><row valign="top"><entry colname="col1" align="left">EDTA</entry><entry colname="col2" align="left">5 mM</entry><entry colname="col3" align="left">500 mM</entry><entry colname="col4" align="left">5 ml</entry></row><row valign="top"><entry colname="col1" align="left">Tris-HCl, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">5 ml</entry></row><row valign="top"><entry colname="col1" align="left">PMSF</entry><entry colname="col2" align="left">1 mM</entry><entry colname="col3" align="left">100 mM</entry><entry colname="col4" align="left">Add fresh</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 500 ml</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display></ce:para><ce:para>Washing buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/L</entry></row><row valign="top"><entry colname="col1" align="left">Triton X-100</entry><entry colname="col2" align="left">0.1%</entry><entry colname="col3"></entry><entry colname="col4" align="left">1 ml</entry></row><row valign="top"><entry colname="col1" align="left">NaCl</entry><entry colname="col2" align="left">300 mM</entry><entry colname="col3" align="left">5 M</entry><entry colname="col4" align="left">60 ml</entry></row><row valign="top"><entry colname="col1" align="left">EDTA</entry><entry colname="col2" align="left">5 mM</entry><entry colname="col3" align="left">500 mM</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">Tris-HCl, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 1 liter</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec9"><ce:section-title>Step 4</ce:section-title><ce:para>Washing buffer<ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="4"><colspec colname="col1"/><colspec colname="col2"/><colspec colname="col3"/><colspec colname="col4"/><tbody><row valign="top"><entry colname="col1" align="left">Component</entry><entry colname="col2" align="left">Final concentration</entry><entry colname="col3" align="left">Stock</entry><entry colname="col4" align="left">Amount/L</entry></row><row valign="top"><entry colname="col1" align="left">Digitonin</entry><entry colname="col2" align="left">0.1%</entry><entry colname="col3"></entry><entry colname="col4" align="left">1 g</entry></row><row valign="top"><entry colname="col1" align="left">NaCl</entry><entry colname="col2" align="left">50 mM</entry><entry colname="col3" align="left">5 M</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">EDTA</entry><entry colname="col2" align="left">2 mM</entry><entry colname="col3" align="left">500 mM</entry><entry colname="col4" align="left">4 ml</entry></row><row valign="top"><entry colname="col1" align="left">Tris-HCl, pH 7.4</entry><entry colname="col2" align="left">10 mM</entry><entry colname="col3" align="left">1 M</entry><entry colname="col4" align="left">10 ml</entry></row><row valign="top"><entry colname="col1" align="left">Add water to 1 liter</entry><entry colname="col2"></entry><entry colname="col3"></entry><entry colname="col4"></entry></row></tbody></tgroup></ce:table></ce:display><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left"><ce:italic>Tip</ce:italic></entry><entry colname="col2" align="left"><ce:italic>Print out recipes of all stock solutions needed to create the buffers online</ce:italic></entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section></ce:section></ce:section></ce:section><ce:section id="sec10"><ce:section-title>Protocol</ce:section-title><ce:section id="sec11"><ce:section-title>Introduction</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left"><ce:italic>Duration</ce:italic></entry><entry colname="col2" align="left"><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left">Preparation</entry><entry colname="col2" align="left">about 1 day</entry></row><row valign="top"><entry colname="col1" align="left">Protocol</entry><entry colname="col2" align="left">about 5-6 hours</entry></row></tbody></tgroup></ce:table></ce:display></entry></row><row valign="top"><entry colname="col1" morerows="1" align="left">Preparation</entry><entry colname="col2" align="left">Isolate mitochondria to use as starting material in order to reduce the background, which might be rather high when starting from whole cells. (Boldogh et al., 2007)</entry></row><row valign="top"><entry colname="col2" align="left">Prepare the [<ce:sup>35</ce:sup>S]-radiolabeled pSu9(1-112)-DHFR fusion protein by in vitro transcription and translation. (Stojanovski et al., 2007)</entry></row><row valign="top"><entry colname="col1" align="left"><ce:italic>Caution</ce:italic></entry><entry colname="col2" align="left"><ce:italic>Consult your institute Radiation Safety Officer for proper ordering, handling, and disposal of radioactive materials</ce:italic>.</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec12"><ce:label>Step 1</ce:label><ce:section-title>Arrest of a Radiolabeled Precursor Protein in the mitochondrial import channel</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left">Overview</entry><entry colname="col2" align="left">Carry out an <ce:italic>in vitro</ce:italic> mitochondrial protein import assay under conditions that will trap the translocating precursor protein within the import channel.</entry></row><row valign="top"><entry colname="col1" align="left">Duration</entry><entry colname="col2" align="left">30 min</entry></row><row valign="top"><entry colname="col1" align="left">1.1</entry><entry colname="col2" align="left">Preincubate mitochondria (150 μg) in 600 μl import buffer in the presence of 1 μM methotrexate for 5 min at 4°C.</entry></row><row valign="top"><entry colname="col1" align="left">1.2</entry><entry colname="col2" align="left">Add 20 μl reticulocyte lysate containing [<ce:sup>35</ce:sup>S]-radiolabeled pSu9(1–112)-DHFR.</entry></row><row valign="top"><entry colname="col1" align="left">1.3</entry><entry colname="col2" align="left">Incubate for 5 min on ice.</entry></row><row valign="top"><entry colname="col1" align="left">1.4</entry><entry colname="col2" align="left">Incubate for 15 min at 25°C.</entry></row><row valign="top"><entry colname="col1" align="left">Tip</entry><entry colname="col2" align="left">Import reaction should be carried out using freshly isolated, not frozen, mitochondria.</entry></row><row valign="top"><entry colname="col1" align="left">Tip</entry><entry colname="col2" align="left">We did not observe any effects of addition of up to 2–4% DMSO (final concentration) on protein import into isolated mitochondria. However, higher concentrations can have harmful effects on membrane integrity.</entry></row><row valign="top"><entry colname="col1" align="left">Tip</entry><entry colname="col2" align="left">ATP-depletion, dissipation of the membrane potential, or low temperature may be used to arrest translocating proteins instead of using DHFR/methotrexate.</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec13"><ce:label>Step 2</ce:label><ce:section-title>Cross-link proteins of translocation intermediate</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left"><ce:italic>Overview</ce:italic></entry><entry colname="col2" align="left">In this step you will covalently cross-link proteins that are in close proximity to one another. The radiolabeled precursor protein will be cross-linked to those subunits of the import channel that it is in closest contact with. An aliquot of the sample will be removed as a control prior to cross-linking.</entry></row><row valign="top"><entry colname="col1" align="left">Duration</entry><entry colname="col2" align="left">45 min</entry></row><row valign="top"><entry colname="col1" align="left">2.1</entry><entry colname="col2" align="left">Remove 50 μl as a ‘Total without cross-linker’ control. Add 0.5 μl DMSO and an equal volume of 2x SDS-PAGE sample buffer.</entry></row><row valign="top"><entry colname="col1" align="left">2.2</entry><entry colname="col2" align="left">Add 5.5 μl DSS to the 550 μl aliquot.</entry></row><row valign="top"><entry colname="col1" align="left">2.3</entry><entry colname="col2" align="left">Incubate for 30 min at 25°C.</entry></row><row valign="top"><entry colname="col1" align="left">2.4</entry><entry colname="col2" align="left">Add Tris–HCl, pH 8.0 to 100 mM final concentration.</entry></row><row valign="top"><entry colname="col1" align="left"><ce:italic>Tip</ce:italic></entry><entry colname="col2" align="left"><ce:italic>It is important to keep the pH above 7–7.5 to reduce protonation of the amino groups. These reagents are rather unstable in water especially at a pH above 8. To prevent hydrolysis of the cross-linkers during storage, they have to be carefully kept dry. It is very important to warm the vials containing the cross-linkers to room temperature before opening to protect them against condensing water. The cross-linker solutions in DMSO should be prepared fresh before use.</ce:italic></entry></row><row valign="top"><entry colname="col1" align="left">Tip</entry><entry colname="col2" align="left">Quenching reagents, such as Tris or glycine, should be present during all steps after the cross-linking reaction.</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec14"><ce:label>Step 3</ce:label><ce:section-title>Lysis of mitochondria</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left">Overview</entry><entry colname="col2" align="left">Mitochodria will be lysed in order to solubilze the proteins for subsequent analysis. The boiling lysis in SDS will disrupt even strong protein-protein interactions while the subsequent dilution in a milder lysis buffer places the sample in conditions more amenable to immunoprecipitation.</entry></row><row valign="top"><entry colname="col1" align="left">Duration</entry><entry colname="col2" align="left">30 min</entry></row><row valign="top"><entry colname="col1" align="left">3.1</entry><entry colname="col2" align="left">Remove 55 μl of the cross-linked sample (total, with cross-linker). Add an equal volume of 2× SDS-PAGE sample buffer.</entry></row><row valign="top"><entry colname="col1" align="left">3.2</entry><entry colname="col2" align="left">Centrifuge at 12,000 × g for 5 min at 4°C.</entry></row><row valign="top"><entry colname="col1" morerows="1" align="left">3.3</entry><entry colname="col2" align="left">Wash in sorbitol buffer.</entry></row><row valign="top"><entry colname="col2" align="left">Centrifuge at 12,000 × g for 5 min at 4°C.</entry></row><row valign="top"><entry colname="col1" align="left">3.4</entry><entry colname="col2" align="left">Resuspend in 80 μl SDS-lysis buffer.</entry></row><row valign="top"><entry colname="col1" align="left">3.5</entry><entry colname="col2" align="left">Vortex sample.</entry></row><row valign="top"><entry colname="col1" align="left">3.6</entry><entry colname="col2" align="left">Boil for 3 min.</entry></row><row valign="top"><entry colname="col1" align="left">3.7</entry><entry colname="col2" align="left">Dilute with 1 ml lysis buffer.</entry></row><row valign="top"><entry colname="col1" align="left">3.8</entry><entry colname="col2" align="left">Centrifuge for 10 min at 30,000 × g at 4°C.</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section><ce:section id="sec15"><ce:label>Step 4</ce:label><ce:section-title>Immunoprecipitation and visualization of cross-linked proteins</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left"><ce:italic>Overview</ce:italic></entry><entry colname="col2" align="left">Solubilized mitochondria will be subjected to immunoprecipitation using antisera directed against one of the subunits of the mitochondrial import channel. If the radiolabeled precursor protein was cross-linked to this subunit, it will also be brought down as a complex. Immunoprecipitated proteins will be fractionated by SDS-PAGE and visualized by autoradiagraphy since the precursor protein is radiolabeled.</entry></row><row valign="top"><entry colname="col1" align="left">Duration</entry><entry colname="col1" align="left"><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><tbody><row valign="top"><entry colname="col1" align="left">3 1/2 hours</entry><entry colname="col2" align="left">Immunoprecipitation, wash</entry></row><row valign="top"><entry colname="col1" align="left">1 hour</entry><entry colname="col2" align="left">SDS-PAGE</entry></row><row valign="top"><entry colname="col1" align="left">Variable</entry><entry colname="col2" align="left">Dry gel, autoradiography</entry></row></tbody></tgroup></ce:table></ce:display></entry></row><row valign="top"><entry colname="col1" align="left">4.1</entry><entry colname="col2" align="left">Add anti-Tim17 antibody or preimmune serum (1:100-1:500 dilution) to 1 ml of the mitochondrial extract.</entry></row><row valign="top"><entry colname="col1" align="left">4.2</entry><entry colname="col2" align="left">Incubate for 2 h on ice.</entry></row><row valign="top"><entry colname="col1" align="left">4.3</entry><entry colname="col2" align="left">Add 100 μl of protein A-sepharose (10 % slurry), previously washed twice in lysis buffer. Incubate for 1 h at 4°C with gentle agitation.</entry></row><row valign="top"><entry colname="col1" align="left">4.4</entry><entry colname="col2" align="left">Wash the beads four times with 1 ml washing buffer, once with 10 mM Tris-HCl, pH 7.4</entry></row><row valign="top"><entry colname="col1" align="left">4.5</entry><entry colname="col2" align="left">Resuspend in gel loading buffer containing SDS.</entry></row><row valign="top"><entry colname="col1" align="left">4.6</entry><entry colname="col2" align="left">Incubate for 3 min at 96°C.</entry></row><row valign="top"><entry colname="col1" align="left">4.7</entry><entry colname="col2" align="left">Separate isolated proteins by SDS-PAGE.</entry></row><row valign="top"><entry colname="col1" align="left">4.8</entry><entry colname="col2" align="left">Dry gel.</entry></row><row valign="top"><entry colname="col1" align="left">4.9</entry><entry colname="col2" align="left">Expose to X-ray film for autoradiography.</entry></row><row valign="top"><entry colname="col1" align="left">Tip</entry><entry colname="col2" align="left">Alternatively, can pre-bind antibodies to proteinA-sepharose CL-4B for 30 min at room temperature in washing buffer, prior to immunoprecipitation.</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section></ce:section><ce:section id="sec16"><ce:section-title>Topics</ce:section-title><ce:para><ce:display><ce:table frame="topbot" colsep="0" rowsep="0"><tgroup cols="2"><colspec colname="col1"/><colspec colname="col2"/><thead><row rowsep="1" valign="top"><entry colname="col1">Topic Class</entry><entry colname="col2">Keyword</entry></row></thead><tbody><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Methods</ce:bold><ce:br/>List the methods used to carry out this protocol (ie., for each step).</entry><entry colname="col2" align="left">1 cross-linking of interacting proteins</entry></row><row valign="top"><entry colname="col2" align="left">2 immunoprecipitation</entry></row><row valign="top"><entry colname="col2" align="left">3 SDS-PAGE</entry></row><row valign="top"><entry colname="col2" align="left">4 <ce:italic>in vitro</ce:italic> mitochondrial protein import assay</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5 radiolabeling protein <ce:italic>in vitro</ce:italic></entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Process</ce:bold><ce:br/>List the biological process(es) addressed in this protocol.</entry><entry colname="col2" align="left">1 protein localization</entry></row><row valign="top"><entry colname="col2" align="left">2 mitochondrial organization</entry></row><row valign="top"><entry colname="col2" align="left">3 intracellular protein transport across a membrane</entry></row><row valign="top"><entry colname="col2" align="left">4 protein import into mitochondria</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5 arrest of precursor protein as a translocation intermediate in the TIM complex</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Organisms</ce:bold><ce:br/>List the primary organism used in this protocol. List any other applicable organisms.</entry><entry colname="col2" align="left">1 Saccharomyces cerevisiae</entry></row><row valign="top"><entry colname="col2" align="left">2 (mammalian cells or tissues)</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Pathways</ce:bold><ce:br/>List any signaling, regulatory, or metabolic pathways addressed in this protocol.</entry><entry colname="col2" align="left">1 protein sorting pathway</entry></row><row valign="top"><entry colname="col2" align="left">2</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Molecule role/function</ce:bold><ce:br/>List any cellular or molecular functions or activities addressed in this protocol.</entry><entry colname="col2" align="left">1 membrane transport activity</entry></row><row valign="top"><entry colname="col2" align="left">2 protein import into mitochondria</entry></row><row valign="top"><entry colname="col2" align="left">3 protein import across mitochondrial inner membrane</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Phenotype</ce:bold><ce:br/>List any developmental or functional phenotypes addressed in this protocol (organismal or cellular level).</entry><entry colname="col2" align="left">1</entry></row><row valign="top"><entry colname="col2" align="left">2</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Anatomy</ce:bold><ce:br/>List any gross anatomical structures, cellular structures, organelles, or macromolecular complexes pertinent to this protocol.</entry><entry colname="col2" align="left">1 mitochondria</entry></row><row valign="top"><entry colname="col2" align="left">2 inner membrane presequence translocase complex</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row rowsep="1" valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Diseases</ce:bold><ce:br/>List any diseases or disease processes addressed in this protocol.</entry><entry colname="col2" align="left">1</entry></row><row valign="top"><entry colname="col2" align="left">2</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row valign="top"><entry colname="col2" align="left">5</entry></row><row valign="top"><entry morerows="4" colname="col1" align="left"><ce:bold>Other</ce:bold><ce:br/>List any other miscellaneous keywords that describe this protocol.</entry><entry colname="col2" align="left">1</entry></row><row valign="top"><entry colname="col2" align="left">2</entry></row><row valign="top"><entry colname="col2" align="left">3</entry></row><row valign="top"><entry colname="col2" align="left">4</entry></row><row valign="top"><entry colname="col2" align="left">5</entry></row></tbody></tgroup></ce:table></ce:display></ce:para></ce:section></ce:sections></body><tail><ce:bibliography><ce:section-title>References</ce:section-title><ce:bibliography-sec><ce:section-title>Source article(s) used to create this protocol</ce:section-title><ce:bib-reference id="bib1"><ce:label>Herrmann, et al., 2007</ce:label><sb:reference><sb:contribution><sb:authors><sb:author><ce:given-name>Johannes M.</ce:given-name><ce:surname>Herrmann</ce:surname></sb:author><sb:author><ce:given-name>Benedikt</ce:given-name><ce:surname>Westermann</ce:surname></sb:author></sb:authors><sb:title><sb:maintitle>Analysis of Protein–Protein Interactions in Mitochondria</sb:maintitle></sb:title></sb:contribution><sb:host><sb:issue><sb:series><sb:title><sb:maintitle>Methods in Cell Biology</sb:maintitle></sb:title><sb:volume-nr>vol. 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Romain Loth