npgrj_nprot_2009-39 753..766 - Uni Marburg

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Analysis of iron–sulfur protein maturation in eukaryotes Antonio J Pierik, Daili J A Netz & Roland Lill Institut fu¨r Zytobiologie und Zytopathologie, Philipps-Universita¨t Marburg, Marburg, Germany. Correspondence should be addressed to R.L. ([email protected]).

© 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Published online 30 April 2009; doi:10.1038/nprot.2009.39

Iron–sulfur (Fe/S) proteins play crucial roles in living cells by participating in enzyme catalysis, electron transfer and the regulation of gene expression. The biosynthesis of the inorganic Fe/S centers and their insertion into apoproteins require complex cellular machinery located in the mitochondria (Fe/S cluster (ISC) assembly machinery systems) and cytosol (cytosolic Fe/S protein assembly (CIA) systems). Functional defects in Fe/S proteins or their biogenesis components lead to human diseases underscoring the functional importance of these inorganic cofactors for life. In this protocol, we describe currently available methods to follow the activity and de novo biogenesis of Fe/S proteins in eukaryotic cells. The assay systems are useful to follow Fe/S protein maturation in different cellular compartments, identify novel Fe/S proteins and their biogenesis factors, investigate the molecular mechanisms underlying the maturation process in vivo and analyze the effects of genetic mutations in Fe/S protein-related genes. Comprehensive analysis of one biogenesis component or target Fe/S protein takes about 10 d.

INTRODUCTION Iron–sulfur (Fe/S) clusters are ubiquitous inorganic cofactors that are present in all forms of life1. Despite the chemical simplicity of Fe/S clusters, their biosynthesis and insertion into apoproteins within cells require dedicated and complex machinery2,3. On the basis of the identification of proteins responsible for the biosynthesis of bacterial Fe/S enzymes,2,4 a related ISC assembly machinery has been discovered in mitochondria of eukaryotes. In Baker’s yeast, the best-studied eukaryote for this process, 15 proteins are known as members of the mitochondrial ISC assembly machinery (for recent reviews, see refs. 3,5). The key players are the cysteine desulfurase complex Nfs1/Isd11, which supplies sulfur to the scaffold protein Isu1 on which an Fe/S cluster is assembled de novo (see Fig. 1) (ref. 3). This biosynthetic step requires the redox chain NADH—ferredoxin reductase (Arh1)—ferredoxin (Yah1) and the putative iron donor frataxin (Yfh1). The preassembled Fe/S cluster is then transferred to apoproteins involving a dedicated chaperone system6 and the monothiol glutaredoxin Grx5. Some mitochondrial Fe/S proteins further depend on Isa1, Isa2 and Iba57 proteins7 or GTP8 for functional assembly. In eukaryotes, Fe/S proteins are localized in the mitochondria, cytosol and nucleus9. Strikingly, maturation of the extra-mitochondrial Fe/S proteins depends on the function of the mitochondrial ISC assembly machinery10. A current working model (Fig. 1) suggests that the mitochondria export a (still unknown) compound synthesized by virtue of the ISC assembly machinery to the cytosol where it is utilized by the so-called CIA machinery11. The export reaction is facilitated by members of the ISC export machinery, which encompass the mitochondrial inner membrane ABC transporter Atm1, the intermembrane space-located sulfhydryl oxidase Erv1 and glutathione. Over the past years, five CIA factors have been identified. Fe/S clusters are first assembled on the P-loop NTPases Cfd1 and Nbp35, which thus serve as scaffolds in the eukaryotic cytosol (Fig. 1) (ref. 12). Later steps require the iron-only hydrogenaselike protein Nar1 (ref. 13) and the b-propeller protein Cia1 (ref. 14). Recently, the Fe/S protein Dre2 was found to be another crucial member of the CIA machinery but its function is still unclear15.

The ISC and CIA proteins found in yeast are highly conserved in eukaryotes,16 suggesting that yeast provides a valuable model for Fe/S protein maturation in eukaryotes. In fact, seven mammalian relatives of yeast ISC and CIA proteins have been experimentally studied to date and shown to serve a similar function in the cell as their yeast counterparts17–23. The crucial importance of Fe/S proteins and their assembly machineries has been recognized by virtue of the association of lesions in ISC machinery genes with three human diseases (for a review, see refs. 3,24). First, a GAA triplet repeat expansion in an intronic region of frataxin is the most frequent cause of Friedreich ataxia, an autosomal recessive neurodegenerative disorder25. Second, a splicing defect in GLRX5 (encoding human Grx5) causes sideroblastic anemia26. Third, a myopathy of patients from northern Swedish descent has recently been shown to derive from a splicing defect in the ISCU gene (encoding human Isu1)27,28. The functional significance of the ISC and CIA machineries has gained progressive interest since a number of essential cytosolic and nuclear Fe/S proteins have been identified, the function of which may critically depend on their Fe/S cofactors29–31. In fact, the presence of essential Fe/S clusters in these proteins may explain the indispensable character of mitochondria and the ISC and CIA machineries for cell viability29. Ribosome maturation and protein translation require the 2 [4Fe–4S] cluster-containing protein Rli1, which resides in both cytosol and nucleus29,32. Other essential Fe/S proteins are involved in the integrity, maintenance and repair of DNA, but the presence of Fe/S clusters in these proteins in vivo has not been verified yet30,31. Replication of the lagging strand critically depends on the primase complex, which includes the Fe/S cluster-containing large subunit Pri2 (ref. 30). The DNA helicase Rad3 (XPD in humans) is one of the ten subunits of the RNA polymerase transcription factor IIH and is vital for nucleotide excision DNA repair33. A prokaryotic homolog of Rad3 has been shown by biochemical and structural studies to contain an essential [4Fe–4S] cluster inserted into its helicase fold31,34. This protocol describes various

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PROTOCOL CIA machinery Figure 1 | Current model for Fe/S protein Dre2 Apo maturation in eukaryotes. After import of reduced Cfd1 Cfd1 Nar1 - Cia1 iron (red circle) into mitochondria by the carrier proteins, Mrs3/4 (mitoferrin), the iron-binding Nbp35 Nbp35 protein frataxin is believed to deliver Fe to the scaffold protein Isu1 (yeast contains a second Cytosolic and nuclear GSH Fe/S proteins highly related protein termed Isu2). Sulfur Erv1 (yellow circle) is released from cysteine by the ISC export Atm1 GTP? cysteine desulfurase complex Nfs1-Isd11 machinery Nfu1? Mitochondrial generating alanine. The sulfur is transferred to Fe/S proteins ? Isu1 and possibly reduced by the electron transfer ISC assembly chain NADH—ferredoxin reductase Arh1— machinery Ssq1-ATP ferredoxin Yah1. These reactions lead to the de Jac1/Mge1 Apo Isu1 Isu1 novo assembly of an Fe/S cluster on Isu1. The Grx5 Iba57 Frataxin cluster is then transferred to apoproteins (Apo), a Isa1-Isa2 reaction facilitated by the glutaredoxin Grx5 and Yah1-Arh1-NADH the chaperone system Ssq1-Jac1-Mge1. Functional Ala Aconitase Nfs1-Isd11 activation of aconitase- and biotin synthase-like biotin synthase Cys- SH Fe/S proteins requires the additional function of Mitochondrion Isa1-Isa2-Iba57. Extra-mitochondrial Fe/S proteins Cytosol Mrs3/4 depend on most of the mitochondrial ISC assembly components for maturation as well as on the ISC 2+ Iron (Fe ) export machinery with the central component Atm1, which exports an unknown (?) compound to the cytosol for use by the CIA machinery. This leads to assembly of Fe/S clusters on the P-loop NTPases Cfd1Nbp35. The Fe/S cluster is then transferred to cytosolic and nuclear apoproteins involving the function of Nar1 and Cia1. The functional step of the essential CIA protein Dre2 has not been identified yet. GSH, glutathione. For further details, see text and ref. 3.

experimental approaches to verify that these proteins in fact contain Fe/S clusters in the living cell. Currently, the majority of novel Fe/S proteins are identified by isolation after heterologous expression in Escherichia coli, chemical reconstitution of Fe/S clusters, cysteine mutagenesis, bioinformatic analysis and/or X-ray crystallography12,13,30,31. An important question is the relevance of the Fe/S cluster in the identified protein for the physiology of the original organism. For example, on synthesis in E. coli metal centers different from those present in the original host can be inserted, for instance due to the lack of dedicated metallochaperones. Well-known examples are the single iron-containing Clostridium pasteurianum rubredoxin35 and the [2Fe–2S]or [4Fe–4S]-containing Haemophilus influenzae IscU36, both of which bind zinc on heterologous expression in E. coli. Thus, sensitive in vivo assays in the original organism are required to complement biochemical and structural approaches used to identify novel Fe/S-containing proteins. Here, we provide several methods intended to verify the presence of Fe/S clusters in proteins of interest in vivo35,36. We describe the use of radiolabeled iron (55Fe) and several enzymatic assays to determine the presence of Fe/S clusters in mitochondrial, cytosolic and nuclear proteins. We further outline how the assays described here serve to unequivocally identify new biogenesis components, i.e., members of the ISC and CIA machineries. The use of genetic depletion methods to diminish the cellular amounts of these usually essential proteins allows the investigation of their role in vivo. For instance, the stages at which these factors perform their function in the biosynthetic pathway can be determined12,37. In turn, this experimental approach opens the way to address the phenotypes associated with the depletion of the ISC and CIA components. A number of additional assay systems for testing the biogenesis of Fe/S proteins in whole cells will not be described here because of their indirect character. The reader is referred to the summary of these approaches in refs. 3,38,39. For the study of maturation of the ferredoxin Yah1 754 | VOL.4 NO.5 | 2009 | NATURE PROTOCOLS

(ref. 40) and aconitase8, specialized protocols have been developed for the use with isolated mitochondria. Aconitase can be measured qualitatively in a gel-based assay (zymogram)21. A non-radioactive method, which also uses regulatable promoters, has recently been developed for a bacterial system, Azotobacter vinelandii41,42. This method allows determination of both Fe/S cluster amount and type, but might not be applicable for eukaryotic systems due to inherently lower protein amounts. Experimental design Measurement of enzymatic activities of Fe/S proteins. There are several assays available to measure Fe/S enzyme activities at their natural abundance (Table 1) for testing the cellular Fe/S protein biogenesis machinery. As the activity of these enzymes depends on the presence of a particular cluster type, i.e., [4Fe–4S] in aconitase43, activity measurements provide information on both the quantity and the Fe/S cluster integrity. It should be noted, however, that this strategy does not necessarily provide information about the maturation efficiency of Fe/S proteins inside the cell, i.e., about de novo Fe/S protein biogenesis activity, as cluster lability, iron status43 and oxidative stress44 can lead to the damage or removal of the Fe/S cluster from the protein of interest. Methods for the determination of two cytosolic Fe/S enzymes (isopropylmalate isomerase and sulfite reductase) and two mitochondrial Fe/S enzymes (succinate dehydrogenase and aconitase) are given in Boxes 1 and 2, respectively. The activity of isopropylmalate isomerase can be detected by the formation of the UVabsorbing double bond of isopropylmaleate on dehydration of 3isopropylmalate10. In a similar manner, formation of cis-aconitate from isocitrate in concentrated aconitase preparations (mitochondria) can be followed10. For diluted aconitase samples (whole-cell extract), this assay cannot be used due to the high UV absorbance from DNA and other proteins. Instead, formation of NADPH at 340 nm can be recorded7, if the aconitase-catalyzed

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TABLE 1 | Fe/S cluster-containing enzymes suitable for monitoring Fe/S protein maturation. Protein Aconitase

Cluster type 4Fe/3Fea

Other cofactors None

Homoaconitase (Lys4) Sulfite reductase

4Fe 4Fe

None Siroheme, FAD, FMN

Isopropylmalate isomerase (Leu1)

4Fe

None

Complex II (succinate dehydrogenase) 4Fe, 3Fe, 2Fe Complex III (cytochrome c reductase)d 2Fe Xanthine dehydrogenase 2 2Fe

FAD, cytochrome b Cytochromes b and c1 MoCo, FAD

Description Reference NADPH formation from cis-aconitate with 23 isocitrate dehydrogenase 10 cis-Aconitate formationb Homoaconitate formation from homoisocitrate 69 Methylene blue formation from HS produced by 14 sulfite reductionc Formation of isopropylmaleate from 10 3-isopropylmalate Succinate-dependent DCPIP reduction 10 Cytochrome c reduction from succinate 10 Production of superoxide as detected by the 48 Amplex Red method

aThe enzymatically active [4Fe-4S]2+ (4Fe) form of aconitase can be converted to the apo- or [3Fe-4S]+ (3Fe) form depending on the conditions used for cell growth, preparation of mitochondria or cell extract. 2Fe, [2Fe-2S] cluster. bThe high UV absorbance of nucleotides prevents the use of this assay for cell extracts. cDiscontinuous assay. dNote that the assay measures both complexes II and III.

isocitrate formation from cis-aconitate is coupled with isocitrate dehydrogenase and NADP+. Succinate dehydrogenase activity in intact mitochondria harboring intrinsic quinones and the bc1 complex is detected by cytochrome c reduction7. If the bc1 complex is lacking or the mitochondria are damaged, reduction of the artificial electron acceptor DCPIP can be used instead. The assay for sulfite reductase employs detection of the acid-labile sulfide formed from NADPH-dependent SO32 reduction. NADPH is (re)generated in situ from NADP+ with glucose-6-phosphate dehydrogenase and glucose-6-phosphate14. In a noncontinuous assay, the enzymatically formed sulfide condenses in acidic environment in an Fe3+-dependent manner with two N,N-dimethyl-pphenylene diamine molecules to the conveniently detectable methylene blue45. Ideally, Fe/S proteins used as biosynthesis markers should be abundant enzymes that are expressed in a largely constitutive manner and the activity of which is not influenced by growth conditions. However, as the majority of the Fe/S proteins are metabolic enzymes or may be subject to regulation similar to any non-Fe/S protein involved in biosynthetic processes, inherent alterations of the protein levels may occur that have to be accounted or corrected for. For instance, decreased levels of sulfur (methionine/cysteine) influence sulfite reductase expression in yeast46. Furthermore, the type of carbon source utilized determines the extent of fermentation and thereby influences the activities of fermentative cytosolic and mitochondrial enzymes, including those of the respiratory chain and aconitase47. The yeast strain used should also be considered. High activity of isopropylmalate isomerase (Leu1) is particularly observed in the yeast W303 genetic background10. Effects by strain and medium differences are not confined to the yeast system. In human cell lines, complications may arise from significant metabolic responses. Finally, cell density affects the cellular iron status, which in turn can complicate Fe/S protein activity measurements, in particular those of cytosolic aconitase/IRP1 (ref. 23). Caution should be taken with the use of enzymes that require multiple cofactors (Table 1). The presence of flavin and molybdenum cofactor (MoCo) is a serious drawback for the use of xanthine dehydrogenase (human and plant cells) and that of flavin and siroheme for sulfite reductase (yeast) as Fe/S protein marker enzymes. Biosynthesis of MoCo requires other Fe/S proteins and

the lack of insertion of other cofactors could hinder Fe/S cluster assembly48. Certain enzymes, which in some organisms serve as convenient indicators for Fe/S protein maturation, are absent in others, and vice versa. For example, Baker’s yeast lacks a respiratory complex I and xanthine dehydrogenase but contains isopropylmalate isomerase and dihydroxyacid dehydratase (Ilv3), whereas human cells have the opposite configuration3. An even more complex situation is found for DNA glycosylases, adenosine-5¢phosphosulfate reductase, ferrochelatase and the AC40 subunit of RNA polymerase II,49 which are present in plants, fungi and humans but, depending on the organism, possess or lack Fe/S clusters. The sensitivity of Fe/S enzyme detection can usually be increased by cell fractionation and isolation of the respective compartments harboring the Fe/S enzyme of interest. This procedure substantially diminishes background values and in many cases is crucial for detection of low levels of activity (e.g., for cytosolic aconitase in human cells19). If necessary, marker enzymes can be artificially introduced into an organism or cellular compartment by ectopic expression. This methodology allows convenient determination of soluble enzyme activities. Examples include cytosolic human aconitase (IRP1) in a yeast deletion mutant lacking mitochondrial aconitase50, cytosolic15 or mitochondria-targeted51 isopropylmalate isomerase (Leu1) in a yeast cell lacking or with low cytosolic Leu1. In addition to the introduction of suitable marker Fe/S proteins, such experiments show that central parts of both the ISC and CIA machineries do not exhibit pronounced target specificity for Fe/S proteins of one given compartment.50,51 Incorporation of radioactive iron into Fe/S proteins in vivo. The pulse radiolabeling of yeast cells with 55Fe provides a faithful assay for the estimation of the de novo biogenesis of Fe/S cluster-containing proteins in vivo10. A similar approach has been employed to study Fe/S protein assembly in isolated or lysed mitochondria in vitro52. Even though the high-energy isotope 59Fe (b-emitter, 1.6 MeV, t1/2 ¼ 45 d) can be utilized in this assay, 55Fe (electron capture, 5.9 keV, t1/2; ¼ 1,005 d) is preferable because of its low radiation energy53. In our hands, Fe/S proteins such as Leu1 (ref. 10) and Aco1 (ref. 7) with an abundance of 410,000 molecules per yeast cell54 can be detected at their natural abundance. Those present at lower abundance (see refs. 7,12–14,55) require NATURE PROTOCOLS | VOL.4 NO.5 | 2009 | 755

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BOX 1 | ASSAYS FOR MEASURING Fe/S ENZYME ACTIVITY IN WHOLE-CELL EXTRACTS MATERIALS Reagents Saccharomyces cerevisiae strain W303-1A (MATa ura3-1 ade2-1 trp1-1 his3-11,15 leu2-3,112 can1-100) (strain 208352 from ATCC) Tris-HCl (AppliChem, cat. no. A2264) Disodium EDTA (Acros Organics, cat. no. 147850010) NaCl (Roth, cat. no. 9652) Glycerol (Sigma-Aldrich, cat. No. G7757) Triton X-100 (Sigma-Aldrich, cat. no. T9284) Phenylmethylsulfonyl fluoride (PMSF) (AppliChem, cat. no. A0999) ! CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. Ethanol (AppliChem, cat. no. A3693) Triethanolamine hydrochloride (Fluka, cat. no. 90290) NaOH (AppliChem, cat. no. A0991) NADP (Roth, cat. no. AE13.3) cis-Aconitic acid (Sigma-Aldrich, cat. no. A-3412) Isocitrate dehydrogenase from porcine heart (IDH; Sigma-Aldrich, cat. no. I-2002) DL-threo-3-Isopropylmalic acid (Wako, cat. no. 096-02681) K2HPO4 (Roth, cat. no. 6878.1) KH2PO4 (AppliChem, cat. no. A1364) Glucose-6-phosphate dipotassium salt (Sigma-Aldrich, cat. no. G7375) Glucose-6-phosphate dehydrogenase (Calbiochem, cat. no. 346774) Na2SO3 (Sigma-Aldrich, cat. no. S-6547) N,N-Dimethyl-p-phenylene diamine hydrochloride (DMPD; Sigma-Aldrich, cat. no. G-6378) ! CAUTION DMPD is neurotoxic; wear suitable protective clothing, gloves and eye/face protection. 25% HCl (7.7 M, Roth, cat. no. 6331-4) FeCl3 (Sigma-Aldrich, cat. no. F-2877) Li2S (Sigma-Aldrich, cat. no. 213241) ! CAUTION Li2S is toxic; wear suitable protective clothing, gloves and eye/face protection. Double-distilled deionized water (ddH2O) produced with a Elix 5 UV Millipore system Equipment Vortex (Genie-2 vortex; Scientific Instruments) Centrifuges: Eppendorf 5810-R (Eppendorf), Beckman Coulter Avanti J-20XP and Optima LE-80K (Beckman Inc.) Spectrophotometer Jasco V-550 (Jasco Inc.) or any suitable double-beam spectrophotometer Glass (type 104B-OS) and Suprasil (type 104B-QS) quartz cuvettes from Hellma 1.5-ml microfuge tubes Reagent setup Unless indicated otherwise, all reagents are prepared on the day of use. Frozen solutions can be stored up to 1 month. TNETG buffer: 10 mM Tris/Cl pH 7.4, 2.5 mM EDTA, 150 mM NaCl, 10% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100: per 500 ml use 5 ml of 1 M Tris-HCl pH 7.4, 2.5 ml of 0.5 M disodium EDTA (pH 7.4), 37.5 ml of 2 M NaCl, 50 ml of glycerol and 25 ml of 10% (wt/vol) Triton X-100 200 mM PMSF in 100% ethanol. m CRITICAL STEP Prepare just before use; PMSF rapidly hydrolyses. ! CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. 0.1 M NADP+ in ddH2O 20 mM cis-aconitic acid in ddH2O Triethanolamine buffer: 100 mM triethanolamine adjusted to pH 8 with 1 M NaOH IDH 40 U ml1 in triethanolamine buffer with 10% (wt/vol) glycerol, store in small aliquots at 80 1C Isopropylmalate isomerase (Leu1) buffer: 20 mM Tris-Cl pH 7.4, 50 mM NaCl 10 mM DL-threo-3-isopropylmalic acid in ddH2O, dilute freshly from a 100 mM stock solution in ddH2O stored frozen at 20 1C Phosphate buffer: mix 1 M K2HPO4 and 1 M KH2PO4 in the right proportion to obtain pH 7.5 100 mM glucose-6-phosphate dipotassium salt in 20 mM phosphate buffer (pH 7.5) 100 U ml1 glucose-6-phosphate dehydrogenase, dissolve 500 U in 5 ml of 0.1 M phosphate buffer (pH 7.5), store in small aliquots at 80 1C 10 mM Na2SO3 in ddH2O, prepare just before use 20 mM DMPD in 7.2 M HCl. ! CAUTION DMPD is neurotoxic; wear suitable protective clothing, gloves and eye/face protection. 30 mM FeCl3 in 1.2 M HCl 1 mM Li2S calibration standard solution in ddH2O, prepared by dilution from a 100 mM solution of Li2S in ddH2O. m CRITICAL STEP Prepare both solutions just before use; Li2S oxidizes. ! CAUTION Li2S is toxic; wear suitable protective clothing, gloves and eye/face protection.

Cell extract preparation TIMING B45 min 1. Grow a 100 ml yeast culture in minimal or complete medium overnight by shaking at 150 r.p.m., 30 1C m CRITICAL STEP For sulfite reductase activity measurements, use minimal medium lacking methionine, cysteine and sulfide.

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BOX 1 | CONTINUED 2. Harvest cells by centrifugation for 5 min at 2,000g, room temperature (20–24 1C) remove the supernatant and resuspend the cell pellet with 10 ml of ddH2O, transfer to a 15-ml Falcon tube and repeat the centrifugation. m CRITICAL STEP All subsequent steps should be carried out at 4 1C. 3. Resuspend the pellet in 500 ml of ice-cold TNETG buffer; add 10 ml of 0.2 M PMSF and 1 ml of glass beads (with a porcelain spoon). Close the lid tightly. Vortex at maximum speed with the lid side of the tubes contacting the rotating plate for 1 min, repeat the vortexing three times with intermittent 1 min cooling periods on ice. m CRITICAL STEP Ensure that there are no glass beads trapped in the lid. 4. Centrifuge for 5 min at 2,000g at 4 1C. Transfer the supernatant to 1.5-ml microfuge tubes and centrifuge for 10 min at 13,000g. Transfer the supernatant to a new microfuge tube. Save 50 ml for protein determination73. 5. The supernatant (typically 3–6 mg ml1) can be used to measure Fe/S enzyme activity using the assays of options A–C. These enzyme measurements can be performed under aerobic conditions but should be carried out immediately after isolation of the cell extracts to prevent deterioration of the enzymes. (A) Aconitase (coupled assay) TIMING B1 h (including cell extract preparation) (i) Pipette 950 ml of triethanolamine buffer, 12 ml of 20 mM cis-aconitic acid, 10 ml of 40 U ml1 IDH, 12 ml of 0.1 M NADP+ and cell extract (50 mg protein) into the sample cuvette. Omit cis-aconitic acid and IDH in the reference cuvette. Mix well. (ii) Measure the increase of absorbance at 340 nm for 2–4 min; a short lag phase is usually observed. De340 nm ¼ 6,200 M1 cm1. (B) Sulfite reductase TIMING B1.5 h (including cell extract preparation) (i) During the cell extract preparation step, prepare a master mix for 10 reactions: 500 ml of 1 M phosphate buffer, pH 7.5, 10 ml of 0.1 M NADP+, 500 ml of 0.1 M glucose-6-phosphate solution, 25 ml of glucose-6-phosphate dehydrogenase (0.1 U ml1) and 2.965 ml of ddH2O. (ii) Aliquot 400 ml of the master mix into 1.5-ml microfuge tubes, rapidly add sample (50 ml buffer for the reagent blank, 10–50 ml Li2S calibration standard solution adjusted to 50 ml with buffer or 50 ml of cell extract (B200 mg of protein)), mix and incubate for 10 min at 30 1C. ! CAUTION Li2S is toxic; wear suitable protective clothing, gloves and eye/face protection. (iii) Add 50 ml of 10 mM Na2SO3 and then immediately add 100 ml of DMPD solution, 100 ml of ferric reagent and mix. This sample serves as t¼0 min control. ! CAUTION DMPD is neurotoxic; wear suitable protective clothing, gloves and eye/face protection. (iv) Incubate the remaining tubes for 10, 20 or 40 min in a thermoblock at 37 1C. At each time point, add 100 ml of DMPD solution and 100 ml of ferric reagent and mix. (v) Incubate for 20 min at room temperature (i.e., 20–24 1C) to allow the color to develop. If sulfide has been formed from sulfite, a clear blue color is visible. (vi) Centrifuge for 2 min at 13,000g at room temperature and measure the absorbance at 670 nm. Typically, one finds e670 nm ¼ 14,000– 25,000 M1 cm1 for the Li2S calibration standard under the conditions described. (vii) For the cell extract, calculate the sulfide formation using the calibration results and determine the sulfite reductase activity from the steepest linear part of the time course. (C) Isopropylmalate isomerase (Leu1) TIMING B1 h (including cell extract preparation) (i) Mix 970 ml of Leu1 buffer, 20 ml of 10 mM DL-threo-3-isopropylmalic acid and 5–10 ml of cell extract in a quartz cuvette. (ii) Measure the increase of absorbance at 235 nm for 90 s. De235 nm ¼ 4,530 M1 cm1.

overproduction by substitution of the chromosomal promoter or expression from high copy plasmids utilizing suitable strong promoters (TDH3, GAL1-10 or MET25)56. The high natural concentration of iron in growth media and inside cells requires the depletion of ambient iron by cell cultivation at submicromolar iron concentrations (‘iron poor’ conditions) before addition of the radioisotope10. This requirement does not pose a limitation in experiments with yeast cells, as their growth is only mildly affected at low iron concentrations and the frequent use of plasmids urges the use of defined (minimal) media. For radiolabeling of human cell lines, however, the depletion of iron severely decreases cell growth23, and an efficient radiolabeling procedure has not yet been described. Our standard radiolabeling procedure is described in this protocol. Radiolabeling of iron-starved yeast cells with 55Fe is performed for 1–4 h in the presence of the reducing agent ascorbate. The presence of ascorbate prevents the formation of ferric precipitates in the growth medium. Following radiolabeling, cells are lysed with glass beads, a soluble cell extract is prepared and the 55Fe/S proteins are immunoprecipitated with specific antibodies

(see Fig. 2). Alternatively, N- or C-terminal epitope tags can be used to affinity purify the Fe/S proteins of interest. In our hands, the tandem affinity purification (TAP), triple hemagglutinin (3HA) or Myc tags work well, provided the proteins are not functionally compromised. The amount of radioactive iron associated with the Fe/S protein of interest is quantified by liquid scintillation counting. The specificity of the 55Fe association as part of an Fe/S cluster may be verified by testing the dependence on members of the ISC and CIA machineries (see below). On depletion of particular ISC and CIA components by growth on glucose of yeast strains harbouring a GAL promotor in front of the respective genes, the 55Fe incorporation into Fe/S proteins usually drops at least fivefold10,13,14,29,51,55. As a control, the cellular uptake of 55Fe during the labeling period is measured, which reflects the extent of contamination with nonradioactive Fe in the medium. Vital 55Fe uptake is also a viability and fitness parameter for the cells as active transport of Fe against a concentration gradient can only be carried out, if the cellular energy status is not compromised. In higher eukaryotes, ferrochelatase is a mitochondrial Fe/S protein3, and the formation of radioactive heme, therefore, can be used to indirectly NATURE PROTOCOLS | VOL.4 NO.5 | 2009 | 757

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BOX 2 | ASSAYS FOR MEASURING Fe/S ACTIVITY IN ISOLATED MITOCHONDRIA MATERIALS Reagents Tris-HCl (AppliChem, cat. no. A2264) NaCl (Roth, cat. no. 9652) DL-Isocitric acid trisodium salt (Sigma-Aldrich, cat. no. I-1252) Dodecylmaltoside (Calbiochem, cat. no. 324356) MgCl2 (Sigma-Aldrich, cat. no. M-0250) K2HPO4 (Roth, cat. no. 6878.1) KH2PO4 (AppliChem, cat. no. A1364) NADP (Roth, cat. no. AE13.3) KCN (Sigma-Aldrich, cat. no. 20.781-0) ! CAUTION KCN is toxic; wear gloves, safety glasses and ensure good ventilation. Sodium succinate (Sigma-Aldrich, cat. no. S-2378) Sodium malonate (Sigma-Aldrich, cat. no. M-1875) Bovine heart cytochrome c (Sigma-Aldrich, cat. no. C-2037) n-Decylubiquinone (Sigma-Aldrich, cat. no. D-7911) Dichlorophenol indophenol (DCPIP; Fluka, cat. no. 33125) Equipment Spectrophotometer Jasco V-550 (Jasco Inc.) or any suitable double-beam spectrophotometer. Glass (type 104B-OS) and Suprasil (type 104B-QS) quartz cuvettes from Hellma 1.5-ml microfuge tubes Reagent setup Unless indicated otherwise, all reagents are prepared on the day of use. Buffer A: 50 mM Tris-HCl pH 8.0, 50 mM NaCl Aconitase buffer: dissolve 516 mg of DL-isocitric acid trisodium salt in 100 ml of buffer A Mitochondria lysis buffer: dissolve 4.8 mg of dodecylmaltoside in 2 ml of buffer A Triethanolamine buffer: 50 mM triethanolamine-Cl pH 8.5, 50 mM NaCl, 1.5 mM MgCl2 100 mM KCN in ddH2O. ! CAUTION KCN is toxic; wear gloves, safety glasses and ensure good ventilation. 20% (wt/vol) sodium succinate in buffer A 20% (wt/vol) sodium malonate in buffer A Bovine heart cytochrome c, 20 mg ml1 in ddH2O 10 mM n-decylubiquinone in 100% ethanol 10 mM DCPIP in ddH2O 1. Isolate yeast mitochondria as described in detail elsewhere74. 2. Determine the protein concentration with the Bradford method73. 3. The enzymatic activity of the mitochondrial Fe/S protein can be measured using the assays described in options A–C. (A) Aconitase (direct assay) TIMING B5 min (i) Add 950 ml of aconitase buffer to a quartz cuvette. (ii) Dissolve mitochondria (10–20 mg of protein) in 60 ml of mitochondria lysis buffer, mix well and directly proceed with the activity measurement. (iii) Add 50 ml of the lysed mitochondria, mix well and measure the absorbance increase at 235 nm for 2 min. De235 nm ¼ 4,950 M1 cm1. (B) Succinate dehydrogenase (DCPIP reduction assay) TIMING B5 min (i) Pipette in the following order (reference cuvette): 950 ml of buffer A, 7 ml of 10 mM n-decylubiquinone, 10 ml of 10 mM DCPIP, 12 ml of 20% (wt/vol) malonate, 12 ml of 20% (wt/vol) succinate and isolated mitochondria (10–20 mg protein). For the sample cuvette, omit the malonate solution. Mix well. (ii) Measure the increase of absorbance at 600 nm of the sample cuvette versus the reference cuvette for 2 min. De600 nm ¼ 21,000 M1 cm1. (C) Succinate dehydrogenase (cytochrome c reduction assay) TIMING B5 min (i) Immediately before beginning the assay, mix 200 ml of 100 mM KCN solution with 19.8 ml of buffer A. ! CAUTION KCN is toxic; wear gloves, safety glasses and ensure good ventilation. (ii) Pipette in the following order (reference cuvette): 920 ml of buffer A (1 mM KCN), 12 ml of 20% (wt/vol) malonate, 12 ml of 20% (wt/vol) succinate, 50 ml of cytochrome c and isolated mitochondria (10–20 mg protein). For the sample cuvette, omit the malonate solution. Mix well. m CRITICAL STEP Ensure that the reaction is measured for a sufficient amount of time: after an initial lag phase (up to 2 min) the linear phase with maximal activity occurs between 2 and 4 min. (iii) Measure the increase of absorbance at 550 nm of the sample cuvette versus the reference cuvette for 5 min. De550 nm ¼ 20,000 M1 cm1.

sample Fe/S cluster biogenesis (Fig. 2). Owing to its high solubility in organic solvents, heme can be quantitatively extracted from the cell lysates into the organic phase after acidification57. The degree of 55Fe incorporation into heme is quantified by liquid scintillation 758 | VOL.4 NO.5 | 2009 | NATURE PROTOCOLS

counting. In a limited number of cases, the inherent lability of protein-bound Fe/S clusters may require specialized experimental conditions such as anaerobic immuno- or affinity purification of the Fe/S protein, shorter and/or less stringent washing conditions

PROTOCOL


Figure 2 | Experimental strategy of the in vivo 55Fe incorporation into Fe/S proteins. In this example, a plasmid drives constitutive expression of a target Fe/S protein (blue) with a C-terminally fused TAP tag (red) in a yeast cell. After radiolabeling with 55Fe and the preparation of a cell extract by glass beads under nondenaturing conditions, the target protein is affinity-isolated through binding of the protein A domain of the TAP tag to the Fc part of IgG (black Y) covalently coupled to Sepharose beads (blue circle). The beads are washed and the 55Fe radioactivity indicative of Fe/S cluster insertion into the apoprotein is quantified by liquid scintillation counting. In parallel controls, the cellular 55Fe uptake may be followed by measuring the radioactivity present in the cell extract. Heme formation can be estimated by organic extraction and scintillation counting, and the amount of protein is evaluated by western blotting.

than those used for stable Fe/S proteins and optimized times for radiolabeling. As 55Fe incorporation does not give any information on the cluster type, ideally radiolabeling assays and methods that detect the cluster type (i.e., enzymatic activity measurement) are performed in parallel12–14,55. The purchase, storage, use and disposal of the 55Fe isotope require an appropriate license, training of staff, planning or construction of facilities (isotope lab) and monitoring of safety and contamination. Depletion of ISC or CIA components. The specificity of the assays described above can best be verified by analyzing the dependence of the Fe/S enzyme activities or 55Fe/S cluster incorporation on the function of known components of the ISC and CIA machineries10,29. Mitochondrial target Fe/S proteins specifically depend on the mitochondrial ISC assembly machinery for maturation, whereas cytosolic and nuclear Fe/S proteins require the activities of the two ISC systems and the CIA machinery3 (Fig. 1). Potential novel members of the three machineries identified by other methods (genetic screens and bioinformatics) can be investigated. Deletion of the majority of the ISC and CIA genes is lethal necessitating genetic means to downregulate synthesis of the encoded proteins by regulated gene expression. A convenient promoter is that of the GAL1-10 gene, which is induced in the presence of galactose and repressed by glucose. To create a Gal-YFG1 (your favorite gene 1) strain, the endogenous YFG1 promoter is exchanged for that of GAL1-10 by generating a suitable PCR product that can be inserted in front of the YFG1 promoter locus by homologous recombination58. Attenuated versions of this promoter termed GALL have been developed avoiding overexpression with galactose and allowing faster depletion in the presence of MATERIALS REAGENTS

. Yeast nitrogen base without amino acids (ForMedium, cat. no. CYN0510) . Yeast nitrogen base without amino acids, iron-free (ForMedium, cat. no. CYN1202)

. (NH4)2SO4 (Roth, cat. no. 3746.1) . Galactose (Sigma-Aldrich, cat. no. G0625) . Glucose (Sigma-Aldrich, cat. no. G7528) . Agar-agar (Roth, cat no. 5210.2) . Yeast extract (Roth, cat. no. 2363.2) . Casein peptone (MP Biomedicals, cat. no. 3066-132) . Phenylmethylsulfonyl fluoride (PMSF) (AppliChem, cat. no. A0999) ! CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. . Ethanol (AppliChem, cat. no. A3693)

Growth in the presence of 55Fe Cellular iron uptake Plasmid Heme extraction

Yeast cell TAP tag Target protein

55 [ Fe-S]

ISC, export and CIA machineries

Immunoblot

Preparation of cell extract Immunoprecipitation Quantitation of 55 Fe by scintillation counting

[55Fe-S] Bead

glucose. It is crucial to test for possible sugar effects that may arise, e.g., from the switch from respiratory to fermentative growth. As general glucose repression is particularly weak in the W303 yeast genetic background, this strain is well suited for the use of the GAL1-10 promoter during (mitochondrial) Fe/S protein biogenesis investigations. Other suitable depletion techniques are the tetracycline-regulated promoter system59, the heat-induced proteolysis with the degron system60, temperature-sensitive strains61 and the decreased abundance by mRNA perturbation approach62. Downregulation of the target genes must be optimized individually to assure the occurrence of phenotypical consequences, yet no viability loss. The specificity of downregulation should be verified by complementation with a plasmid-borne YFG1 gene. For the Gal-YFG1 strains, depletion to physiologically critical levels is achieved by several passages in liquid media under repressive conditions (see Fig. 2b in ref. 29). Once the behavior of the Gal-YFG1 strain has been determined, the yeast cells can be used for the assays described above. If necessary, the yeast strain can be transformed with a plasmid encoding a potential Fe/S protein using a non-sugar-dependent promoter. Depletion of the ISC and CIA proteins in human cells such as HeLa cell lines is achieved by the RNA interference technology (see e.g., ref. 19). Depletion of the proteins under study is followed by western blotting. To avoid off-target effects of the RNA interference treatment, it is recommended to validate the specificity of the physiological depletion effects by complementing the cells by synthesis of the native protein using genes with silent mutations that are resistant to RNA interference. In many cases, one round of RNA interference treatment is not sufficient to reach critical levels of depletion. Therefore, it is useful to repeat the treatment until a reasonable depletion is achieved without losing cell viability17.

. NaOH (AppliChem, cat. no. A0991) . 2-Mercaptoethanol (Fluka, cat. no. 63689) . Trichloroacetic acid (TCA) (Roth, cat. no. 8789.2) ! CAUTION TCA is corrosive, causes severe burns; wear suitable protective clothing, gloves and eye/face protection. . Acetone (Roth, cat. no. 9372.5) . 55FeCl3 (NEN/Perkin Elmer, cat. no. NEZ043-110711B) ! CAUTION Causes cancer, designate area for handling 55Fe and clearly label all containers. Store mCi quantities of 55Fe behind thin lead shielding. Wear disposable lab coats, wrist guards and gloves for secondary protection. For more detailed information, see instructions of occupational limits in the NRC regulations (10 CFR) part 20, Standards for protection against radiation (http:// www.nrc.gov/reading-rm/doc-collections/cfr/part020/). . 25% HCl (7.7 M; Roth, cat. no. 6331.4)


PROTOCOL . Sodium ascorbate (Fluka, cat. no. 11140) . Trisodium citrate dihydrate (Roth, cat. no. 3580.3) . Disodium EDTA (Acro´sOrganics, cat. no. 147850010) . HEPES (Sigma-Aldrich, cat. no. H3375) . KOH (Roth, cat. no. 6751.1) . Tris-HCl (AppliChem, cat. no. A2264) . NaCl (Roth, cat. no. 9652) . Glycerol (Sigma-Aldrich, cat. no. G7757) . Triton X-100 (Sigma-Aldrich, cat. no. T9284) . Glass beads (+ 0.7-1.0 mm; Roth, cat. no. A554.1) . IgG Sepharose 6 Fast Flow beads (GE Healthcare, cat. no. 17-0969-01) . Protein A-Sepharose CL-4B (GE Healthcare, cat. no. 17-0780-01) . Anti-hemagglutinin (F-7) agarose beads (Santa Cruz, cat. no. © 2009 Nature Publishing Group http://www.nature.com/natureprotocols

Sc-7392AC)

. Anti-Myc (A-14) agarose beads (Santa Cruz, cat. no. Sc-789AC) . Antibodies specifically raised against target proteins . Ultima gold liquid scintillation fluid (Perkin Elmer, cat. no. 6013329) ! CAUTION irritant, wear suitable protective clothing, gloves and eye/face protection. . Reagents for SDS–polyacrylamide gel electrophoresis (PAGE) electrophoresis and western blotting . Double-distilled deionized water (ddH2O) produced with an Elix 5 UV Millipore system EQUIPMENT . Simple spectrophotometer suitable for the measurement of optical density (OD) at 600 nm (Genesys 20, Thermo Scientific) and double-beam spectrophotometer for accurate absorbance measurements (Jasco V-550 spectrophotometer, Jasco Inc.) . Vortex (Genie-2 vortex, Scientific Industries) . pH meter (C6840, Schott Instruments) . Rotary shaker (type Reax2, Heidolph) . Incubator (Thermo Forma Scientific Incubator Stericult 3035, Thermo Forma Scientific) and shaking incubator (Multitron, Infors HT) for growth of yeast cells . Centrifuges: for 1.5-ml microfuge tubes (Eppendorf 5810-R; Eppendorf), swing out centrifuge for harvest of yeast cells and separation of glass beads in 50- and 15-ml Falcon tubes (type 5810R, Eppendorf) and fix-angle highspeed centrifuge for fractionation of cell extracts (Beckman Coulter Optima LE-80K, Beckman Inc.) . Scintillation counter (LS 6500, Beckman Coulter Inc.) . Disposable plastic tubes (15 and 50 ml, Falcon) and 1.5-ml microfuge tubes . Electrophoresis and western blotting supplies (see ref. 63) REAGENT SETUP All reagents are stored at room temperature (i.e., 20–24 1C) and are stable for 1 month, unless indicated otherwise. Minimum essential medium (SC) (1 l) Dissolve 1.7 g of yeast nitrogen base without amino acids and 5 g of (NH4)2SO4 in 900 ml of ddH2O (add 20 g of agar-agar for solid agar plates). After autoclaving, add 100 ml of sterile 20% (wt/vol) galactose or glucose stock solution and auxotrophic markers appropriate for the yeast strains and plasmids used64. For SC medium without iron, use iron-free yeast nitrogen base. Galactose and glucose stock solutions Galactose and glucose stock solutions of 20% (wt/vol) in ddH2O, dissolve and autoclave. m CRITICAL The galactose stock solution should be slightly warmed (40 1C) in case galactose does not dissolve or has formed a crust after cooling. For iron-free SC medium, confirm that the iron concentration in the SC medium is below 0.1 mM by colorimetry65. Galactose from some suppliers is contaminated with iron. YP medium (1 l) Dissolve 10 g of yeast extract and 20 g of casein peptone in 900 ml of ddH2O (add 20 g of agar-agar for solid agar plates). After autoclaving,

add 100 ml of sterile 20% (wt/vol) galactose or glucose stock solution and auxotrophic markers appropriate for the yeast strains and plasmids used64. PMSF PMSF (200 mM) in 100% ethanol was used. m CRITICAL Prepare just before use, PMSF rapidly hydrolyses. ! CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. Alkaline lysis mix Mix on the day of use 6.91 ml of ddH2O, 1.85 ml of 10 M NaOH (40% (wt/vol) in ddH2O) and 0.74 ml of 2-mercaptoethanol. Add 0.5 ml PMSF solution just before use. ! CAUTION The alkaline lysis mix is caustic, irritant and toxic; wear suitable protective clothing, gloves and eye/face protection. ! CAUTION PMSF is toxic; wear suitable protective clothing, gloves and eye/face protection. TCA solution (50% wt/vol) Per 100 ml use 50 g of TCA. TCA solution (30% wt/vol) Per 100 ml use 30 g of TCA. ! CAUTION TCA is corrosive, causes severe burns; wear suitable protective clothing, gloves and eye/face protection 55FeCl with a specific radioactivity of 155–190 Gbq mg1 (76–94 mCi mg1) 3 Aliquots of 1 mCi in 15–25 ml 0.5 M HCl are delivered by NEN/Perkin Elmer in individual containers, to which 0.1 M of HCl is added to obtain 1 mCi ml1 (B250 mM Fe). ! CAUTION Causes cancer, designate area for handling 55Fe and clearly label all containers. Store mCi quantities of 55Fe behind thin lead shielding. Wear disposable lab coats, wrist guards and gloves for secondary protection. For more detailed information, see instructions of occupational limits in the NRC regulations (10 CFR) part 20, Standards for protection against radiation (http://www.nrc.gov/reading-rm/doc-collections/ cfr/part020/). Ascorbate solution (0.1 M) Dissolve 0.198 g in 10 ml of ddH2O on the day of use. Citrate buffer (50 mM citrate, 1 mM EDTA, pH 7.0) Dissolve 14.7 g of trisodium citrate and 0.372 g of disodium EDTA in 800 ml of ddH2O, adjust the pH to 7.0 with 1 M HCl, bring up to a final volume of 1 l with ddH2O and autoclave. HEPES buffer (20 mM HEPES/KOH pH 7.4) Per 100 ml, use 4 ml 0.5 M HEPES adjusted to pH 7.4 with 2 M KOH, and autoclave. TNETG buffer (10 mM Tris/Cl pH 7.4, 2.5 mM EDTA, 150 mM NaCl, 10% (vol/vol) glycerol, 0.5% (vol/vol) Triton X-100) Per 500 ml, use 5 ml of 1 M Tris-HCl pH 7.4, 2.5 ml of 0.5 M disodium EDTA (pH 7.4), 37.5 ml of 2 M NaCl, 50 ml of glycerol, and autoclave. On the day of use, add Triton X-100 to a final concentration of 0.5% (vol/vol). Beads for immunoprecipitation All steps should be carried out at 4 1C. Beads for immunoprecipitation of TAP-tagged proteins: Suspend the commercial IgG Sepharose 6 Fast Flow beads by inversion, transfer 500 ml to a 1.5-ml microfuge tube and centrifuge for 5 min at 800g at 4 1C to remove the supernatant. Wash three times with 500 ml of TNETG buffer and finally adjust the volume to 500 ml with TNETG buffer. Beads for immunoprecipitation with serum raised against a protein of interest: Swell 50 mg of Protein A-Sepharose CL-4B in 0.5 ml cold TNETG buffer in a 1.5-ml microfuge tube by incubation for at least 30 min in a rotary shaker. Centrifuge for 5 min at 800g at 4 1C, remove the supernatant and add 500 ml of antibody serum. After incubation for at least 1 h in a rotary shaker, centrifuge for 5 min at 800g at 4 1C and remove the supernatant. Wash five times with 500 ml of TNETG and centrifuge as before. Adjust the volume to 500 ml with TNETG buffer. Beads for immunoprecipitation with antibodies against hemagglutinin or Myc epitopes coupled to agarose: The 25% (wt/vol) commercial suspension can directly be used after repeated inversion. m CRITICAL Resuspend the beads by gentle swirling or inverting. Do not vortex, use a magnetic stirrer or spatula. Use blue or yellow pipette tips where 4 mm of the end have been cutoff with scissors, otherwise beads fracture and distribute unevenly.

PROCEDURE Transform yeast cells with plasmid TIMING 5 h 1| Grow yeast cells overnight in 50 ml of YP medium; on the next day, make competent yeast cells66, transform with 0.5–5 mg of plasmid of choice and plate onto SC agar containing 2% (wt/vol) galactose and the appropriate auxotrophy selection markers64. Incubate at 30 1C until colonies of the transformants of 1–2 mm have grown, this usually takes 3–5 d. m CRITICAL STEP Also, transform cells with a control plasmid lacking the gene of interest. ? TROUBLESHOOTING


PROTOCOL

Grow yeast transformants on plate TIMING 30 min 2| Plate individual transformants from Step 1 onto a quadrant of an SC agar plate with 2% (wt/vol) galactose and appropriate auxotrophy selection markers64. Incubate at 30 1C until a rather continuous but not overgrown lawn has developed, which usually takes 2–3 d. m CRITICAL STEP For stronger protein depletion, some galactose-regulatable mutants have to be downregulated by additional growth on glucose-containing SC agar plates before growth in liquid medium. Cell densities should always remain lower than an OD of 2.


Confirm the presence of the protein of choice in the transformants (optional) TIMING B8 h including SDS–PAGE and western blot 3| Take a few milligrams of cells from each quadrant (Step 2) using a yellow tip, transfer the cells to a 1.7-ml microfuge tube filled with 1 ml of ddH2O, centrifuge for 3 min at 13,000g, 4 1C. Add 75 ml of alkaline lysis mix and incubate for 10 min on ice. Add 575 ml of 50% (wt/vol) TCA, vortex and incubate for 10 min on ice and centrifuge for 3 min at 13,000g, 4 1C. Remove the supernatant and wash the pellet twice with 1 ml of ice-cold acetone, dissolve in 50 ml sample buffer63 and perform western blot after SDS–PAGE63. ! CAUTION TCA is corrosive, causes severe burns, wear suitable protective clothing, gloves and eye/face protection.

Pre-culture of yeast cells TIMING 15 min 4| Inoculate 50 ml of SC medium supplemented with a final concentration of 2% (wt/vol) galactose (or 2% (wt/vol) glucose) and the appropriate auxotrophy selection markers64 with a small scoop of yeast cells from the plate (Step 2). Incubate at 30 1C for 24 h. m CRITICAL STEP Some galactose-regulatable mutants have to be downregulated by repeating this pre-culturing step in SC glucose medium. Cell densities should always remain lower than an OD of 2 (see Step 5). ? TROUBLESHOOTING

Culture of yeast cells in iron-free medium TIMING B1 h 5| Determine the OD of the pre-culture from Step 4 at 600 nm. 6| Remove an appropriate volume of the pre-culture from Step 4 for the inoculation of 100 ml of iron-free SC medium at an OD of 0.2 (i.e., 20 divided by the OD measured in Step 5 (in milliliters)). 7| Transfer the culture to 50-ml Falcon tubes and centrifuge for 5 min at 2,000g at room temperature. Remove the supernatant and add 10 ml of sterile ddH2O, vortex the cell pellet and repeat the centrifugation as before. 8| Prepare sterile 300 ml Erlenmeyer flasks with 100 ml of iron-free SC medium with 2% (wt/vol) galactose (or 2% (wt/vol) glucose) and the appropriate auxotrophy selection markers64. Use 10 ml of this medium to resuspend the pellets obtained in Step 7 and transfer the cells to the same flask. Grow the cells overnight (16 h) in a shaking incubator at 150 r.p.m., 30 1C.

Radiolabeling with 55FeCl3 TIMING B3 h 9| Harvest the cells from the 100 ml cultures into a single 50-ml Falcon tube by two successive centrifugations for 5 min at 2,000g at room temperature. 10| Add 10 ml of ddH2O and resuspend the cell pellet, transfer to a 15-ml Falcon tube and centrifuge for 5 min at 1,500g at room temperature. 11| Determine the wet weight of the cell pellet (0.5–0.75 g). Resuspend the cells in 10 ml of iron-free SC medium with 2% (wt/vol) galactose (or 2% (wt/vol) glucose). ? TROUBLESHOOTING 12| Remove the appropriate volume that corresponds to 0.5 g of cells and place in a 15-ml Falcon tube. Adjust the volume to 10 ml with iron-free SC medium with 2% (wt/vol) galactose (or 2% (wt/vol) glucose). 13| Incubate the cell suspension for 10 min in a shaking incubator at 150 r.p.m., 30 1C. 14| Mix 10 ml of 55FeCl3 solution (10 mCi) with 100 ml of 0.1 M sodium ascorbate and add to the cells. Incubate for 2 h at 150 r.p.m., 30 1C. ! CAUTION Causes cancer; designate area for handling 55Fe and clearly label all containers. Store mCi quantities of 55Fe behind thin lead shielding. Wear disposable lab coats, wrist guards and gloves for secondary protection. For more detailed information,


PROTOCOL see instructions of occupational limits in the NRC regulations (10 CFR) part 20, Standards for protection against radiation (http://www.nrc.gov/reading-rm/doc-collections/cfr/part020/). ! CAUTION From this point onward, all liquid and solids have to be disposed of according to radiation safety regulations or decontaminated and sterilized for repeated use by washing with citrate buffer, water and ethanol.


Cell extract preparation TIMING B1 h 15| Transfer the 55Fe-labeled cells to a 15-ml Falcon tube and harvest by centrifugation for 5 min at 2,000g at room temperature. Remove the supernatant and wash the cell pellet with 10 ml of citrate buffer to remove residual 55Fe from the medium and the outside of cells. Centrifuge for 5 min at 2,000g at room temperature, remove the supernatant and wash the cell pellet with 2 ml of 20 mM HEPES-KOH, pH 7.4 to remove the citrate buffer. 16| Centrifuge the cells for 5 min at 2,000g at room temperature, remove the supernatant by pipetting and determine the wet weight of the cells (typically 0.45–0.55 g). 17| Resuspend the cell pellet in a volume of TNETG buffer equal to the mass of the cell pellet and place the tubes on ice. Add 10 ml of 0.2 M PMSF and 1 ml of glass beads. Close the lid tightly. 18| Invert the tube by flicking the contents to the lid side of the tube in one motion. Vortex at maximum speed with the lid side of the tubes contacting the rotating plate for 1 min, repeat a total of three times with intermittent 3 min cooling periods on ice. m CRITICAL STEP Ensure that there are no glass beads trapped in the lid. All subsequent Steps (19–23) should be carried out at 4 1C. 19| Centrifuge the cells for 5 min at 2,000g at 4 1C to pellet the glass beads and unlysed cells. Transfer the supernatant to 1.5-ml microfuge tubes, centrifuge for 10 min at 13,000g at 4 1C and carefully transfer 450–500 ml of the supernatant to a fresh tube. m CRITICAL STEP Avoid the transfer of the lipid-containing surface layer. 20| Remove 5 ml of the extract for the determination of the cellular iron uptake: mix with 45 ml of ddH2O in a 1.5-ml microfuge tube, add 1 ml of scintillation fluid, vortex for 30 s and determine the radioactivity by scintillation counting. For western blotting of Fe/S and non-Fe/S marker proteins, take a 25-ml sample of the extract and add 175 ml of ice-cold 30% (wt/vol) TCA, vortex and incubate on ice for 10 min. ! CAUTION TCA is corrosive, causes severe burns, wear suitable protective clothing, gloves and eye/face protection 21| Centrifuge the TCA-treated extract from Step 20 for 10 min at 13,000g, 4 1C and remove the supernatant. Add 500 ml of ice-cold acetone, repeat the centrifugation step and wash again with 500 ml of ice-cold acetone. Remove the supernatant and air dry the protein pellet. Dissolve the pellet in 50 ml of sample buffer.63 ’ PAUSE POINT The sample can be stored up to 1 month at –20 1C before SDS–PAGE and western blotting (see Step 25).

Immunoprecipitation TIMING B1.5–2 h 22| Add either 20–50 ml of self-made antibody Protein A-Sepharose beads, 20–40 ml of commercially available IgG Sepharose beads, or 10–15 ml of anti-hemagglutinin or anti-Myc (A-14) beads to 200–250 ml of cleared cell extract (from Step 19) and incubate the mixtures in 1.5-ml microfuge tubes for 1 h on a rotary shaker at 4 1C. 23| Collect the beads by centrifuging for 5 min at 800g at 4 1C and carefully remove the supernatant by repeated pipetting with a 200 ml pipette. Wash the beads three times in 500 ml of ice-cold TNETG and collect the beads by centrifugation. m CRITICAL STEP The first two removals of supernatant are critical for a successful experiment (i.e., low background). Use a black background to improve visibility of the beads during pipetting. 24| Add 50 ml of ddH2O and 1 ml of scintillation cocktail to the beads, vortex for 30 s, place the microfuge tube in a plastic counting vial and measure the 55Fe radioactivity associated with the beads in a scintillation counter with settings appropriate for 3H (ref. 67). ? TROUBLESHOOTING

Protein analysis by western blotting TIMING 1 d 25| Analyze protein extracts from Step 21 by SDS–PAGE and western blotting63.

TIMING Step 1, transform yeast cells with plasmid: 5 h plus 3–5 d for cell growth Step 2, grow yeast transformants on plate: 30 min plus 2–3 d for cell growth



PROTOCOL Step 3, confirm the presence of protein of choice in transformants (optional): B8 h including SDS–PAGE and western blot Step 4, pre-culture of yeast cells: 15 min plus 24 h for cell growth Steps 5–8, culture of yeast cells in iron-free medium: B1 h plus 16 h for cell growth Steps 9–14, radiolabeling with 55FeCl3: B3 h Steps 15–21, cell extract preparation: B1 h Steps 22–24, immunoprecipitation: B1.5–2 h Step 25, protein analysis by western blotting: 1 d Box 1, culture of cells: 1–10 d (depends on the availability of pre-culture and necessity of transformation); cell extract preparation: B45 min; protein determination: 30 min; activity measurements: 5 min for aconitase, 5 min for isopropylmalate isomerase, 45 min for sulfite reductase Box 2, culture of cells: 1–10 d (depends on the availability of pre-culture and necessity of transformation); preparation of mitochondria: B6 h; protein determination: 30 min; activity measurements: 5 min for aconitase and 5 min for succinate dehydrogenase ? TROUBLESHOOTING Troubleshooting advice can be found in Table 2. TABLE 2 | Troubleshooting table. Step 1

Possible reason Wrong auxotrophic marker(s)

Solution Check medium, yeast genotype and plasmid

Toxic plasmid

Use a weaker promotor

Few or too many transformants

Plasmid quantity inappropriate

Vary the length of heat shock in transformation protocol and modify the plasmid quantity

4

Incomplete protein depletion

Insufficient downregulation on glucose

Screen downregulation: try depletion on glucose agar plate and/or vary culture time in liquid medium (16, 40 and 64 h) until target protein cannot be detected on western blot

11

Insufficient cell mass

Slow growth

Grow a larger volume of pre-culture, use a fresher preculture, inoculate with slightly higher OD

Too high expression level

Use moderate expression level by use of low-copy plasmid and/or weaker promoter

Tag interferes with biological function

Change tag or tag position (N-/C-terminal), use untagged protein and antibodies against the protein

Weak binding of proteins to beads

Try other tag, change tag to N- or C-terminal position, raise new antibodies, buy fresh beads, check binding of antibody to Protein A

Oxygen lability of Fe/S

Perform cell extract preparation and further steps in an anaerobic chamber; all buffers should be anaerobic

Protein is degraded

Add protease inhibitors. PMSF should be prepared freshly. If the tag is cleaved off, try another tag, change to N- or C-terminal position or co-synthesize interaction partner that might stabilize the target12

Cells lysed inefficiently

Determine protein content of whole-cell extracts (should be 3–6 mg/ml), optimize glass bead/vortex procedure with nonradioactive cells

24

Problem No transformants

Low radioactivity in the immunoprecipitate

(continued)


PROTOCOL TABLE 2 | Troubleshooting table (continued).

Step

Problem


High or variable background

Possible reason Protein not expressed

Solution Check several transformants by western blot, change to high-copy plasmid and/or use stronger promoter

Iron contamination of medium

Check iron content of medium components, change supplier

Target is not an Fe/S protein

Confirm that the protocol functions for a known Fe/S protein (Table 3)

Inefficient washing of beads

Remove all liquid, especially in the first wash step Centrifuge cell extracts for longer, remove supernatant more carefully. If yeast cells loose viability, the background may vary. Try shorter downregulation times

Triton or chelator concentration too low

Try other detergents, use EDTA or citrate

ANTICIPATED RESULTS 55Fe incorporation into Fe/S proteins in yeast is a powerful method to determine (1) the presence of an Fe/S cluster in the analyzed protein in vivo, (2) new members of the ISC assembly, ISC export or CIA machineries, (3) the target specificity of ISC and CIA components by the effect of their downregulation on various target Fe/S proteins, (4) the presence of Fe/S clusters in scaffold and other proteins of the ISC and CIA machineries, (5) the order of in vivo events during de novo assembly of Fe/S clusters by downregulation of the ISC and CIA components. Using this approach, we previously were able to define the scaffold function of Isu1 in vivo and distinguish early and late components of the ISC assembly machinery37. Further, we identified the P-loop NTPases Cfd1 and Nbp35 as cytosolic scaffold proteins and thus were able to stage the requirement of the TABLE 3 | Summary of expected results for yeast Fe/S proteins studied by 55Fe/S cluster incorporation and enzymatic activity. Protein Gene Cytosolic and nuclear Fe/S proteins Isopropylmalate isomerase Leu1 Sulfite reductase Ecm17

55Fe (pmol per g cells)a

Activity (U per mg of protein)a Biological function

15–23 NDb

0.1–0.3 0.0035

ABC protein Rli1

Rli1

27–35

ND

P-loop NTPase Nbp35

Nbp35

11–13

ND

P-loop NTPase Cfd1

Cfd1

8–12

ND

Hydrogenase-like protein

Nar1

6–10

ND

DNA glycosylase

Ntg2

9–11

ND

Aco1

13

6–11

0.5c 2.4–3.5d ND 0.021–0.071e ND ND 0.15–0.3f 0.15–0.3g ND

13

ND

Mitochondrial Fe/S proteins Aconitase Homoaconitase Glutamate dehydrogenase Lipoate synthase Biotin synthase Complex II Ferredoxin

Lys4 Glt1 Lip5 Bio2 Sdh2 Sdh2-Rip1 Yah1

Scaffold protein Isu1

Isu1

aValues

12–21 ND 10 80–300 ND

Reference

Biosynthesis of leucine Biosynthesis of methionine and cysteine Biogenesis of ribosomes, translation initiation Maturation of cytosolic and nuclear Fe/S proteins Maturation of cytosolic and nuclear Fe/S proteins Maturation of cytosolic and nuclear Fe/S proteins DNA repair

14,55 14

Citric acid cycle

7 10,55 7 70 7 14,71 10,55 10 37,51,72

Biosynthesis of lysine Biosynthesis of glutamate Biosynthesis of lipoate Biosynthesis of biotin Succinate dehydrogenase Maturation of Fe/S proteins, heme A synthesis Maturation of Fe/S proteins

29,55 12,55 12 12,13 55

37

refer to yeast cells grown in SC medium supplemented with 2% (wt/vol) galactose. Here 1.07 103 c.p.m. correspond to 1 pmol 55Fe under the conditions used in the procedure. All radiolabeling data refer to overproduction of the respective Fe/S proteins from plasmids except for Leu1 and Aco1. Enzyme activities correspond to wild-type levels. bNot determined. cSpecific activity in whole-cell extract with the coupled assay (Box 1). dSpecific activity in mitochondria with the direct assay (Box 2). eValues refer to yeast cells grown in medium supplemented with 2% (wt/vol) glucose and various nitrogen sources. fSpecific activity expressed as DCPIP reduction (Box 2). gSpecific activity expressed as cytochrome c reduction (Box 2), which requires functional complex III.


+

40 h Gal

16 h

+

–

α-Cfd1 α-Nfs1 α-Leu1 40 h Glc Gal-NFS1

40 h

0

Bio2 Leu1 Rli1- Ntg2HA HA

α-Bio2 α-Leu1 α-HA α-HA α-Cfd1 G lc

0 +

25

G lc

0 Cfd1-TAP

5

50

G al

4

75

G lc

10

G al

Leu1

8

100

G lc

15

b

G al

Cfd1

Fe incorporation (%) (Glc/Gal)

12

55

55 Fe incorporation into Cfd1 (103 c.p.m. per g cells)

a

G al


Figure 3 | Incorporation of 55Fe into yeast Fe/S proteins in vivo. Gal-NFS1 (a) and Gal-CFD1 (b) cells were grown in galactose (Gal) or glucose (Glc) to induce or repress the synthesis of Nfs1 and Cfd1, respectively. Cells were grown for the indicated time periods (a) or 40 h (b), radiolabeled for 2 h with 55Fe according to the Protocol and cell extracts were prepared. An aliquot of the extract was immediately TCA-precipitated for detection of the proteins by western blotting (lower panels). The remainder (B250 ml) was added to IgG (Cfd1-TAP), anti-HA antibodies (Rli1-HA and Ntg2-HA) or home-made antibodies (against yeast Leu1 and Bio2) coupled to beads. After 1 h of incubation, the beads were extensively washed, and the amount of 55Fe associated with the beads was determined by scintillation counting (upper panels). Reproduced from ref. 12 previously published in Nature Chemical Biology.

55 Fe incorporation into Leu1 (103 c.p.m. per g cells)

PROTOCOL

Gal-CFD1

CIA proteins in the pathway of cytosolic Fe/S cluster assembly12. Typical results for 55Fe incorporation into reporter Fe/S proteins in wild-type cells are given in Table 3. Except for Aco1 and Leu1, the Fe/S proteins were overproduced from expression plasmids. Likewise, Table 3 provides representative specific enzyme activities in galactose-grown wild-type cells. Appropriate non-Fe/S enzymes should be measured in parallel as controls10,19,68. Figure 3 shows typical results for an in vivo 55Fe incorporation experiment in yeast. In Figure 3a, the mitochondrial cysteine desulfurase Nfs1 was synthesized or depleted in Gal-NFS1 cells by growth in galactose- or glucose-containing media, respectively. These cells carry the NFS1 gene under the control of the GAL1-10 promoter, which is induced by galactose and repressed by glucose. The incorporation of 55Fe into the scaffold Cfd1 (carrying a TAP tag) and the cytosolic Fe/S target protein Leu1 was determined as described above. Incorporation of 55Fe into both Cfd1 and Leu1 strongly decreased on depletion of Nfs1 verifying that the associated 55Fe is part of an Fe/S cluster. Detection of 55Fe on Cfd1 depended on its overproduction, whereas wild-type levels of Leu1 were sufficient for the measurement of 55Fe/S cluster assembly. The background levels of 55Fe assay can be determined, e.g., by omitting production of Cfd1 (Fig. 3a) or by the use of pre-immune serum instead of specific antiserum. Typically, the background values vary between 2 and 15% of the total signal depending on the Fe/S protein and antiserum. In Figure 3b, we used the yeast mutant Gal-CFD1 with the CFD1 gene under the control of the GAL1-10 promoter to deplete Cfd1 (see western blot in Fig. 3b). After 55Fe radiolabeling, cell extracts were prepared as described under Protocols. Cytosolic (Leu1) and mitochondrial (Bio2) Fe/S proteins were immunoprecipitated with specific antibodies coupled to Protein A-Sepharose beads, whereas the HA-tagged Fe/S proteins Rli1 (cytosol/nucleus) and Ntg2 (nucleus) were immunoprecipitated using commercially available HA beads. The latter three Fe/S proteins require overproduction from appropriate plasmids due to their low cellular abundance. The radioactivity associated with mitochondrial Bio2 remained almost unchanged on depletion of cytosolic Cfd1 (Fig. 3b). In contrast, the nuclear and cytosolic Fe/S proteins had only 15–25% of the radioactivity associated compared with galactose-grown cells. These data, in conjunction with western blotting experiments assessing the presence or absence of the relevant proteins, show the specific function of Cfd1 for Fe/S cluster assembly on extra-mitochondrial proteins.

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