Supporting Information Kropat et al. 10.1073/pnas.1422492112 SI Text Inductively Coupled Plasma Mass Spectrometry. The 63Cu, 65Cu, and Fe content of the samples was quantified from response curves generated from a dilution series of a certified standard solution of known concentration. Linear responses (r2 > 0.999) were observed over a dynamic range of four orders of magnitude of metal concentration that encompassed the concentrations of metal found in the samples. Individual analyses of the standards, reagent blanks, and samples were corrected for variations in the response for the internal standards (Ga, Y, and Tl), and the content of metals in the samples was normalized to cell count where appropriate after subtraction of the reagent blank. Accuracy and precision of the preparative and analytical procedures were confirmed by the analysis of five replicate samples of certified reference material. Overall accuracy was determined to be typically better than 5%, and quantitative precision was greater than 3%. Isotopic ratio analyses of samples were compared with certified Cu standards prepared in 1% HNO3 to confirm the absence of polyatomic interferences due to 63NaAr+ formation. No interferences were observed, and precision of the isotopic ratio measurements of 63Cu/ 65Cu was typically better than 1%. Quality control of accuracy during the analysis of a batch of samples was ensured by quantification of the reference material and a 100-μg/L certified Cu standard after every 10 samples. Detection limits for Cu, as determined by the 3δ value obtained from the analysis of multiple reagent blanks, were typically better than 0.5 nM. The association and distribution of metals in soluble supernatant extracts were determined using chromatography interfaced inductively coupled plasma mass spectrometry (ICP-MS) as described previously (1). 1. Mason AZ, Moeller R, Thrippleton KA, Lloyd D (2007) Use of stable isotopically enriched proteins and directly coupled high-performance liquid chromatography inductively coupled plasma mass spectrometry for quantitatively monitoring the transfer of metals between proteins. Anal Biochem 369(1):87–104. Fig. S1. Preferential accumulation of copper-containing proteins in copper-deficient cells. Cells of strain CC-4532 were grown to a density of 1.2 × 107 cells per milliliter in tris-phosphate (TP) medium containing the indicated concentrations of copper and either 1 μM (Left) or 18 μM (Right) iron. The abundances of plastocyanin (PC), Fox1, COX2B, and the α/β subunit of CF1 (α/β CF1) were monitored by immunoblotting. Samples were loaded based on equal cell amounts. Kropat et al. www.pnas.org/cgi/content/short/1422492112 1 of 7 Fig. S2. Copper requirement for plastocyanin maintenance is dependent on the ferroxidase and Cyt oxidase quota. Strain CC-4532 was grown photoheterotrophically in TAP medium or phototrophically in TP medium containing various amounts of copper under either iron-replete (20 μM Fe) or irondeficient (1 μM Fe) conditions. Cells were collected at a density of 12 × 106 cells per milliliter and analyzed by immunoblotting for plastocyanin, Cyt oxidase, or Fox1 abundance. The α/β CF1 is used as a loading control. A dilution series of samples with the highest protein expression (*) is shown for reference. All samples were blotted onto one membrane so that signal intensities could be compared between samples. Fig. S3. Loss of plastocyanin in copper-deficient cells is not attributed to dilution by cell division. Copper-deficient cells were inoculated into TAP medium containing 20 nM copper at a density of 1 × 104 cells per milliliter. Cells were collected at the indicated cell density for analysis of plastocyanin (A) and Cyt c6 (B–D) abundance. The lanes were loaded on the basis of equal culture volume to give an indication of the amount of plastocyanin per milliliter of culture. Lanes 7 through 12 show a dilution of the sample in lane 3 for plastocyanin and lane 6 for Cyt c6. Kropat et al. www.pnas.org/cgi/content/short/1422492112 2 of 7 Fig. S4. Plastocyanin is the most abundant copper protein in the soluble fraction. Freshly prepared soluble protein extract from WT (CRR1:crr1) cells grown under copper-replete conditions was prepared as described in Materials and Methods. Around 100 μg of extract was loaded on an anion exchange column (BioSep-DEAE; PEI) and eluted by a gradient of 0–500 mM ammonium chloride, pH 8.2, over 30 min. The metal content of the eluate was directly analyzed by inductively coupled plasma mass spectrometry (ICP-MS). Purified plastocyanin and a 10 mM CuCl2 solution were used as standards to identify their corresponding peaks. Kropat et al. www.pnas.org/cgi/content/short/1422492112 3 of 7 Fig. S5. Genes that coexpress with CYC6 during the transition from the copper-replete condition to the copper-deficient condition. As in Fig. 5 and Fig. S6, RNA abundance estimates were determined at the indicated cell densities by RNA-seq from WT cells grown with either 20 or 400 nM copper. Presented here are the 20 genes with expression profiles most similar to the expression profile of CYC6. Log10 transformations of RNA abundance estimates in terms of FPKMs were used to generate a clustered heat map on a scale ranging from blue (lowest expression) to yellow (highest expression). Kropat et al. www.pnas.org/cgi/content/short/1422492112 4 of 7 Fig. S6. Genome-wide changes in transcript abundances during the transition from the copper-replete to copper-depleted condition. WT strain CC-4532 was inoculated at 1 × 104 cells per milliliter into TAP medium supplemented with either 20 or 400 nM copper. RNA was purified from cells of each culture once the cell density reached 0.5 × 106, 1 × 106, 2 × 106, 4 × 106, and 8 × 106 cells per milliliter. RNA was quantified in terms of FPKMs by RNA-seq. Genes whose expression was at least 10 FPKMs and also increased eightfold or greater at any point relative to the first time point were determined with cummeRbund and plotted as a clustered heat map with R. Log10 transformations of FPKMs are presented on a scale ranging from blue (lowest expression) to yellow (highest expression). AOX2, alternative oxidase 2; CAH8, carbonic anhydrase 8; FEA2, Fe-assimilation protein 2; HYDEF, hydrogenase assembly protein; LHCBM8, light harvesting chlorophyll a/b binding protein, major subunit 8; LHCSR3, light harvesting chlorophyll a/b binding protein, stress related 3; MME3, malic enzyme 3; PPD1, pyruvate phosphate dikinase 1; TEF13, thylakoid-enriched fraction protein 13. Kropat et al. www.pnas.org/cgi/content/short/1422492112 5 of 7 Fig. S7. Cu- and CRR1-dependent growth. Growth of WT (solid line) and crr1 (dashed line) mutant Chlamydomonas cells in copper-supplemented (blue) or copper-deficient (red) medium containing acetate (heterotrophic) or not containing acetate (phototrophic) was monitored by cell counting. (A) Photoheterotrophic, (B) phototrophic, (C) heterotrophic growth. Kropat et al. www.pnas.org/cgi/content/short/1422492112 6 of 7 Fig. S8. Plastocyanin accumulation in a strain lacking Cyt oxidase. (A) Growth of WT (CC-124) and dum19 mutant (CC-3400) Chlamydomonas cells in the dark. Cultures were inoculated with 4 × 106 cells per milliliter, and the image was taken after 6 d of growth in the dark. (B) Rate of cyanide-sensitive oxygen consumption in WT and dum19. Measurements were performed as described in Table S1. (C) Plastocyanin abundance is monitored by immunoblotting of soluble protein extracts from copper-supplemented (+) and copper-deficient (−) WT and dum19 cells. Another lumen-located protein, OEE1, is monitored as a loading control. The abundance of Cyt c6 is an indication of the copper nutrition status of the cells. OEE1, oxygene-evolving enhancer protein 1. Table S1. Oxygen consumption in copper-replete and copperdeficient conditions Condition No inhibitor +5 mM KCN +5 mM SHAM No inhibitor +5 mM SHAM +5 mM KCN +Cu, nmol of O2 per minute per 107 cells 20.9 8.2 1.9 21.6 14.5 2.0 ± ± ± ± ± ± 1.4 0.4 0.2 1.9 1.9 0.3 −Cu, nmol of O2 per minute per 107 cells 8.5 5.2 1.8 8.3 4.5 1.8 ± ± ± ± ± ± 0.2 0.8 0.2 0.7 0.8 0.4 WT strain CC-4532 was grown in copper-deficient or copper-replete (6 μM Cu) medium to a density of 6 × 106 or 8 × 106 cells per milliliter. Two milliliters of culture was removed; after addition of 34 mM acetate, oxygen consumption in the dark was analyzed using a ChloroLab2 electrode from Hansatech. After 7 min of a stable respiratory rate, potassium cyanide was added to a final concentration of 5 mM. After about 7 min, the second inhibitor, salicyl hydroxamic acid (SHAM), was added to a final concentration of 5 mM and oxygen consumption was observed for another 7 min. The measurement was repeated with 2 mL of the same culture but with a reversed order of inhibitor addition. The values represent the average and SD of six independent experiments with all measurements adjusted to 107 cells per milliliter. Kropat et al. www.pnas.org/cgi/content/short/1422492112 7 of 7