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SUPPLEMENTARY MATERIALS for:

Manganese co-localizes in Chlamydomonas acidocalcisomes with Ca and P and is mobilized in Mn-deficient situations

Munkhtsetseg Tsednee a, 1, Madeli Castruita a, 1, Patrice A. Salomé a, b, 1, Ajay Sharma,c Brianne

Elizabeth Lewis d, Stefan R. Schmollinger a, b, Daniela Strenkert a, b, Kristen Holbrook a, e, Marisa S. Otegui f, Kaustav Khatua g, Sayani Das g, Ankona Datta g, Si Chen h, Christina Ramon i, Martina Ralle j, Peter K. Weber i, Timothy L. Stemmler d, Jennifer Pett-Ridge i, Brian M. Hoffman c and

Sabeeha S. Merchant a, b, k

From the a Department of Chemistry and Biochemistry, and b Institute for Genomics and Proteomics, University of California, Los Angeles, CA 90095; c Department of Chemistry, Northwestern University, Evanston, IL 60208; d Department of Pharmaceutical Sciences, Wayne State University, Detroit, MI 48201; e Present address: Amgen, 1 Amgen Center Dr, Thousand Oaks, CA 91320; f Departments of Botany and Genetics, University of Wisconsin-Madison, WI 53706; g Department of Chemical Sciences, Tata Institute of Fundamental Research, Mumbai, 400005, India; h Advanced Photon Source, Argonne National Laboratory, Lemont, IL 60439; i Physical and Life Sciences Directorate, Lawrence Livermore National Laboratory, Livermore, CA 94550; j Department of Molecular and Medical Genetics, Oregon Health and Science University, Portland, OR 97239; k Present address: Departments of Plant and Microbial Biology, Molecular and Cell Biology, UC Berkeley, Berkeley, CA 94720 1: equal contribution

Running title: Robust Mn homeostasis in Chlamydomonas is mediated by the acidocalcisome

* to whom correspondence should be addressed: Sabeeha S Merchant: 176 Stanley Hall QB3, UC Berkeley, Berkeley, CA 94720; sabeeha@chem.ucla.edu

Keywords: algae, antioxidant, calcium, Chlamydomonas, H+-PPase, histidine, imaging, lysosomal acidification, lysosome, manganese, metal homeostasis, NRAMP, organelle, photosynthesis,

polyphosphate

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SUPPLEMENTARY METHODS: X-ray Fluorescence

Chlamydomonas cultures for XFM analysis were grown in TAP medium with replete Mn (6 µM Mn-EDTA) in Fe limitation (0.1 µM Fe-EDTA). Cells were collected at mid-log phase at a density of 1x106 cells/ml, washed briefly in Phosphate buffered saline (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), before being fixed in 4% paraformaldehyde in PBS for 30 min at 37 C on a 0.01% Poly-L-Lysine pre-treated silicon nitride window (Silson Ltd). Windows were washed twice each with PBS, freshly made 100 mM ammonium acetate solution and finally with doubly distilled water. Windows were air-dried overnight before being shipped to the beamline for spatial elemental analysis. XFM data were collected on the Bionanoprobe (BNP) (Vogt et al., 2015; Chen et al., 2014) at the Life Sciences Collaborative Access beamline 21-ID-D (now at 9-ID-B) at the Advanced Photon Source, Argonne National Laboratory, Argonne, IL. The BNP was operated below 110K under high vacuum (10-7 – 10-8 Torr). The incident X-ray energy was tuned to 10 keV using a Si(111) double-crystal monochromator. The monochromatic beam was focused to ~100 nm x 100 nm using stacked Fresnel zone plates. Cells were selected using an optical microscope with 10x lens integrated in the BNP. For each scan, the sample was placed at 15º to the incident X-ray beam and the resulting X-ray fluorescence was collected at 90º with regards to the incident beam using an energy dispersive 4-element detector (Vortex ME-4, SII Nanotechnology, Northridge, CA). Elemental maps were obtained by extracting the raw data, background subtracting, and fitting the fluorescence counts for each element at each point using the program MAPS (Vogt, 2008). The fluorescent photon counts were translated into µg/cm2 using calibrated X-ray standards (AXO products, Dresden, Germany). Quantitative analysis was performed using a 2-element angular cluster analysis function in MAPS. By doing this unbiased ROIs for background, the cell, and the acidocalcisomes were obtained. Exported images were assembled in Adobe Illustrator. TEM-EDXS analysis

Chlamydomonas cells grown in medium containing high concentration of Mn were high-pressure frozen in a Baltec HPM 010. For structural analysis, high-pressure frozen cells were freeze-substituted in 2% OsO4 in acetone during 14 h, embedded in Eponate 12, sectioned, and stained with 2% uranyl acetate in 70% methanol and lead citrate (2.6% lead nitrate and 3.5% sodium citrate, pH 12). For EDX analysis, high-pressure frozen cells were freeze-substituted for 24 h in 2% glutaraldehyde in acetone and embedded in Lowicryl HM20 (Electron Microscopy Sciences). Unstained 100-thick sections mounted on single slot gold grids were loaded in a beryllium holder and imaged in a Tecnai T12 transmission electron microscope equipped with an EDS probe (Thermo Scientific) with a resolution of 139 eV. Proteomics analysis of whole cell proteins

Protein digestion and peptide detection by LC-MS/MS We collected 108 cells by centrifugation at 1,450 g at 4ºC for 4 min. The cell pellet was washed once with 1 ml 10 mM Phosphate buffer, pH 7.0, resuspended in 200 µl 10 mM Phosphate buffer, pH 7.0, flash frozen in liquid nitrogen and stored in -80° until processing. Samples were thawed and Urea was added to a final concentration of 8 M urea and the protein lysate was transferred to 2mL snap-cap centrifuge tubes (Eppendorf, Hamburg, Germany) with 0.1 mm zirconia beads and bead beat in a Bullet Blender (Next Advance, Averill Park, NY) at speed 8 for 3 minutes at 4°C. After bead beating, the lysate was spun into a 15mL Falcon tube at 2000 g for 10 minutes at 4°C. The supernatant was removed to a clean tube. Protein concentration was determined by BCA assay (Thermo Scientific, MA, USA). Urea and dithiothreitol were added to all samples at a final concentration of 8 M and 10 mM, respectively before incubation at 60ºC for 30 min with constant shaking (800 rpm). All samples were then diluted 8-fold with 100 mM NH4HCO3 and 1 mM CaCl2, and digested with sequencing-grade modified porcine trypsin (Promega, WI, USA) provided at a 1: 50 [w/w] trypsin-to protein ratio for 3 h at 37ºC. Digested samples were desalted using a 4-probe positive pressure Gilson GX-274 ASPEC™ system (Gilson Inc., WI, USA) with Discovery C18 100 mg/mL solid phase extraction tubes (Supelco, MO, USA) as follows: columns were pre-conditioned with 3 ml methanol, followed by 2 ml 0.1%trifluoroacetic acid (TFA) in water. Samples were then loaded onto

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columns, followed by 4 ml 95:5 water: acetonitrile (ACN) 0.1% TFA. Samples were eluted with 1 ml 20:80 water:ACN 0.1% TFA, and concentrated to a final volume of ~100 µl in a Speed Vac. After determination of peptide concentration by BCA assay, samples were diluted to 0.25 µg/µl with nanopore water for LC-MS/MS analysis (LC part: LC column of fused silica [360 µm x 70 cm] handpacked with Phenomenex Jupiter derivatized silica beads of 3 µm pore size (Phenomenex, CA, USA); HPLC part: HPLC NanoAcquity UPLC system (Waters, MA, USA); MS part: Q Exactive Plus mass spectrometer (Thermo Fisher, MA, USA)). Samples were loaded onto LC columns with 0.05% formic acid in water and eluted in 0.05% formic acid in ACN over 100 min. Twelve high resolution (17.5K nominal resolution) data-dependent MS/MS scans were recorded in centroid mode for each survey MS scan (35K nominal resolution) using normalized collision energy of 30, isolation width of 2.0 m/z, and rolling exclusion window lasting 30 seconds before previously fragmented signals are eligible for re-analysis. Unassigned charge and singly charged precursor ions were ignored.

MS/MS data search The MS/MS spectra from all LC-MS/MS datasets were converted to ASCII text (.dta format) using MSConvert (http://proteowizard.sourceforge.net/tools/msconvert.html) which precisely assigns the charge and parent mass values to an MS/MS spectrum as well as converting them to centroid. The data files were then interrogated via target-decoy approach (Elias and Gygi, 2010) using MSGFPlus (Kim and Pevzner, 2014) using a +/- 20 ppm parent mass tolerance, partial tryptic enzyme settings, and a variable posttranslational modification of oxidized Methionine. All MS/MS search results for each dataset were collated into tab separated ASCII text files listing the best scoring identification for each spectrum. SUPPLEMENTARY REFERENCES: Chen, S. et al. (2014). The Bionanoprobe: Hard X-ray fluorescence nanoprobe with cryogenic

capabilities. J. Synchrotron Radiat.

Elias, J.E. and Gygi, S.P. (2010). Target-decoy search strategy for mass spectrometry-based proteomics. In Methods in molecular biology (Clifton, N.J.), pp. 55–71.

Kim, S. and Pevzner, P.A. (2014). MS-GF+ makes progress towards a universal database search tool for proteomics. Nat. Commun. 5.

Vogt, S. (2008). MAPS : A set of software tools for analysis and visualization of 3D X-ray fluorescence data sets. J. Phys. IV 104: 635–638.

Vogt, S., Paunesku, T., Flachenecker, C., Finney, L., Jin, Q., Woloschak, G., Hornberger, B., Brister, K., Yuan, Y., Lai, B., Chen, S., and Jacobsen, C. (2015). The Bionanoprobe: Synchrotron-Based Hard X-ray Fluorescence Microscopy for 2D/3D Trace Element Mapping. Micros. Today 23: 26–29.

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SUPPLEMENTARY FIGURES:

Figure S1. Varying Mn supply does not affect Chlamydomonas physiology. Cultures were inoculated from a preculture containing 6 µM Mn in TAP medium to an initial cell density of 104 cells/mL (PFD, ~100 µmol m-2s-1); samples were collected after 72 h to determine chlorophyll content (A) and chlorophyll fluorescence (B). The experiment was from 4 independent experimental replicates. The horizontal bars represent the mean for each sample, ± standard deviation.

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Figure S2. Mn hyper-accumulation does not impact the uptake of other metals. Cultures were inoculated from a 6 µM MnEDTA pre-culture at an initial cell density of 104 cells ml-1

and grown in medium supplied with the indicated amount of MnEDTA (PFD, ~100 µmol m-2s-1). Intracellular content was quantified via ICP-MS for Fe (A), Cu (B), Zn (C), P (D), and Ca (E) from 4 independent experiment replicates. Corresponding Mn content from the same cultures is shown in Figure 1B of the main manuscript. The horizontal bars represent the mean for each sample, ± standard deviation.

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Figure S3. Mn co-localizes with P and Ca in Mn replete conditions. Shown are X-ray differential phase contrast and false color 2 D elemental maps for Ca (blue), Mn (green), P (red) as well as an overlay for all three elements (merge). Minimum and maximum area concentration are displayed above each element. Cells grown in 6 μM EDTA and fixed on X-ray sample supports were raster scanned at the APS Bionanoprobe. X-ray fluorescence data were measured in flyscan mode using an energy dispersive detector, fluorescence spectra fitted at each point of the scan, and the elemental area concentration determined from measuring X-ray standards. The scan area was 13 x 12 um; pixel size: 70 nm; dwell time: 200 ms.

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Figure S4. Structural and EDX analysis of high-pressure frozen/freeze-substituted Chlamydomonas cells grown in medium containing 1000μM MnEDTA. (A) Micrograph of a cell with acidocalcisomes (ac) containing electron-dense inclusions. For structural analysis, high-pressure frozen cells were freeze-substituted in 2% osmium tetroxide in acetone and stained with 2% uranyl acetate in 70% methanol and lead citrate. Ch = chloroplast. (B) Semi quantitative EDX analysis of two areas of a cell corresponding to an electron dense inclusion inside an acidocalcisome (red line and red asterisk on micrograph) and cytoplasm (black line and black asterisk on micrograph). For EDX analysis, high-pressure frozen cells were freeze-substituted in 2% glutaraldehyde. Note the presence of P, Mn, Ca, Mg, and Fe in the electron dense inclusion. The EDX spectrum is representative of one of the 10 electron dense particles analyzed. The area close to 0.5Kev is not well resolved and multiple possible spectral lines are assigned to this region.

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Figure S5. Defining regions of interest (ROIs) for quantification of NanoSIMS data. Cells were primarily identified based on the 12C+ images shown in (A), and to a lesser extent the 40Ca+ and 31P+ images (not shown). Each cell was individually outlined by hand (B) to serve as reference for ROIs generated by an automated subroutine that sub-divided cells into hexagons (C). Ion ratios (31P+/12C+, 40Ca+/12C+ and 55Mn+/12C+) were quantified for each ROI based on ion counts per scan (= cycle).

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Figure S6. Ca and P content is independent from Mn supply. Correlative quantification of 12C+-normalized 40Ca+ and 31P+ from NanoSIMS-imaged cells grown in 1000 μM (3 replicates) and 6 μM MnEDTA (2 replicates). Note that 12C+-normalized Ca and P content do not change between cells grown in the presence of 6 µM or 1000 µM MnEDTA.

1

0

-1

-1-2-3

1

0

-1

-1-2-3

1

0

-1

-1-2-3

31P / 12C (log10)

6 µM Mn

1000 µM Mn

10.750.50.25

31P / 12C (log10)

1000 µM Mn

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Figure S7. Mn accumulation increases in low Ca condition. Cultures were inoculated from a Ca replete culture into medium supplemented with 6 or 600μM MnEDTA and with different Ca concentrations (34, 42.5, 85, 170, and 340 μM CaCl2) to a cell density of 104 cells ml-1. Total Mn (A), Ca (B) and P (C) content was measured via ICP-MS from 2 independent experiment replicates. The horizontal bars represent the mean for each sample.

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Figure S8. The vtc1-1 phenotype is recapitulated in P deficient cells. Cultures were inoculated from a (+P) pre-culture into medium with different Mn concentrations (6, 100, and 600μM MnEDTA) and with (+P) or without (–P) addition of phosphorous to a cell density of 105 cells ml-1. Total Mn (A), P (B) and Ca (C) content was measured via ICP-MS from 3-4 independent experiment replicates, shown ± standard deviation. The horizontal bars represent the mean for each sample, ± standard deviation.

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Figure S9. A Chlamydomonas mutant in VTC1 does not compromise the acidocalcisome membrane. The vtc1-2 allele (LMJ.RY0402.126297) was ordered from the Chlamydomonas Resource Center (CRC, chlamycollection.org) and its genotype was confirmed with insertion-specific primers. The mutant and its corresponding wild-type strain (CC-4533) were grown in TAP medium containing 6 µM Mn, and samples were collected for ICP (A) and whole-cell proteomics analysis (B, C). ICP samples were collected as technical duplicates, while proteomics samples were from three independent experiment replicates. The horizontal bar represents the mean for each sample. *:significantly different evaluated by t-test with equal variance (itself tested by F-test) with p-value < 0.05; **: t-test p-value < 0.01.

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Figure S10. Reduced Mn accumulation in vtc1-1 mutant revealed by EPR spectroscopy. EPR spectra were collected from CC-4532, VTC1 and vtc1-1 cells grown in 6 µM or 600 µM MnEDTA, and their height adjusted to facilitate comparison of the high-symmetry peak around 12 kG. The y-axis scale of the low Mn spectra were increased 6x (VTC1) and 4x (vtc1-1). Inset: original scale of spectra. The EPR spectra of all strains are similar to various possible cellular Mn-metabolites (Pi, polyp, imidazole, bicarbonate) as shown here suggesting EPR is not sensitive to cellular Mn-speciation. CW EPR conditions: MW frequency 34.8 GHz, T = 2K, magnetic field scan rate 2 kG/min, modulation amplitude 1G.

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Figure S11. Detection of Mn in cells with the M1 probe. A. Strain CC-4532 was grown in 6 (left panels) or 600 (right panels) µM MnEDTA until mid-log before being processed for microscopy. Mn was detected with the Bodipy-based probe M1 (Bakthavatsalam et al., 2015). Note that the probe does not detect cellular Mn under standard conditions of 6 µM, but readily detects Mn concentrated inside acidocalcisomes. Images were collected in channel mode using a Zeiss LSCM 880 Airyscan. B and C. Quantification of fluorescence derived from the M1 probe, lysosensor-DND 167 and chorophyll (blue squares and traces), collected in spectral imaging mode on a Zeiss LSCM 880 Airyscan confocal microscope for the vtc1-1 mutant and its VTC1 complemented strain grown in 600 µM MnEDTA. Individual cells are shown in B, and the associated fluorescence spectra from regions of interest in C. The red and green squares in B denote acidocalcisomes. The Lysosensor DND-167 dye will show a peak emission around 450 nm, while Mn bound to the M1 probe will emit maximally around 500-550 nm. Note that the vtc1-1 mutant exhibits lower fluorescence emenating from the M1 probe than VTC1, but remains above chlorophyll autofluorescence background, suggesting some accumulation of Mn in the mutant.

400 450 500 550 600 650wavelength

protons Mn2+ chl protons chl

400 450 500 550 600 650wavelength

5 µm 5 µm

6 µM Mn 600 µM Mn

M1 probe M1/chl overlay M1 probe M1/chl overlayA

B

C

vtc1-1VTC1

5 µm 5 µm

CC-4532

M1 probe / lysosensor chlorophyll

0

25

50

75

100 Mn2+

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Figure S12 : 31P couplings of Mn-metabolite complexes observed intracellularly. 31P (I=1/2) ENDOR spectra of Mn-P complexes primarily show two peaks labelled as (n+/n) centered around the larmor frequency nL. The Mn-Pi/polyP complexes show similar 31P hyperfine coupling (APi/polyP = 5 MHz), but are distinguished from each other by different 31P ENDOR amplitude, The Mn-Phy complex shows two different n+ hyperfine peak; one with 31P coupling (Aphy = 5 MHz) similar to as observed for Mn-Pi, and a second peak which corresponds to a stronger 31P coupling (8 MHz). Davies ENDOR conditions: magnetic field ~ 12.5 kG, tp/2 = 60 ns, t = 400 ns, Trf = 160µs, repetition time 10ms.

RF frequency (MHz)14 16 22 24 2818 20 26

31P

CC-4532

VTC1

vtc1-1

Mn-Phy

Mn-Pi

Mn-polyP

Aphy

6 µM600 µM

L

APi/polyP

+–

+–

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Figure S13 : Partitioning of cellular Mn-P complexes into Mn-Pi/polyP, Mn-Phy. The n+ peak of the 31P ENDOR response is baseline-corrected and then used as a representative of 31P ENDOR response.

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Figure S14. Conservation of amino acids in Vtc4p and Cre VTC4 coordinated with Mn2+. Yeast and Chlamydomonas VTC4 protein sequences were aligned with the global alignment tool EMBOSS Needle (https://www.ebi.ac.uk/Tools/psa/emboss_needle/). Amino acids shown in yeast to be coordinated with Mn2+ in the active site are shown in green, and conservation with Chlamydomonas highlighted in gray.

Vtc4p 1 MKFGEHLSKSLIRQYSYYYISYDDLK---------------------TEL 29 ||:|:::...:..::..|||.|..|| |.| CreVtc4 1 MKYGKYIESKVKPEWKDYYIDYKGLKDLIKACQKEAETGEASFSPRTTSL 50

Vtc4p 30 EDNLSKNNGQWTQELETDFLESLEIELDKVYTFCKVKHSEVFRRVKEVQE 79 ......|....:||| |...||.:::||..| ..::.| CreVtc4 51 TVQRYNNTKDSSQEL---FFRRLERDVEKVNKF-----------TNKLVE 86

Vtc4p 80 QVQHTVRLLDSNNPPTQLDFEILEEELSDIIA-------DVHDLAKFSRL 122 :::.:::.|:| :.:.|..:::..|::. |...|.|:..: CreVtc4 87 EMRASLKSLNS-----KAEKETDQDKKDDLLKEAQRIGDDFLGLEKYVNI 131

Vtc4p 123 NYTGFQKIIKKHDKKTGFI-LKPVFQVRLDSKPFFKENYDELVVKISQLY 171 ||.||.||:|||||..... .:..:...|..:|:.:.||.:|:|.:|.|| CreVtc4 132 NYLGFHKILKKHDKCLPHAPCRQFYVAHLHQQPWVQGNYSDLLVSLSNLY 181

Vtc4p 172 DIARTSGRPIKGDSSAGGKQ----QNFVRQTTKYWVHPDNITELKLIILK 217 . .::|||| |.|. |.|||.||||||..::::.:|..:|: CreVtc4 182 S-------KLRGDSS-GEKNEDAAQGFVRSTTKYWVRNEDVSTVKHHVLQ 223

Vtc4p 218 HLPVLVFNTNKEFEREDSAITSIYFDNENLDLYYGRLRKDEGAEAHRLRW 267 ||||..|:.| .|..:...|.|:|.||.||:||:|||.|..||.|.|:|| CreVtc4 224 HLPVFQFDKN-IFSGDAQLINSVYMDNSNLELYHGRLDKKPGAIALRIRW 272

Vtc4p 268 YGGMSTDTIFVERKTHREDWTGEKSVKARFALKERHVNDFLKGKYT---- 313 ||....:.:|.|||||:|.|.||:|||.||.|....|..|:.|:|: CreVtc4 273 YGSNPPNMVFFERKTHKESWKGEESVKERFTLPSDKVVAFMDGEYSLEAA 322

Vtc4p 314 -VDQVFAKMRKEGKKPMNEIENLEALASEIQYVMLKKKLRPVVRSFYNRT 362 |||..|..||.......|.:....|.:|:...:..|:|:|:||:.|.|| CreVtc4 323 LVDQEAAAARKGKSLSQEEKDAYSQLFTEVYKAVDSKQLKPLVRTQYMRT 372

Vtc4p 363 AFQLPGDARVRISLDTELTMVREDNFDGVD-RTHKNWRRTDIGVDWPFKQ 411 |||:|.||.|||||||.|.|::|:..||.. .....|.|. |... CreVtc4 373 AFQIPFDATVRISLDTNLCMIKENPEDGPSCAATGRWYRD------PSLP 416

Vtc4p 412 LDDKDICRFPYAVLEVKLQTQLGQEPPEWVRELVGSHLVEPVPKFSKFIH 461 :...:|.|||:|||||||..|.||..|.||:||:.|..:..|.||||||| CreVtc4 417 VTRTEITRFPHAVLEVKLSLQEGQTAPAWVQELLDSGYLTEVHKFSKFIH 466

Vtc4p 462 GVATLLNDKVDSIPFWLPQMDVDIRKPPLPTNIEITRPGRSDNEDNDFDE 511 |.|||....|.::|:|: CreVtc4 467 GSATLFPGDVRAVPYWV--------------------------------- 483

Vtc4p 512 DDEDDAALVAAMTNAP---GNSLDIEESVGYGATSAP-TSNTNHVVESAN 557 |||...|.:.|...:| |:.|..:.||..|:...| |||.:.|..:|. CreVtc4 484 DDESVRASMLASAPSPLEGGSPLPEDGSVAGGSAGGPSTSNGSPVAAAAA 533

Vtc4p 558 AAYYQR---KIRNAENPISKKYYEIVAFFDHYFNGDQ--ISKIPKGTTFD 602 ||...| :.|:|: ..|....|: .|...|:| :..:| | CreVtc4 534 AASAARSKPRTRHAK-AASPGLDEL----QHPLLGEQATLKLMP-----D 573

Vtc4p 603 TQ-----------------------------------IRAPP----GKTI 613 .: :..|| |... CreVtc4 574 REQIAGFRNAGLSAAGGAGAGRGGSGHPGFFARLLGLVPPPPPAGGGSLR 623

Vtc4p 614 CVPVRVEPKVYFATERTYLSWLSISILLGGVSTTLLTYGSPT-------- 655 ..|:|:|||.:||.|||:|:||.:::.||.||..||.:.:.| CreVtc4 624 STPMRIEPKTFFANERTFLAWLHMAVTLGSVSAALLGFAAGTDDDSESTG 673

Vtc4p 656 ------AMIGSIGFFITSLAVLI--RTVMVYAKRVVNIRLKRAVDYEDKI 697 .::..|...:..|.|.: ..:.|:..|..||..||||.::|:: CreVtc4 674 GAAISRHLVELIALILLPLGVAMCGYALHVFVWRATNIAKKRAVHFDDRV 723

Vtc4p 698 GP----GMVSVFLILSILFSF--FCNLVAK-------------------- 721 || |.|.|.|:...|.|. |..|:|. CreVtc4 724 GPLALCGAVVVALVAITLLSLVDFFELLAAADAAAPAPPPPAAVLAGATG 773

Vtc4p 722 ----- 721 CreVtc4 774 TLSLF 778

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