A proteomic view of the response of Paracoccidioides yeast cells to zinc deprivation

Accepted Manuscript A proteomic view of the response of Paracoccidioides yeast cells to zinc deprivation Ana Flávia Alves Parente, Tereza Cristina Vieira de Rezende, Kelly Pacheco de Castro, Alexandre Melo Bailão, Juliana Alves Parente, Clayton Luiz Borges, Luciano Paulino Silva, Célia Maria de Almeida Soares PII:

S1878-6146(13)00055-X

DOI:

10.1016/j.funbio.2013.04.004

Reference:

FUNBIO 385

To appear in:

Mycological Research

Received Date: 5 December 2012 Revised Date:

9 April 2013

Accepted Date: 11 April 2013

Please cite this article as: Alves Parente, A.F., de Rezende, T.C.V., de Castro, K.P., Bailão, A.M., Parente, J.A., Borges, C.L., Silva, L.P., Soares, C.M.d.A., A proteomic view of the response of Paracoccidioides yeast cells to zinc deprivation, Fungal Biology (2013), doi: 10.1016/ j.funbio.2013.04.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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A proteomic view of the response of Paracoccidioides yeast cells to zinc deprivation Ana Flávia Alves Parentea; Tereza Cristina Vieira de Rezendea; Kelly Pacheco de

Luciano Paulino Silvac; Célia Maria de Almeida Soaresa,*

a

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Castroa, b; Alexandre Melo Bailãoa; Juliana Alves Parentea; Clayton Luiz Borgesa;

Laboratório de Biologia Molecular, Instituto de Ciências Biológicas, Universidade

b

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Federal de Goiás, Goiânia, Goiás, Brazil.

Programa de Pós Graduação em Patologia Molecular, Faculdade de Medicina,

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Universidade de Brasília, Brasília, Distrito Federal, Brazil.

Laboratório de Espectrometria de Massa, Centro Nacional de Pesquisa de Recursos

Genéticos e Biotecnologia, Empresa Brasileira de Pesquisa Agropecuária, Brasília,

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Distrito Federal, Brazil.

* Corresponding author: C. M. A. Soares, Laboratório de Biologia Molecular, ICB II,

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Campus II, Universidade Federal de Goiás, 74001-970, Goiânia-Goiás, Brazil. Tel/fax:

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55-62-3521-1110. e-mail: [email protected]

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Abstract Zinc plays a critical role in a diverse array of biochemical processes. However, an excess of zinc is deleterious to cells; therefore, cells require finely tuned homeostatic

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mechanisms to balance the uptake and the storage of zinc. There is also increasing evidence supporting the importance of zinc during infection. To understand better how Paracoccidioides adapts to zinc deprivation, we compared the two-dimensional (2D)

gel protein profile of yeast cells during zinc starvation to yeast cells grown in a zinc rich condition. Protein spots were selected for comparative analysis based on the protein

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staining intensity, as determined by image analysis. In response to zinc deprivation, a total of 423 out of 845 protein spots showed a significant change in abundance. Quantitative RT-qPCR analysis of RNA from Paracoccidioides grown under zinc

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restricted conditions validated the correlation between the differentially regulated proteins and transcripts. According to the proteomic data, zinc deficiency may be a stressor to Paracoccidioides, as suggested by the upregulation of a number of proteins related to stress response, cell rescue and virulence. Other process induced by zinc deprivation included gluconeogenesis. Conversely, the methylcitrate cycle was downregulated. Overall, the results indicate a remodeling of the Paracoccidioides

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response to the probable oxidative stress induced during zinc deprivation.

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Keywords: Paracoccidioides, zinc deprivation, proteomic analysis

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1. Introduction Zinc is an essential nutrient because it is a required cofactor for many enzymes and transcription factors. Like other metals, it is vital in trace amounts, but toxic at high

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concentrations (Finney and O’Halloran 2003). Zinc homeostasis is maintained by

transcriptional and posttranslational homeostatic regulatory mechanisms (Eide 2003; Lyons et al. 2000). In Saccharomyces cerevisiae, the best studied organism for zinc

homeostasis, the uptake of this micronutrient is mediated by two systems. The first is a

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high-affinity system that is active in zinc-limited conditions (Zhao and Eide 1996a) and a lower affinity uptake system that is not highly regulated by zinc concentrations (Zhao and Eide 1996b). The expression of the high-affinity zinc transporter, Zrt1p, and the

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low-affinity zinc transporter, Zrt2p, is regulated by the transcription factor, Zap1p, which plays a central role in zinc homeostasis (Zhao and Eide1997). ZAP1 encodes a transcriptional activator with seven carboxy-terminal C2H2 zinc finger domains and two amino terminal activation domains. Rutherford and Bird (2004) reported that in S. cerevisiae, under conditions of limited zinc, Zap1p induces the expression of genes coding for the transporters, Zrt1p and Zrt2p. The second mechanism by which S.

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cerevisiae regulates zinc transporter activity is at the post-translational level. In zinclimited cells, Zrt1p is a stable plasma membrane protein. The exposure to high levels of extracellular zinc triggers the rapid loss of Zrt1p via its increased uptake, thereby decreasing Zrt1p protein levels. This inactivation occurs through the zinc-induced

1998).

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endocytosis of the protein and its subsequent degradation in vacuoles (Gitan et al.

Aspergillus fumigatus has three genes encoding for zinc transporters belonging

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to the ZIP family. The expression of these transporters is regulated by both pH and the environmental concentration of zinc. The ZRFA and ZRFB genes of A. fumigatus are transcribed at higher levels and are required for fungal growth under acidic, zinc-limited conditions, whereas they are dispensable for growth in neutral or alkaline, zinc-limited media (Amich et al. 2009; 2010). The transporter for the zinc uptake system that functions when A. fumigatus is grown in neutral or alkaline environments is encoded by ZRFC (Amich et al. 2010). It has been suggested that ZrfB represents a high-affinity zinc permease because its cognate transcript is downregulated in high zinc conditions (Vicentefranqueira et al. 2005). 3

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Paracoccidioides, a complex of several phylogenetic species (Matute et al. 2006; Carrero et al. 2008; Teixeira et al. 2009), is the causative agent of paracoccidioidomycosis (PCM), a human systemic mycosis that is prevalent in South America (Restrepo et al. 2001). The fungus is thermo dimorphic, that is, it grows as a

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yeast-like form in host tissues or when cultured at 35 - 37 ºC, and as mycelium in saprobe condition or when cultured at ambient temperature (Brummer 1993). After penetrating the host, Paracoccidioides differentiates into the yeast form, which is a

fundamental step for the successful establishment of the disease (San-Blas et al. 2002).

The temperature-dependent cellular differentiation to the parasitic yeast cell takes place

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in the lungs and from the primary pulmonary infection site, the fungus eventually

disseminates to other organs by hematogenic and/or lymphatic routes (Brummer 1993).

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A careful analysis of the Paracoccidioides genome available at

http://www.broad.mit.edu/annotation/genome/Paracoccidioides_brasiliensis/MultiHom e.html revealed that it has orthologues to the zinc transporters that have been previously described in fungi and that are localized in the plasmatic, vacuolar and endoplasmic reticulum membranes (Silva et al. 2011; Bailão et al. 2012). Importantly, genes encoding zinc transporters of the ZIP family, with homology to S. cerevisiae Zrt1p or Zrt2p, are present in the Paracoccidioides genomic database (Silva et al. 2011). The

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expression of these transcripts could be addressed by the transcriptional analysis of Paracoccidioides yeast cells after incubation in human blood and plasma (Bailão et al. 2006, 2007) and in yeast cells subjected to zinc depletion (Bailão et al. 2012). The Paracoccidioides isolate, Pb01, has two vacuolar membrane zinc transporters, encoded

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by the ZRC1 and COT1 genes, whereas the isolates Pb03 and Pb18 only contain the COT1 homolog. An orthologue to the transcription factor Zap1p of S. cerevisiae is also

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present in the three Paracoccidioides isolates. Therefore, zinc assimilation in Paracoccidioides is expected to be similar to that of S. cerevisiae (Silva et al. 2011; Bailão et al.2012). The ZRT2 transcript, but not ZRT1, was highly expressed in neutral to alkaline pH during zinc deprivation, as observed to the ZRFC transcript in A.

fumigatus that was expressed in both conditions, suggesting that in Paracoccidioides, the expression of this gene may be regulated by both zinc and pH (Bailão et al. 2012). Studies have shown that zinc is an essential micronutrient for the proliferation of

pathogenic fungi. Accordingly, it has been demonstrated that zinc deprivation is a host defense mechanism utilized by macrophages during Histoplasma capsulatum infection. It has been demonstrated that upon H.capsulatum infection, granulocyte-macrophage 4

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colony stimulating factor (GM-CSF) activated macrophages reduce intracellular zinc concentration to kill the pathogen (Winters et al. 2010). In Candida albicans, a novel zinc acquisition system has also been described during endothelial cell invasion. Analogous to siderophore-mediated iron acquisition, C. albicans utilizes an

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extracellular zinc scavenger for acquiring this essential metal. The system is composed of a secreted protein encoded by the gene PRA1 and a transporter encoded by ZRT1. C. albicans also secretes the scavenger protein, a ‘‘zincophore’’, Pra1p. This component binds host cellular zinc. Pra1p then reassociates with the fungal cell via a membrane transporter, Zrt1p, to deliver its zinc load. The deletion of PRA1 prevented the

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utilization of host zinc and resulted in the damage of host cells in the absence of

exogenous zinc (Citiulo et al. 2012). In Cryptococcus gattii, the zinc finger protein,

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Zap1p, which is induced by zinc deprivation, has been functionally characterized. The inactivation of ZAP1 compromises the growth of the fungus under limited zinc conditions and reduces C. gattii virulence in a murine model of cryptococcosis infection (Schneider et al. 2012).

Due to the relevance of micronutrients to fungal homeostasis and pathogenesis, our group had been employing proteomic approaches to study the Paracoccidioides response to metal starvation. Parente et al. (2011) previously demonstrated that iron

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deprivation promotes an increase in the amount of proteins of the glycolytic pathway, whereas the proteins of the tricarboxylic acid, glyoxylate and methylcitrate cycles, as well as the electron transport chain, decreased in abundance under conditions of limited iron. These data suggest a remodeling of Paracoccidioides metabolism toward

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prioritizing iron independent pathways. To address the question of what proteins and processes are important for Paracoccidioides in a zinc limited condition, we utilized 2D

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gel electrophoresis coupled to mass spectrometry to identify proteins sensitive to low zinc levels. The image analysis allowed us to identify 423 differentially regulated spots, of which 135 were identified. The 135 identified spots allowed the distinction of 100 different proteins that were differentially regulated in response to zinc starvation, 46 were induced and 54 were repressed, rendering an integrated view of the reorganization of metabolic and cellular processes during zinc deprivation. The screen of the metabolic cell status, as determined by proteomics, reflected a shift in cellular metabolism during zinc deprivation, as indicated by the increase in oxidative stress response and gluconeogenesis and the repression of the methylcitrate cycle.

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2. Materials and Methods 2.1. Paracoccidioides isolate and growth conditions Paracoccidioides, Pb 01 (ATCC MYA-826), was used in all experiments. The

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yeast phase was maintained in vitro by subculturing at 36 °C in Fava Netto’s semisolid medium [1% (w/v) peptone, 0.5% (w/v) yeast extract, 0.3% (w/v) proteose peptone,

0.5% (w/v) beef extract, 0.5% (w/v) NaCl, 4% (w/v) glucose, 1.2% (w/v) agar, pH 7.2] every 7 days.

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2.2. Zinc depletion experiments

For the experiments of intra and extracellular zinc depletion, Paracoccidioides yeast cells were incubated in McVeigh/Morton medium (MMcM) (Restrepo and

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Jiménez 1980) in the presence and absence of zinc. The zinc depleted medium was prepared without the addition of ZnSO4 and supplemented with the zinc chelator N,N,N_,N_-tetrakis (2-pyridyl-methyl) ethylenediamine (TPEN 0.05 mM; Sigma Aldrich, Co., St. Louis, MO). The cultures were allowed to grow at 36 °C, 150 rpm, and the number of viable cells was determined at each specific time interval (1, 2, 3, 4, 6, 8 and 24 h) by counting living cells using trypan blue as vital dye. The viability results

assays.

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were used to determine the time of cell exposure to zinc starvation for the proteomic

To obtain protein extracts, yeast cells were prepared by inoculating 50 mL of Fava Netto’s liquid medium with 108 cells/mL of Paracoccidioides. The cultures were

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maintained for 72 h at 36 °C with gentle shaking. Thereafter, the cultures were centrifuged and the cells were washed in sterile phosphate buffered saline solution 1 X

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(PBS; 1.4 mM KH2PO4, 8 mM Na2HPO4, 140 mM NaCl, 2.7 mM KCl; pH 7.3). The cells were then resuspended in modified MMcM culture medium (Restrepo and Jiménez 1980), followed by incubation for 18 h at 36 ºC with agitation. The cells were centrifuged at 5000x g for 5 min and washed in PBS 1X. Then, 2 x 106 cells were

introduced into MMcM medium supplemented with zinc chelate-specific TPEN (Sigma Aldrich, Co) at a concentration of 0.05 mM. The cultures were incubated with gentle shaking at 36 °C for 6 and 24 h following the deprivation of zinc. As a control, yeast cells of Paracoccidioides were incubated in MMcM medium containing 0.03 mM of ZnSO4 for 6 and 24 h. To obtain RNA for quantitative RT-qPCR analysis, cells were incubated as described above for 3, 6 and 24 h. 6

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2.3. Preparation of protein extracts Yeast cells were collected following 6 and 24 h of zinc deprivation and submitted for total protein extraction. The cells were centrifuged at 10,000 x g for 15

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min at 4 ºC and disrupted by vigorous mixing with glass beads in a solution containing 20 mM Tris-HCl, pH 8.8, 2 mM CaCl2 (Fonseca et al. 2001) and a mixture of nuclease

and protease inhibitors (serine, cysteine and calpain; GE Healthcare, Uppsala, Sweden). After centrifugation, the supernatant was collected and the protein concentrations were determined using the Bradford reagent (Sigma Aldrich), using bovine serum albumin

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2.4. Two-dimensional gel electrophoresis

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(BSA) as a standard (Bradford 1976). The samples were stored in aliquots at -80 ºC.

This experiment was performed as previously described (Parente et al. 2011; Rezende et al. 2011). Briefly, protein samples (300 µg) were treated with 2-D Clean-up Kit (GE Healthcare) according to the manufacturer’s instructions. The precipitate was solubilized in a rehydration buffer {7 M urea, 2 M thiourea, 2% (w/v) [(3cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 65 mM dithiothreitol (DTT), 0.5% (v/v) ampholyte-containing buffer (IPG) and 0.001% (w/v)

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bromophenol blue}. This solution was then applied onto 13 cm long immobilized nonlinear Dry Strips, pH 3-11 (GE Healthcare). Subsequently, the IPG strips were rehydrated for 14 h at 30 V using the Ettan IPGphor III Isoelectric Focusing System (GE Healthcare). The isoelectric focusing was performed with a limiting current of 50

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µA/strip under the following steps: 500 V for 1 h; 500-1000 V for 1 h; 1000-8000 V over 12 h and 30 min and 8000 V for 2 h and 30 min. The IPG strips were reduced with

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0.5% (w/v) DTT for 40 min by gentle agitation and then alkylated with 2.5% (w/v) iodoacetamide for 40 min by gentle agitation in the dark, in equilibration buffer [6 M urea, 0.5 M Tris–HCl, pH 8.8, 30% (v/v) glycerol, 2% (w/v) SDS and 0.001% (w/v) bromophenol blue]. The second dimension electrophoresis was performed in a Hoefer SE 600 electrophoresis (GE Healthcare) system at 15 °C at 100 V for 1 h, followed by 200 V until the indicator reached the bottom of the gel. The proteins were stained using Coomassie brilliant blue (PlusOne Coomassie Tablets PhastGel Blue R-350, GE Healthcare) according to the manufacturer’s instructions.

2.5. 2D-gel image analysis 7

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The gel images were produced by the Image Scanner III (GE Healthcare) at 300 dpi resolution and the digitalized images were analyzed by the Image Master Platinum 6.0 software (GE Healthcare) for spot detection, quantification and matching. To refine automatic spot matching, mismatched spots were corrected manually. The spot values

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were normalized by using the following equation: intensity of each spot value/total intensity, in which the total intensity refers to the sum of all spots belonging to the same gel. Thus, the spot intensity values are indicated as a percentage of the total volume of the gel (%vol). The spots found in all 3 gels were used to determine statistical

significance. One-way ANOVA was applied to compare %vol values of matched spots.

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P values ≤ 0.05 were considered statistically significant. The spots presenting a fold

change higher than 20% were considered to be differentially expressed. The spots found

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in only one experimental condition were also considered differentially expressed. To compare proteins with multiple isoforms, the sum of the %vol corresponding to the same protein was first obtained for each gel. Then, the sum of the %vol values was used for statistical analysis which was performed to determine the significance of differences in the expression profile, p values ≤ 0.05 were considered statistically significant.

2.6. In-gel digestion

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This experiment step was performed as previously described (Parente et al. 2011; Rezende et al. 2011). Briefly, protein spots were manually excised from the 2Dgel and the gel pieces were incubated with a solution containing 50 mM sodium thiosulfate and 15 mM potassium ferricyanide for 5 min. The gel pieces were

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dehydrated in acetonitrile (ACN) and dried in a speed vacuum. The gel pieces were then reduced using 10 mM DTT and alkylated using 55 mM iodoacetamide. The supernatant

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was removed and the gel pieces were dehydrated with a solution containing 25 mM ammonium bicarbonate/50% (v/v) ACN solution. The gel pieces were dried and a 10 ng/µL trypsin solution (sequencing grade modified trypsin, Promega, Madison, WI, USA) was added, followed by rehydration on ice at 4 °C for 10 min. After the supernatant was removed, 25 mM ammonium bicarbonate solution was added to the gel pieces, followed by incubation at 37 °C for 16 h. After the digestion, the supernatant was placed in a clean tube. A solution containing 50% (v/v) ACN and 0.1% (v/v) trifluoroacetic acid (TFA) was added to the gel pieces. The samples were mixed for 10 min, sonicated for 3 min and combined with the aqueous extraction. The samples were dried in a speed vacuum and the peptides were solubilized in water. Two microliters of 8

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each sample were delivered to a target plate and dried at room temperature. Subsequently, the peptide mixtures were covered with 2 µL of MALDI matrix solution [10 ng/mL alphacyano-4-hydroxycinammic acid in 50% (v/v) ACN and 5% (v/v) TFA]. Prior to mass spectrometry (MS), the samples were concentrated and purified using a

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pipette tip with a bed of chromatographic media (ZipTips® C18 Pipette Tips, Milipore, Bedford, MA, USA).

2.7. Mass spectra analysis

MALDI-MS and MALDI- MS/MS were performed using a MALDI Synapt

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MS™ spectrometer (Waters-Micromass, Manchester, UK) and an Ultra Flex III

MALDI-TOF/TOF mass spectrometer (Bruker, Bremen, Germany). All spectra were

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obtained in positive reflector mode. The mass spectrometric data analysis was performed using Masslynx 4.0 software (Waters-Micromass, Manchester, UK) and Flex Analysis (Bruker Daltonics version 2.4) software. The protein identification was performed as previously described (Rezende et al. 2011). Briefly, the monoisotopic peak lists were submitted using an in-house Mascot server (Version 2.1.04, Matrix Sciences, London, UK) to identify candidate proteins. For the MS data, the following parameters were selected: only tryptic peptides with up to one missed cleavage site were

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allowed; carbamidomethyl cysteine as a fixed modification and oxidized methionine as a variable modification. For the MS data, a mass tolerance of 25-100 ppm and 0.2-0.6 Da for MS/MS fragment ions were used. For both MS and MS/MS, only proteins with statistical significance (p≤ 0.05), as determined by the MASCOT algorithm, were

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accepted as protein sequence matches. The analysis by MS/MS confirmed the proteins identified on the basis of the PMFs, validating the identifications.

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The ORF sequences of the identified proteins were analyzed by using the Pedant-Pro Sequence Analysis Suite of Biomax GmbH (http://pedant.gsf.de.) and all identified proteins were categorized according to the Functional Catalogue (FunCat2). The investigation of post-translational modifications (PTM) was carried out in

differentially regulated proteins identified in more than one spot from the 2D-gel analysis. This analysis was performed by PMF database search using the MASCOT algorithm, adding the variable modifications of lysine acetylation and serine, threonine and tyrosine phosphorylation, and verifying the presence of mass values corresponding to modified peptides.

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2.8. RNA extraction, cDNA synthesis and RT-qPCR These experiments were performed as previously described (Rezende et al. 2011). Briefly, the cells were disrupted by vigorous mixing with glass beads for 10 min in the presence of Trizol (GIBCO™ Invitrogen Corporation), according to the

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manufacturer's instructions. The cDNAs were prepared using the high capacity RNA-tocDNA kit (Applied Biosystems, Foster City, CA, USA). Quantitative RT-PCR analysis was performed on a Step One Plus™ real-time PCR system (Applied Biosystems,

Foster City, CA, USA). The PCR thermal cycling was performed at 40 cycles of 95 °C for 15 s followed by 60 °C for 1 min. In each set of qRT-PCR experiments, the data

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were normalized to transcripts encoding α-tubulin. A non-template control was also

included to eliminate contamination or nonspecific reactions. Samples of each cDNA

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were pooled and serially diluted 1:5 to generate a relative standard curve. The relative expression levels of the genes of interest were calculated using the standard curve method for relative quantification (Bookout et al. 2006). The specificity of each oligonucleotide pair was analyzed by checking the melting curve of the reaction. The oligonucleotides used in the real-time PCR analyses are listed in Table S1 (see

3. Results

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“Supplementary information”).

3.1. The expression of Paracoccidioides zinc acquisition genes during zinc starvation. We used real-time RT-qPCR to investigate the transcriptional profile of zinc

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responsive genes in Paracoccidioides. The analyzed genes included the Paracoccidioides orthologues to the zinc transporters, ZRT11 and ZRT2 (Fig. 1 and

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Table S1, see “Supplementary information”). Although the two orthologues for these zinc transporters were induced after 3 h to 24 h of zinc deficiency, after 6 h and 24 h of zinc deprivation, a higher induction of ZRT2 was observed, with induction values higher than 20-fold (Fig. 1). The zinc transporter ZRT1 was induced by 10-fold 24 h after zinc deprivation. From these results, timepoints of 6 h and 24 h were chosen for protein extraction and proteomic analyzes.

3.2. The 2D-gel analysis of Paracoccidioides during zinc starvation. Up to 24 h after zinc deprivation, approximately 90% of the cells remained viable, as assessed by trypan blue staining (data not shown). Next, 2-D gel analysis was 10

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used to separate total soluble protein extracts, while image analysis was used to quantify proteins and isoforms. Three independent experiments were carried out to generate three replicates, which included 6 h control, 6 h in zinc depletion, 24 h control and 24 h in zinc depletion (Fig. 2A-D, respectively). Using the gel image software, a total of 845

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spots were successfully matched between the control yeast cells and the zinc depletion conditions (Fig. 2 and Fig. S1, see “Supplementary information”). Statistical analysis

revealed that 127 and 296 proteins/isoforms were differentially accumulated after 6 and 24 h of zinc deprivation, respectively, yielding a total of 423 differentially regulated

3.3. The identification of zinc-regulated proteins.

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proteins/isoforms (Fig. S1, see “Supplementary information”).

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To determine the identities of the differentially regulated protein spots, in-gel digestion using trypsin was performed and was followed by MS analysis. Mass spectrometry analysis followed by protein database sequence matching resulted in the identification of 135 differentially expressed proteins/isoforms (Fig. 2, Fig. S1 and Table S2, see “Supplementary information). Eighty-seven proteins/isoforms were identified by peptide mass fingerprinting (PMF) and confirmed by MS/MS analysis, while 30 protein/isoforms spots yielded identification by PMF and 18 by MS/MS. All

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the spots identified are depicted in Table S2 (see “Supplementary information”). The GenBank general information identifiers (gi), PMF and MS/MS mascot scores, protein molecular mass, and isoelectric points (pI) of each spot are also listed in Table S2 (see “Supplementary information”). Twenty eight proteins were observed in more than one

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spot, due to shifting in the 2D gel position caused by pI or molecular mass changes. Because modifications of protein characteristics are often related to post-translational

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modifications (PTM; Baumann and Meri 2004), a databank search analysis was carried out by MASCOT to verify putative PTMs to correlate the protein displacement in the 2D gel to possible PTMs. The 28 referred proteins corresponded to 73 spots. The same protein was identified in 2 to 8 different spots. In synthesis, among the analyzed spots belonging to the 28 different proteins, 50 showed possible PTMs, as determined by mass spectrum analysis (Table S3).

3.3.1. Proteins with increased expression upon zinc deprivation. Proteins differentially expressed were considered for further analysis. In this way, as described in item 2.5, the sum of the vol% for each protein was considered for 11

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statistical analysis.We identified 46 proteins that were preferentially expressed upon zinc depletion, in comparison to the control condition (Table 1). The proteins induced in Paracoccidioides were mainly involved in cell rescue, defense and virulence, representing a total of 15 proteins and corresponding to 32.6% of the identified induced

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proteins (Fig. S2, see “Supplementary information”). This functional category of proteins induced 24 h after zinc deprivation comprised components of the antioxidant response, such as thioredoxin, glutathione reductase, glutathione synthetase, disulfide isomerase and Y20, and ranged in fold change from 1.58 to 3.17. Additionally,

family were over expressed (Table 1).

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following 24 h of zinc deprivation, five proteins belonging to the heat shock protein

Enzymes involved in glycolysis and gluconeogenesis, including phosphoenol

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pyruvate carboxykinase, phosphoglycerate kinase, glyceraldehyde-3-phosphate dehydrogenase and fructose-1,6-bisphosphate aldolase were upregulated. The increased regulation was mainly observed 24 h after zinc deprivation (Table 1). The enzymes involved in the metabolism of amino acids, such as aspartate aminotransferase and amino methyltransferase, were induced 24 h after zinc deprivation (Table 1). An overview of the processes induced during zinc deprivation is presented in Fig. S2, panel

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A (see “Supplementary information”).

3.3.2. The proteins repressed by zinc deprivation. We identified a total of 54 proteins that were repressed in response to zinc deprivation (Table 2). These ranged in fold change from 1.32 to 4.25. After 6 h of zinc

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deprivation, some antioxidant proteins, such as glutathione reductase, Y20 and peroxisomal catalase, were repressed. It is important to note that glutathione reductase

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and Y20 were induced at 24 h, as described above. Three Paracoccidioides enzymes involved in the methylcitrate cycle, aconitase, 2-methycitrate dehydratase and 2methylcitrate synthase, were decreased following zinc restriction. Additionally, alcohol dehydrogenase, which is involved in fermentation, was downregulated in the yeast cells upon zinc deprivation (Table 2). The enzymes involved in amino acid metabolism, especially those involved in the metabolism of valine and isoleucine, such as acetolactate synthase and methylmalonate-semialdehyde dehydrogenase, were also decreased following zinc starvation (Table 2). This regulation occurred 6 h following zinc deprivation. The

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proteins related to lipid metabolism, acetyl-coA acetyltransferase and peroxisomal multifunctional enzyme, were also decreased in abundance during zinc deprivation. An overview of the processes repressed during zinc deprivation is presented in

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Fig. S2, panel B (see “Supplementary information”).

3.4. The correlation between the proteomic and transcriptional data.

To validate the significance of our proteomic results, we next sought to

determine if the observed changes in protein levels correlated with changes in transcript levels. Using quantitative RT-PCR, we validated that the differences observed in the

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proteomic assay were in agreement with the transcriptional changes. Specifically, we measured the transcript levels of isocitrate lyase (ICL), citrate synthase (CS),

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peroxisomal catalase (CATP) and alcohol dehydrogenase (ADH) (Fig. 3). The protein and transcript levels of ADH and CATP decreased upon zinc limitation, as depicted in Fig. 3 and Table 2. In contrast, the CS and ICL transcript levels were increased and this correlated with the changes in their protein levels (Fig. 3 and Table 1).

4. Discussion

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It is know that the cellular response to zinc deprivation is regulated mainly at the transcriptional level (Amich et al. 2010; Zhao and Eide 1996a; 1996b). Zinc restriction caused the induction of Paracoccidioides orthologues of several zinc dependent transcripts. By RT-qPCR of cDNAs derived from yeast cells of Paracoccidioides grown

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in zinc deprived medium, we demonstrated that the expression of the PbZRT2 gene was induced by zinc deprivation, suggesting that Zrt2p behaves as a high-affinity

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transporter. Bailão et al. (2012) previously demonstrated that the ZRT2 transcript, but not ZRT1, was highly expressed in neutral to alkaline pH during zinc depletion, as observed in A. fumigatus transcript ZRFC. This suggests that likewise, in Paracoccidioides, the expression of this gene may also be regulated by both zinc and pH.

Here, we analyzed the proteome of Paracoccidioides yeast cells following zinc deprivation and identified 135 differentially regulated proteins/isoforms. Of these, 28 proteins were detected in more than one spot. The descriptions presented here, although not exhaustive, provide the first information regarding Paracoccidioides behavior during zinc deprivation. 13

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Our results demonstrated that proteins related to stress response were over expressed 24 h after exposure to zinc starvation. The same response was not observed following 6 h of treatment. Reactive oxygen species (ROS), including the superoxide anion, hydrogen peroxide (H2O2), and hydroxyl radical, can cause various types of

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biological damage. Both in vitro and in vivo studies have established that zinc deficiency leads to increased oxidative stress in mammalian cells (Powell 2000; Ho and Ames 2002). It has also been demonstrated that S. cerevisae yeast cells experience increased oxidative stress when grown under low zinc conditions. In S. cerevisiae, Zap1p activates the expression of the TSA1 gene, encoding for a cytosolic

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peroxiredoxin, which metabolizes hydrogen peroxide (Wu et al. 2007). The cysteine

residues in Tsa1p become oxidized during this reaction and require thioredoxin to be

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reduced back to their active state. Thioredoxin, in turn, requires thioredoxin reductase to be restored to its active, reduced state (Rhee et al. 2005). According to the proteomic analysis presented here, the thioredoxin, glutathione reductase, and thioredoxin reductase proteins were induced after 24 h of zinc deprivation treatment. This strongly suggests that Paracoccidioides was subjected to oxidative stress induced by zinc deprivation and that the fungal response to this stress condition is evident after 24 h. The genes and proteins in Paracoccidioides that have been identified to be related to

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oxidative stress and the activation of antioxidant defenses mediated by the enzymes catalase, superoxide dismutase (SOD), peroxiredoxin, cytochrome C peroxidase, glutathione and thioredoxin have been described. This indicates that Paracoccidioides uses several antioxidant systems to combat ROS (Campos et al. 2005; Chagas et al.

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2008; Dantas et al. 2008). Additionally, by proteomic analysis, the adaptative response of Paracoccidioides to oxidative stress has been shown to involve a prominent

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activation of antioxidant enzymes, such as thioredoxin and superoxide dismutases, suggesting that these enzymes may be relevant to detoxifying ROS (Grossklaus et al., 2013).

Zinc deficiency leads to increased oxidative stress, as cited above. Under long

term oxidative stress, the fungi Aspergillus niger reduces its glucose uptake (Li et al.

2008) and induces enzymes involved in the gluconeogenesis pathway, such as phosphoenolpyruvate carboxykinase and phosphoglycerate kinase. We observed a similar pattern of induction in Paracoccidioides 24 h after zinc deprivation, which may be due to the oxidative stress caused by zinc deprivation.

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The alcohol dehydrogenase enzyme catalyzes the oxidation of ethanol to acetaldehyde. In S. cerevisiae, this enzyme is zinc-dependent and it is highly expressed in zinc-replete cells, but is repressed in zinc-deficient cells (Bird et al. 2006). We have demonstrated that alcohol dehydrogenase is downregulated in Paracoccidioides after 6

in the adaptation of Paracoccidioides to zinc deficiency.

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and 24 h of zinc starvation, suggesting again that this differential gene expression aids

Three Paracoccidioides enzymes involved in the methylcitrate cycle were

decreased in abundance following zinc restriction. Two of them, methylcitrate synthase and methylcitrate dehydrogenase, were decreased after 6 h of zinc depletion, while

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aconitase was decreased after 24 h of zinc depletion. The methylcitrate cycle provides an alternative source of carbon through pyruvate production (Bramer et al. 2002) and

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one of the major pathways for propionyl-CoA metabolism is the methylcitrate pathway. Propionyl-CoA is generated by the breakdown of odd-chain fatty acids and of the amino acids valine and isoleucine (Fleck and Brock 2008). As such, the reduced abundance of enzymes involved in the metabolism of valine and isoleucine after 6 h of zinc starvation corroborates the hypothesis that the methylcitrate pathway is down regulated under zinc limiting conditions.

Molecular chaperones are conserved and abundant proteins that guard the

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conformational homeostasis of proteins (Hartl 1996). They maintain signaling and regulate pathways involved in proliferation, differentiation and apoptosis (Sõti et al. 2005). The chaperones, or stress proteins, confer cytoprotection and assure survival upon various stresses. In this study of Paracoccidioides, 13 proteins belonging to the

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heat shock protein family (HSP), including isoforms, were altered in abundance following zinc limitation. Nine different proteins related to the HSP family were

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increased in abundance in zinc limiting conditions. This result could be an indication of the involvement of chaperones in protecting the fungus from the stress generated by zinc deprivation.

In conclusion, this proteomic analysis of Paracoccidioides revealed that the

major cellular response affected by zinc restriction was related to the oxidative stress response. Our data suggest also that cell rescue, defense and virulence was the most favored pathway during zinc deprivation. Our results provide the first view of the proteome response of Paracoccidioides to zinc starvation.

5. Acknowledgments 15

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This work at Universidade Federal de Goiás was supported by grants from Conselho Nacional de Desenvolvimento Científico e Tecnológico- CNPq (Pronex, 558923/2009-7, 563398/2010-5 and 473277/2011-5) and Fundação de Amparo à

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Pesquisa do Estado de Goiás-FAPEG.

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Cellular Biology 17: 5044–5052.

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Fig. 1. The quantification of the mRNA expression of selected genes in Paracoccidioides by quantitative real time RT-PCR. Quantitative RT-PCR data showing the transcript levels of ZRT1 and ZRT2 during zinc deprivation. The data were normalized to the α-tubulin protein transcript and are presented as fold change.

deviation of three biological replicates and * represents p ≤ 0.05.

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Student’s t test was used for statistical comparisons. Error bars represent the standard

Fig. 2. The 2D-gel analysis of Paracoccidioides proteins extracted from yeast cells

grown in zinc depleted media for 6 h (B) and 24 h (D). Gels A and C represent yeast

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in table S2 (see “Supplementary information”).

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cells grown in zinc rich conditions. The identified protein spots are numbered and listed

Fig. 3.The validation of proteomic data by quantitative real time RT-PCR. The transcript levels of the Paracoccidioides genes encoding isocitrate lyase (ICL), citrate synthase (CS), peroxisomal catalase (CATP) and alcohol dehydrogenase (ADH) were measured using quantitative RT-qPCR. The data were normalized to the α-tubulin protein transcript and are presented as fold change. Student’s t test was used for statistical comparisons. Error bars represent standard deviation from three biological

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replicates while * represents p ≤ 0.05.

Fig. S1. The graphic summary of the proteomic analysis in response to zinc restriction in Paracoccidioides. The number of differentially expressed spots was determined using

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2D-gel image analysis software. The statistical analyses of the matched proteins and

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spots were performed using ANOVA.

Fig. S2. The categorical representation of the differentially regulated proteins in Paracoccidioides following zinc starvation. The identified proteins were classified according FunCat2. The classification of proteins that were induced (A) and repressed (B) in zinc limited condition

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Table 1: Paracoccidioides proteins with increased expression upon 6 and 24h of zinc starvation and their predicted biological function -FunCat2† Average of amount of isoform abundances in controle

ANOVA (p-value)f

Fold Changeg

6h 6h 6h 6h 24h 24h 24h 24h 24h 24h 24h 24h 24h 24h 24h

1 1 1 2 1 1 1 1 1 1 4 2 3 1 2

0.14 1.75 0.14 0.20 0.52 0.19 0.07 0.16 0.89 0.94 1.31 1.06 1.68 0.11 0.99

** 1.41 ** ** ** 0.12 0.04 ** 0.28 0,33 0.71 0.53 1.00 0.09 0.61

** 0.044 ** ** ** 0.033 0.006 ** 0.001 0.003 0.0031 0.0014 0.0013 0.020 0.0052

** 1.24 ** ** ** 1.58 1.61 ** 3.17 2.83 1.84 2.00 1.68 1.81 1.64

1 1 1 2 1

0.06 0.05 0.08 0.13 0.07

** ** ** 0.10 **

** ** ** 0.001 **

** ** ** 1.22 **

24h 24h 24h 24h

3 1 2 1

3.95 0.37 0.33 0.13

2.14 0.25 0.19 0.05

0.0061 0.032 0.05 0.000

1.85 1.50 1.73 2.59

6h 24h 24h

1 1 1

0.16 0.39 0.41

** 0.10 0.17

0.018 0.0008 0.003

1.86 3.84 2.42

24h

1

0.31

0.20

0.002

1.60

gi|295657024 - Puromycin-sensitive aminopeptidase gi|295674319 - Polyadenylate-binding protein gi|295669794 - Elongation factor gi|295675019 - Elongation factor 2 gi|295660511 - Glycyl-tRNA synthetase

Glycolysis and Gluconeogenesis

Citric acid cycle

gi|295669416 - 2-oxoglutarate dehydrogenase E1 gi|295658897 - Citrate synthase gi|295669416 - 2-oxoglutarate dehydrogenase E1 gi|295658595 - Pyruvate dehydrogenase E1 component subunit alpha

Glyoxylate cycle

6h 6h 6h 24h 24h

AC C

gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase gi|295669690 - Phosphoglycerate kinase gi|295671120 - Fructose-1,6-bisphosphate aldolase gi|295658778 - Phosphoenolpyruvate carboxykinase

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Protein synthesis and Fate

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gi|295658865 - Heat shock protein gi|4164594 - Heat shock protein 70 gi|295659116 - Hsp70-like protein gi|295671569 - Heat shock protein SSC1 gi|295670221 - Thioredoxin gi|295664022 - Glutathione reductase gi|295674755 - Glutathione synthetase gi|295661107 - Thioredoxin reductase gi|17980998 - Y20 protein gi|295673162 - Disulfide isomerase Pdi1 gi|14538021 - Heat shock protein 70 gi|295673716 - Hsp70-like protein gi|295659116 - Hsp70-like protein gi|295659837 - Heat shock protein SSB1 gi|295659787 - Heat shock protein HSP88

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Cell rescue, defense and virulence

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Timeb

Average of amount of isoform abundances in zinc starvationd

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Accession numbera/Protein description

Number of isoforms in Paracoccidioidesc

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1

gi|295661432 - UTP-glucose-1-phosphate-uridylyl transferase gi|295672968 - Phosphomannomutase gi|295663567 - 6-phosphogluconolactonase

6h 24h 24h

1 1 1

0.11

0.001

2.36

0.14 0.25 0.15

** 0.05 0.08

** 0.000 0.016

** 4.74 1.87

24h

2

0.37

0.17

0.004

2.17

6h 6h 24h 24h 24h

1 1 1 1 1

0.16 0.19 0.10 0.19 0.16

** 0.07 0.05 0.15 **

** 0.033 0.014 0.028 **

** 2.67 1.96 1.30 **

1 1 3

0.14 0.17 1.22

** 0.05 0.15

** 0.003 0.0002

** 3.31 8.25

1 1

0.25 0.18

0.13 0.10

0.011 0.046

1.88 1.69

1 1

0.26 0.23

** **

** **

** **

Nucleotide metabolism gi|295672652 - Bifunctional purine biosynthesis protein ADE17 gi|295669240 - Kynurenine-oxoglutarate transaminase gi|295669670 - Adenosyl homocysteinase gi|295667902 - Amino methyltransferase gi|295658698 - Fumaryl acetoacetase gi|295662426 - Aspartate aminotransferase

Lipid, fatty acid and isoprenoid metabolism gi|295657225 - Peroxisomal multifunctional enzyme gi|295664927 - ATP citrate lyase gi|295665123 - Aldehyde dehydrogenase

6h 6h 24h

Metabolism of vitamins, cofactors and prosthetic groups gi|295657369 - Nicotinate-nucleotide pyrophosphorylase gi|295660716 - UDP-galactopyranose mutase

24h 24h

Cell growth / morphogenesis Unclassified Proteins

6h 24h

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gi|295657091 - Tropomyosin-1 gi|295673184 - Actin-interacting protein

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Amino acid and nitrogen metabolism

0.25

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24h

Carbohydrate metabolism

SC

gi|295660969 - Isocitrate lyase

AC C

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gi|295661500 - Conserved hypothetical protein 24h 1 0.11 0.07 0.016 **Spots visualized only in zinc-starvation condition; a GenBank general information identifier; b Time of exposure to zinc starvation; c Number of identified isoforms of protein in Paracoccidioides. Pb01 during zinc starvation d,e The average of amount of values of abundances of all identified isoforms used to statistical test f p≤ 0.05 was used to considerer statistically significant differences g Fold change increase in protein expression in zinc starvation. † Functional classification by FunCat2 (http://pedant.helmholtzmuenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01 )

1.62

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Table 2: Paracoccidioides proteins with decreased expression upon 6 and 24h of zinc starvation and their predicted biological function -FunCat2† Time

Average of amount of isoform abundances in zinc starvationd

Average of amount of isoform abundances in controle

ANOVA (p-value)f

Fold Changeg

6h 6h 6h 6h 6h 6h 24h 24h 24h

1 1 1 1 1 1 2 1 1

0.19 0.12 0.13 0.22 0.80 0.29 0.49 0.06 **

0.34 0.21 0.30 0.36 1.31 0.59 0.88 0.13 0.52

0.024 0.027 0.003 0.013 0.025 0.03 0.00072 0.009 **

1.83 1.69 2.35 1.66 1.64 2.02 1.80 2.28 **

1 1 1

0.45 ** **

0.90 0.47 0.53

0.007 ** **

2.01 ** **

1 1

1.56 0.78

2.35 2.18

0.0054 0.001

1.5 2.80

1 3 1

0.36 0.47 0.10

0.77 1.17 0.28

0.004 0.005 0.029

2.12 2.48 2.7

6h

1

0.03

0.07

0.022

2.49

6h

1

0.14

0.20

0.042

1.41

6h

1

0.82

0.52

0.001

3.12

6h

1

0.17

0.34

0.006

2.00

24h

1

**

0.32

**

**

6h

1

0.11

0.23

0.005

2.15

b

Accession number /Protein description

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a

Number of isoforms in Paracoccidioidesc

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gi|295664022 - Glutathione reductase gi|225681400 - Peroxisomal catalase gi|295662873 - Mitochondrial co-chaperone GrpE gi|295673162 - Disulfide isomerase Pdi1 gi|17980998 - Y20 protein gi|295672932 - 30 kDa heat shock protein gi|295672932 - 30 kDa Heat shock protein gi|295658865 - Heat shock protein gi|295664909 - 10 kDa heat shock protein. mitochondrial

Protein synthesis and Fate gi|295663887 - 40S ribosomal protein S19 gi|295663887 - 40S ribosomal protein S19 gi|295664112 - 40S ribosomal protein S22

6h 24h 24h 6h 6h

Citric acid cycle gi|295673937 - Malate dehydrogenase gi|295664721 - Aconitase gi|295665542 - Fumarate reductase

6h 24h 24h

gi|295665123 - Aldehyde dehydrogenase

Carbohydrate metabolism Nucleotide metabolism

AC C

gi|295662360 - Mannitol-1-phosphate 5-dehydrogenase gi|295666938 - Nucleoside diphosphate kinase gi|295658312 - L-PSP endoribonuclease family protein (Hmf1) gi|225681397 - Xanthine phosphoribosyl transferase 1

Amino acid and nitrogen metabolism gi|295661139 - Methylmalonate-semialdehyde dehydrogenase

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Oxidation of fatty acids

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Glycolysis and Gluconeogenesis gi|295671120 - Fructose-1,6-bisphosphate aldolase gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase

SC

Cell rescue, defense and virulence

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1 1 1 1 1 1 1

0.08 0.04 0.27 0.03 0.08 0.05 0.75

0.15 0.12 0.38 0.02 0.13 0.10 1.21

0.009 0.031 0.014 0.047 0.025 0.009 0.020

1.83 3.17 1.40 1.77 1.7 2.2 1.6

6h 6h 6h 24h 24h

1 1 1 1 1

0.09 0.55 0.52 ** 0.06

0.24 1.10 0.82 0.09 0.11

0.002 0.004 0.028 ** 0.013

2.65 1.99 1.58 ** 1.7

1 1

0.20 **

0.39 0.09

0.019 **

1.9 **

1 1 1

0.07 ** 0.20

0.11 0.03 0.28

0.005 ** 0.039

1.69 ** 1.4

2

0.33

1.01

0.00073

3.10

1 1 1

0.22 0.12 0.56

0.32 0.16 0.96

0.037 0.026 0.013

1.48 1.42 1.7

1 1

0.44 0.37

0.81 1.0

0.002 0.001

1.85 2.7

1 2

0.19 0.041

0.25 0.32

0.044 0.002

1.32 7.93

6h

1

0.13

0.29

0.002

2.24

6h 24h

1 1

0.13 0.11

0.18 0.18

0.003 0.006

1.42 1.7

6h

1

0.41

0.76

0.011

1.8

gi|295668707 - Acetyl-coA acetyltransferase gi|295666179 - 2-Methylcitrate synthase gi|295666197 - 2-Methylcitrate dehydratase gi|295657225 - Peroxisomal multifunctional enzyme gi|295670601 - 3-hydroxyisobutyryl-CoA hydrolase

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Lipid, fatty acid and isoprenoid metabolism

Metabolism of vitamins, cofactors and prosthetic groups gi|295661741- 3-demethylubiquinone 9,3-methyltransferase gi|295660455 - Pyridoxine biosynthesis protein PDX1

24h 24h

Protein/peptide degradation gi|295657201 - Glutamate carboxypeptidase gi|295674421 - Ubiquitin carboxyl-terminalhydrolase gi|295660102 - Dipeptidyl- peptidase

6h 24h 24h

Transcription 24h

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gi|295665468 - Nucleic acid-binding protein

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6h 6h 6h 6h 24h 24h 24h

SC

gi|295662426 - Aspartate aminotransferase gi|295674273 - Acetolactate synthase gi|225678712 - Ketol-acid reductoisomerase gi|295674767 - 4-aminobutyrate aminotransferase gi|295668370 - Aminopeptidase gi|295672027 - Glycine dehydrogenase gi|295668479 - Formamidase

Electron transport and membrane-associated energy conservation gi|295658821 - ATP synthase subunit beta gi|295658923 - Citochrome b-c1 complex subunit 2 gi|295669073 - 12-oxophytodienoate reductase

6h 6h 24h

Fermentation

gi|295672504 - Inorganic pyrophosphatase gi|295672504 - Inorganic pyrophosphatase

Signal transduction gi|295662102 - Rab GDP-dissociation inhibitor

Cytoskeleton/structural proteins gi|295669061 - Arp2/3 complex subunit Arc16 gi|295669061 - Arp2/3 complex subunit Arc16

DNA synthesis and replication gi|295660405 – ssDNA binding protein

EP

Phosphate Metabolism

6h 24h

6h 24h

AC C

gi|295674635 - Alcohol dehydrogenase gi|295674635 - Alcohol dehydrogenase

ACCEPTED MANUSCRIPT

Unclassified Proteins

AC C

EP

TE D

M AN U

SC

RI PT

gi|295673506 - Conserved hypothetical protein 24h 1 0.10 0.21 0.008 gi|295659253 - Conserved hypothetical protein 24h 1 ** 0.21 ** **Spots visualized only in zinc replete condition; a GenBank general information identifier; b Time of exposure to zinc starvation; c Number of identified isoforms of protein in Paracoccidioides. Pb01 in zinc replete conditions d,e The average of amount of values of abundances of all identified isoforms used to statistical test f p≤ 0.05 was used to considerer statistically significant differences g Fold change increase in protein expression in zinc availability. † Functional classification by FunCat2 (http://pedant.helmholtzmuenchen.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01 )

2.0 **

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

Table S1: Oligonucleotides used in RT-qPCR Oligonucleotide

Sequence

Amplicon size (bp)

Accession numbera

AC C

EP

TE D

M AN U

SC

RI PT

Tubulin sense 5’ ACAGTGCTTGGGAACTATACC 3’ 95 PAAG_01647.1 Tubulin anti-sense 5’ GGGACATATTTGCCACTGCC 3’ Zrt1 sense 5’ CTATCCGCTGTGTTCGTCAT 3’ 141 PAAG_08727.1 Zrt1 anti-sense 5’ GGAGATGGATGAAAGCTGTG 3’ Zrt2 sense 5’ GCAAAATCCCCCAATGGTAGT3’ 112 PAAG_03419.1 Zrt2 anti-sense 5’ GGGTAAGGCCGATTATGATAG3’ Alcohol dehydrogenase sense 5’ ACCTTGTTGTGCTGGAGTAGA 3’ 84 PAAG_06715.1 Alcohol dehydrogenase anti-sense 5’ GGAGTCTGGAATCGGGGTG 3’ Isocitratelyase sense 5’ ATGGGAACCGACCTCCTGG 3’ 127 PAAG_06951.1 Isocitratelyase anti-sense 5’ CGTTCTTGCCTGCTTGCTCA 3’ Citrate synthase sense 5’ ACTGAGCACGGCAAGACGG 3’ 126 PAAG_08075.1 Citrate synthase anti-sense 5’ TTCCCAATGCACGGTCGATAA 3’ Peroxisomal catalase sense 5’ AGGTGCAGGAGCTTACGGTG 3’ 160 PAAG_01454.1 Peroxisomal catalase anti-sense 5’ CCCAATTTCCTTGCTCGGTG 3’ a Accession number – accession number of the protein identified in the databaseBroad Institute of MIT and Harvard: (http://www.broadinstitute.org/annotation/genome/Paracoccidioides_brasiliensis/MultiHome.html)

ACCEPTED MANUSCRIPT

Table S2:Paracoccidioides identified proteins/isoforms upon 6 and 24h of zinc starvation Time

gi|295673162 - Disulfide isomerase Pdi1

32

gi|295673162 - Disulfide isomerase Pdi1

c

MS/MS

RI PT

PMF Spot numberb

a

Seq. Cov (%)e

Matched Peptidesf

6h

195

49

29

24h

265

55

gi|295664022 - Glutathione reductase

55

6h

154

44

gi|295664022 - Glutathione reductase

61

24h

94

23

gi|295674755 - Glutathione synthetase

48

24h

gi|295671569 - Heat shock protein SSC1

27

24h

gi|295671569 - Heat shock protein SSC1

23

24h

gi|295671569 - Heat shock protein SSC1

26

24h

gi|295671569 - Heat shock protein SSC1

13

6h

gi|295671569 - Heat shock protein SSC1

73

gi|295671569 - Heat shock protein SSC1

76

gi|295658865 - Heat shock protein

112

gi|4164594 - Heat shock protein 70 gi|14538021 - Heat shock protein 70 gi|14538021 - Heat shock protein 70 gi|14538021 - Heat shock protein 70 gi|14538021 - Heat shock protein 70 gi|295659116 - Hsp70-like protein

34

4.44/4.80

63.67/59.31

4

4.53/4.80

69.67/59.3

15

8.42/6.74

50.83/51.96

2

8.06/6.74

49.67/51.9

SC

14

158

35

1

7.09/6.14

53.33/56.7

239

43

8

5.32/5.92

72.83/73.82

168

49

12

5.12/5.92

74.33/73.82

175

59

7

5.21/5.92

73.0/73.82

173

60

7

5.21/5.92

97.33/73.82

65

6

4.99/5.92

76.0/73.82

24h

104

98

20

1

5.40/5.92

67.0/73.82

24h

86

16

2

5.69/5.92

44.67/73.8

24h

**

**

2

6.41/5.92

43.83/73.8

24h

106

47

**

4.24/5.51

35.0/62.27

177

52

4

4.87/5.51

63.33/62.27

6h

30

6h

**

**

5

4.82/5.43

68.5/65.3

24

24h

143

26

5

4.84/5.05

74.0/70.9

84

24h

100

21

1

7.52/5.05

42.67/70.9

82

24h

183

25

5

5.92/5.05

42.67/70.9

77

24h

207

35

**

6.09/5.05

43.17/70.9

20

6h

182

49

11

4.72/5.08

76.0/70.92

AC C

gi|295658865 - Heat shock protein

6h

Exp/Theo MWh

TE D

22 31

EP

gi|295671569 - Heat shock protein SSC1 gi|295671569 - Heat shock protein SSC1

Exp/Theo pIg

M AN U

Scored

Accession number /Protein description

ACCEPTED MANUSCRIPT

109

24h

**

**

2

gi|295659116 - Hsp70-like protein

75

24h

187

28

3

4.05/5.08 5.79/5.08

43.83/70.9

gi|295659116 - Hsp70-like protein

79

24h

148

23

1

5.67/5.08

43.0/70.92

gi|295673716 - Hsp70-like protein

21

24h

234

36

5

4.73/5.39

76.0/68.8

RI PT

gi|295659116 - Hsp70-like protein

36.67/70.9

80

24h

85

23

1

7.34/5.39

43.0/68.86

gi|295659787 - Heat shock protein HSP88

10

24h

201

35

4

4.69/4.92

97.83/80.7

gi|295659787 - Heat shock protein HSP88

11

24h

157

30

4

4.85/4.92

95.5/80.7

gi|295659837 - Heat shock protein SSB1

57

24h

96

22

2

4.85/5.47

50.5/60.6

gi|295672932 - 30 kDa Heat shock protein

120

6h

109

53

11

7.06/9.75

24.83/28.64

gi|295672932 - 30 kDa Heat shock protein

117

24h

170

48

**

6.80/9.75

27.0/28.64

gi|295672932 - 30 kDa Heat shock protein

119

24h

120

57

**

7.28/9.75

26.5/28.64

gi|295664909 - 10 kDa Heat shock protein, mitochondrial

134

24h

102

58

1

9.41/8.79

12.67/11.19

gi|295662873 - Mitochondrial co-chaperone GrpE

118

6h

**

**

6

5.30/8.89

26.5/28.51

gi|225681400 - Peroxisomal catalase

41

6h

121

41

**

7.73/6.42

57.0/57.66

131

gi|17980998 - Y20 protein

124

gi|17980998 - Y20 protein

123

gi|295669794 - Elongation factor gi|295675019 - Elongation factor 2 gi|295675019 - Elongation factor 2 gi|295674319 - Polyadenylate binding protein gi|295660511 - Glycyl-tRNAsynthetase gi|146762537 - Enolase

M AN U

24h

87

29

1

6.77/5.51

41.0/38.19

24h

77

56

1

5.21/5.24

13.33/12.9

6h

60

33

1

7.19/6.09

22.83/21.64

24h

**

**

2

6.90/6.09

23.0/21.6

15

6h

283

58

12

6.31/5.65

90.0/100.71

67

6h

73

44

3

5.57/6.11

45.67/48.71

100

24h

116

16

2

7.08/6.46

39.17/92.6

104

24h

**

**

2

7.85/6.46

37.83/92.7

14

6h

74

37

**

6.99/6.31

92.0/86.92

28

24h

**

**

2

6.40/5.77

70.67/74.9

62

24h

89

27

**

5.82/5.67

49.5/47.4

AC C

gi|295657024 - Puromycin sensitive aminopeptidase

TE D

91

gi|295670221 - Thioredoxin

EP

gi|295661107 – Thioredoxin reductase

SC

gi|295673716 - Hsp70-like protein

ACCEPTED MANUSCRIPT

gi|146762537 - Enolase

60

24h

gi|295669690 - Phosphoglycerate kinase

71

gi|295658778 - Phosphoenolpyruvate carboxykinase

37

gi|295671120 - Fructose-1,6-bisphosphate aldolase

64

6

24h

91

27

2

7.51/6.48

44.83/45.3

24h

128

27

2

6.99/6.10

60.5/63.9

89

24h

133

43

1

7.48/6.09

41.67/39.72

gi|295671120 - Fructose-1,6-bisphosphate aldolase

101

6h

190

69

7

7.08/6.09

38.67/39.72

gi|295671120 - Fructose-1,6-bisphosphate aldolase

83

24h

153

58

**

6.33/6.09

42.67/39.72

gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase

108

6h

286

85

8

9.13/8.26

36.83/36.62

gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase

99

24h

88

50

**

8.47/8.26

39.67/36.62

gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase

96

24h

8.95/8.26

40.17/36.6

24h

72

24h

gi|295669416 - 2-oxoglutarate dehydrogenase E1

7

6h

gi|295669416 - 2-oxoglutarate dehydrogenase E1

5

24h

gi|295673931 - Pyruvate dehydrogenase protein X complex

47

6h

gi|295673931 - Pyruvate dehydrogenase protein X component

51

gi|295673937 - Malate dehydrogenase

113

gi|295664721 - Aconitase

17

gi|295664721 - Aconitase

16

SC

M AN U

95

gi|295658897 - Citrate synthase

42

**

50.17/47.41

193

69

3

8.28/8.26

40.17/36.6

84

26

2

7.99/8.75

44.83/52.2

122

20

1

7.11/6.68

103.5/121.6

221

27

4

7.30/6.68

108.67/121.6

98

33

6

5.46/6.45

53.67/52.71

TE D

gi|295658119 - Glyceraldehyde-3-phosphate dehydrogenase

140

5.66/5.67

RI PT

205

79

55

5

5.31/6.45

51.83/52.71

6h

185

49

10

8.10/8.99

34.17/36.02

24h

146

47

13

7.64/6.49

86.5/79.20

24h

109

24

12

7.51/6.49

88.17/79.20

19

24h

131

50

13

7.75/6.49

85.5/79.20

63

24h

159

50

**

9.37/6.90

49.33/68.04

3

24h

84

39

**

6.79/6.68

114.0/121.63

gi|295658595 - Pyruvate dehydrogenase E1 component subunit alpha

70

24h

130

28

2

6.63/8.62

44.83/45.3

gi|295660969 – Isocitrate lyase

33

6h

215

74

12

8.05/6.79

63.67/60.17

39

24h

161

27

2

8.10/6.79

57.83/60.2

EP

6h

gi|295664721 - Aconitase

gi|225682695 - 2-oxoglutarate dehydrogenase

gi|295660969 – Isocitrate lyase

AC C

gi|295665542 - Fumarate reductase

ACCEPTED MANUSCRIPT

38

6h

364

46

13

gi|295665123 - Aldehyde dehydrogenase

50

24h

170

41

2

6.19/5.87

52.33/54.5

gi|295665123 - Aldehyde dehydrogenase

53

6h

106

55

7

6.51/5.87

51.5/54.56

gi|295665123 - Aldehyde dehydrogenase

54

24h

140

38

3

6.46/5.87

51.17/54.5

gi|295665123 - Aldehyde dehydrogenase

135

24h

**

**

3

5.08/5.87

12.0/54.5

gi|295672968 - Phosphomannomutase

107

24h

**

**

2

5.57/5.60

37.17/30.6

gi|295663567 - 6-phosphogluconolactonase

111

24h

148

40

3

6.70/5.86

36.17/29.3

gi|295661432 - UTP-glucose-1-phosphate uridylyl transferase

43

6h

93

42

6h

gi|295674635 - Alcohol dehydrogenase

98

24h

gi|295662360 - Mannitol-1-phosphate 5 dehydrogenase

90

6h

gi|295666179 - 2-Methylcitrate synthase

65

6h

gi|295666197 - 2-Methylcitrate dehydratase

52

6h

gi|295672652 - Bifunctional purine biosynthesis protein ADE17

36

24h

gi|295672652 - Bifunctional purine biosynthesis protein ADE17

42

gi|295665468 - Nucleic acid-binding protein

115

gi|295665468 - Nucleic acid-binding protein

116

gi|295666938 - Nucleoside diphosphate kinase

126

gi|295672027 - Glycine dehydrogenase gi|295668479 - Formamidase gi|295674273 - Acetolactate synthase gi|295674767 - 4-aminobutyrate aminotransferase gi|295668370 - Aminopeptidase

SC

178

84

7

134

61

6

8.5/7.55

39.83/38.00

138

26

1

6.44/5.66

41.5/43.12

127

66

12

9.23/9.02

47.17/51.52

**

**

3

7.72/8.55

51.67/62.26

98

20

1

8.15/6.70

62.83/67.2

24h

153

29

2

7.26/6.70

56.17/67.2

24h

79

26

**

6.70/9.40

30.0/30.41

24h

101

41

**

7.18/9.40

30.0/30.41

6h

187

71

**

7.89/6.84

17.5/16.88

EP

gi|225683481 - Cysteinyl-tRNAsynthetase

55.0/58.87 38.33/38.00

8.72/7.55

114

24h

78

50

**

4.93/5.25

32.33/23.11

12

24h

81

22

**

2

24h

106

36

5

AC C

gi|225681397 - Xantine phosphoribosyl transferase

9.37/9.11

M AN U

103

59.67/60.17

7

TE D

gi|295674635 - Alcohol dehydrogenase

8.01/6.79

RI PT

gi|295660969 - Isocitrate lyase

6.18/6.09 7.64/8.84

95.5/89.20 115.0/129.88

64

24h

**

**

5

7.02/6.06

47.17/46.10

35

6h

95

35

5

7.81/8.93

62.83/74.16

58

6h

75

29

**

8.98/9.21

50.5/32.28

8

24h

158

58

6

5.41/6.20

100.67/73.39

40

6h

168

51

8

gi|295658698 - Fumaryl acetoacetase

66

24h

99

26

2

6.36/5.95

45.83/46.7

gi|295667902 - Aminomethyl transferase

68

24h

110

31

**

8.44/9.59

45.5/53.1

gi|295669670 - Adenosyl homocysteinase

69

6h

129

26

1

6.76/5.83

45.17/49.0

gi|295669240 - Kynurenine-oxoglutarate transaminase

74

6h

82

39

6

6.54/7.05

44.0/50.88

gi|295662426 - Aspartate aminotransferase

85

6h

88

30

5

8.46/8.39

42.5/50.91

gi|295662426 - Aspartate aminotransferase

88

24h

89

26

**

8.83/8.39

42.0/50.91

gi|295672504 - Inorganic pyrophosphatase

1

24h

99

59

4

7.0/5.13

115.67/33.55

gi|295672504 - Inorganic pyrophosphatase

86

24h

gi|295672504 - Inorganic pyrophosphatase

110

6h

gi|225678712 - Ketol-acid reductoisomerase

92

6h

gi|295658312 - L-PSP endoribonuclease family protein (Hmf1)

130

6h

gi|295670601 - 3-hydroxyisobutyryl-CoA hydrolase

49

24h

gi|295657225 - Peroxisomal multifunctional enzyme

9

24h

gi|295657225 - Peroxisomal multifunctional enzyme

18

gi|295668707 - Acetyl-coA acetyltransferase

87

gi|295664927 - ATP-citrate lyase

56

gi|295658821 - ATP synthase subunit beta

59

gi|295658923 - Citochrome b-c1 complex subunit 2

78

6h

138

45

5

8.92/9.10

43.17/49.01

81

24h

250

71

13

9.31/8.69

42.83/43.25

105

24h

101

33

1

7.56/6.55

37.67/33.7

24h

100

22

**

7.82/6.81

54.5/58.3

gi|295660716 - UDP-galactopyranose mutase

45

SC

121

61

**

4.82/5.13

42.0/33.55

187

63

11

4.84/5.13

36.5/33.55

192

60

10

7.92/9.12

40.83/44.86

77

46

1

5.98/8.96

15.17/18.72

80

62

**

6.30/7.09

53.17/57.35

93

38

**

9.37/8.98

100.33/97.15

**

**

2

9.59/8.98

86.33/97.15

6h

**

**

4

8.09/8.98

42.0/46.65

6h

**

**

3

6.91/5.99

50.67/52.9

6h

233

57

18

4.54/5.28

50.33/55.18

TE D

AC C

gi|295657369 - Nicotinate-nucleotide pyrophosphorylase

57.67/63.11

6h

EP

gi|295669073 - 12-oxophytodienoate reductase

8.17/8.99

RI PT

gi|295661139 - Methylmalonate-semialdehyde dehydrogenase

M AN U

ACCEPTED MANUSCRIPT

gi|295661741 - 3- demethylubiquinone 9,3-methyltransferase

128

24h

96

40

**

4.74/4.93

17.0/22.42

gi|295660455 - Pyridoxine biosynthesis protein PDX1

93

24h

94

70

**

6.26/6.04

40.67/34.41

gi|295663887 - 40S ribosomal protein S19

125

6h

129

80

5

10.45/9.69

17.67/16.41

127

24h

121

77

**

10.32/9.69

17.33/16.41

gi|295663887 - 40S ribosomal protein S19

ACCEPTED MANUSCRIPT

24h

94

92

**

6

24

242

50

10

5.67/5.52

gi|295657201 - Glutamate carboxypeptidase

44

6h

**

**

5

5.81/6.23

54.83/64.62

gi|295674421 - Ubiquitin carboxyl-terminal hydrolase

4

24h

154

46

4

5.21/5.33

111.67/88.03

gi|295660102 - Dipeptidyl peptidase

25

24h

226

66

**

7.21/7.99

73.50/86.55

gi|295662102 - Rab GDP-dissociation inhibitor

46

6h

158

64

9

5.64/5.44

54.33/52.54

gi|295657091 - Tropomyosin-1

122

6h

80

50

gi|295673184 - Actin interacting protein

97

24h

93

23

gi|295669061 - Arp2/3 complex subunit Arc16

106

6h

gi|295669061 - Arp2/3 complex subunit Arc16

94

24h

gi|295660405 - Hypothetical protein

133

6h

gi|295661500 - Conserved hypothetical protein

102

24h

gi|295673506 - Conserved hypothetical protein

132

24h

gi|295659253 - Conserved hypothetical protein

121

24h

AC C

EP

**Spots visualized only in zinc-depleted or zinc replete conditions; a GenBank general information identifier; b Spot numbers as depicted in Fig 2; c Time of exposure to zinc starvation; d Mascot score; e Amino acid sequence coverage for the identified protein; f Number of matched peptides on MS/MS searching; g Experimental/theoretical isoelectric point; h Experimental/theoretical molecular weight;

10.50/9.99

16.0/14.73 104.33/108.48

1

4.52/4.99

23.0/18.83

1

7.53/6.48

40.0/65.9

M AN U

SC

RI PT

129

gi|295672445 - Alanyl-tRNA synthetase

**

**

5

7.13/5.87

37.5/36.15

**

**

2

6.87/5.87

40.5/36.15

152

64

7

9.26/10.06

13.33/14.97

**

**

2

7.28/6.36

38.5/33.1

113

95

6

5.3/5.36

13.33/13.55

86

61

**

4.81/5.15

23.67/17.35

TE D

gi|295664112 - 40S ribosomal protein S22

ACCEPTED MANUSCRIPT

Table S3: Isoform analysis of proteins identified in more than one spot during the proteomic response of Paracoccidioides submitted to zinc restriction.

Acession numbera

Time b

Exp/Theo pIc

Exp/Theo MWd

Spot numbere

PTMf

Number of mass values matched

RI PT

Protein

Sequence coverage g %

Number of PTMs

Peptidei

Peptide sequence

h

Disulfide isomerase Pdi1

gi|295673162

6h

4.44/4.80

63.67/59.31

-

32

Acetyl (K) Phospho (ST)

24h

4.53/4.80

69.67/59.3

-

29

M AN U

Acetyl (K)

6h

8.06/6.74

8.42/6.74

49.67/51.9

EP

24h

50.83/51.96

AC C

gi|295664022

TE D

Phospho (ST)

Glutathione reductase

61

55

14

-

-

-

31

13

-

-

-

31

13

-

-

-

31

14

-

-

-

55

21

-

-

-

61

23

1

6-39

2

121-131

KSSSITSYMVK

1

353-370

SEPIPEKQEGPVTVVVAR

2

150-165

TLDKVTIIGFFAQDDK

3

214-230

TVYKGELTQEQVTSFIK

1

443-459

LFAAGSKDKPFDYQGLR LFAAGSKDKPFDYQGLR

SC

Phospho (Y)

31

57

23

HFAFGLAGLGIAVLASAADEAA SDVHALKGAAFK

Phospho (Y)

56

22

1

443-459

-

23

13

-

-

Acetyl (K)

25

14

1

1-9

Phospho (ST)

23

13

-

-

Phospho (Y)

23

13

-

-

-

44

35

-

-

Acetyl (K)

44

40

1

44-56

1

220-231

FDPMIQATITKR

1

248-260

QVELISDGKGSDR

2

245-255

LFGPPELKSSK

1

10-27

YDYIVIGGGSGGSGAARR

2

58-70

MTWNFSSIAETLR

2

123-139

Phospho (ST)

44

40

MAPIDEVKK

SGGCCVNVGCVPK

FTGQKEVEVQLQDGSGR

ACCEPTED MANUSCRIPT

72.83/73.82

27

36

1

10-27

-

43

29

-

-

Acetyl (K)

48

34

2

510-533

GVPQIEVTFDIDADSIVHVHAK DK

1

594-601

EFEDKLDK

1

535-550

AKVDELQNASLTLFDK

1

537-553

VDELQNASLTLFDKMHK

2

82-95

TTPSVVAFTKDGER

2

104-117

QAVVNPENTLFATK

1

172-183

ETAEAYLGKPVK

1

337-344

HINSKMTR

2

573-590

AAIEAANRADSVLNDTEK

1

637-650

VDELQNASLTLFDK ETAEAYLGKPVK

45

74.33/73.82

23

M AN U

SC

Phospho (ST)

5.12/5.92

EVEVQLQDGSGR

44

RI PT

5.32/5.92

128-139

Phospho (Y)

35

YDYIVIGGGSGGSGAARR -

Phospho (Y)

43

30

1

172-183

-

49

35

-

-

Acetyl (K)

56

44

2

281-288

NVVQQFKK

2

510-533

GVPQIEVTFDIDADSIVHVHAK DK

1

581-593

ADSVLNDTEKALK

2

599-608

LDKTEADQIK

1

637-653

VDELQNASLTLFDKMHK

1

13-23

AVPSFARSSSR

1

20-28

SSSRSSAYK

1

29-36

LPATPFRR

1

49-69

GQVIGIDLGTTNSAVAVMEGK

2

82-95

TTPSVVAFTKDGER

2

104-117

QAVVNPENTLFATK

2

157-169

YSPSQIGGFVLQK

2

288-298

KESGIDLSNDR

1

337-344

HINSKMTR

TE D

24h

EP

gi|295671569

AC C

Heat shock protein SSC1

1

Phospho (ST)

58

49

-

5.21/5.92

73.0/73.82

-

7

SQTFSTAADFQTAVEIK

2

573-590

AAIEAANRADSVLNDTEK

2

616-634

EVVAKSQSGEGSITADELK

3

621-636

SQSGEGSITADELKAK

2

635-650

AKVDELQNASLTLFDK

3

656-678

SEEGQQQQSQQSNEGQQGGE GEK

1

20-28

59

46

-

-

66

61

1

37-46

WNSTEGGEEKVK

1

73-91

IIENAEGARTTPSVVAFTK

1

104-117

QAVVNPENTLFATK

1

104-118

QAVVNPENTLFATKR

1

170-183

MKETAEAYLGKPVK

2

306-312

EACEKAK

1

358-364

TIEPVRK

1

388-396

MPKVGESVK

1

453-463

LINRNTTIPTK

1

465-481

SQTFSTAADFQTAVEIK

2

510-533

GVPQIEVTFDIDADSIVHVHAK DK

1

581-593

ADSVLNDTEKALK

2

581-593

ADSVLNDTEKALK

1

6-12

FSRALPR

1

20-28

SSSRSSAYK

1

29-36

LPATPFRR

1

49-72

GQVIGIDLGTTNSAVAVMEGKT PR

2

82-95

TTPSVVAFTKDGER

4

82-95

TTPSVVAFTKDGER

2

104-117

QAVVNPENTLFATK

TE D EP AC C

Phospho (ST)

465-481

35

M AN U

Acetyl (K)

2

51

SC

Phospho (Y)

RI PT

ACCEPTED MANUSCRIPT

65

68

SSSRSSAYK -

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

Phospho (Y)

67.0/73.82

31

TE D

5.40/5.92

EP

AC C

44.67/73.8

6.41/5.92

6h

5.21/5.92

43.83/73.8

97.33/73.82

73

76

13

124-131

FTDPECQR

1

184-198

NGVVTVPAYFNDSQR

2

184-198

NGVVTVPAYFNDSQR

1

262-280

STNGDTHLGGEDFDITLVR

1

337-344

HINSKMTR

3

342-357

MTRSQLEALVDPLISR

1

391-401

VGESVKSIFGR

2

465-481

SQTFSTAADFQTAVEIK

3

465-481

SQTFSTAADFQTAVEIK

2

573-590

AAIEAANRADSVLNDTEK

1

599-608

LDKTEADQIK

1

611-620

IASLREVVAK

1

20-28

SSSRSSAYK

1

184-198

NGVVTVPAYFNDSQR

1

482-491

VYQGERELVR

-

20

15

-

-

Acetyl (K)

21

17

1

199-114

QATKDAGQIAGLNVLR

2

281-288

NVVQQFKK

2

82-95

1

337-344

HINSKMTR

2

373-590

AAIEAANRADSVLNDTEK

Phospho (ST)

5.69/5.92

1

26

18

-

TTPSVVAFTKDGER

Phospho (Y)

20

15

-

-

-

-

**

**

-

-

-

Acetyl (K)

-

-

-

-

-

Phospho (ST)

-

-

-

-

-

Phospho (Y)

-

-

-

-

-

-

**

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

60

47

-

-

-

ACCEPTED MANUSCRIPT

64

62

M AN U

SC

RI PT

Acetyl (K)

AC C

EP

TE D

Phospho (ST)

69

70

1

20-28

SSSRSSAYK

2

37-48

WNSTEGGEEKVK

1

157-171

YSPSQIGGFVLQKMK

1

170-183

MKETAEAYLGKPVK

1

199-214

QATKDAGQIAGLNVLR

1

231-255

EQDRVVAVYDLGGGTFDISILEI QK

2

281-288

NVVQQFKK

1

391-401

VGESVKSIFGR

1

464-481

KSQTFSTAADFQTAVEIK

1

581-593

ADSVLNDTEKALK

1

616-634

EVVAKSQSGEGSITADELK

1

621-636

SQSGEGSITADELKAK

1

635-650

AKVDELQNASLTLFDK

1

637-653

VDELQNASLTLFDKMHK

1

9-19

ALPRAVPSFAR

1

82-95

TTPSVVAFTKDGER

2

82-95

TTPSVVAFTKDGER

1

104-117

QAVVNPENTLFATK

2

104-117

QAVVNPENTLFATK

1

157-169

YSPSQIGGFVLQK

1

170-183

MKETAEAYLGKPVK

1

184-202

NGVVTVPAYFNDSQRQATK

2

184-202

NGVVTVPAYFNDSQRQATK

1

289-303

ESGIDLSNDRMAIQR

1

345-357

SQLEALVDPLISR

2

345-363

SQLEALVDPLISRTIEPVR

1

391-401

VGESVKSIFGR

1

465-481

SQTFSTAADFQTAVEIK

2

510-531

GVPQIEVTFDIDADSIVHVHAK

2

573-590

AAIEAANRADSVLNDTEK

Phospho (Y)

4.99/5.92

76.0/73.82

-

22

Acetyl (K)

60

4.24/5.51

35.0/62.27

112

6h

4.87/5.51

63.33/62.27

34

581-593

ADSVLNDTEKALK

2

535-550

AKVDELQNASLTLFDK

2

537-550

VDELQNASLTLFDK

1

537-553

VDELQNASLTLFDKMHK

1

170-183

MKETAEAYLGKPVK

1

184-202

NGVVTVPAYFNDSQRQATK

50

-

-

-

**

**

-

-

-

**

**

-

-

-

68

53

1

184-198

NGVVTVPAYFNDSQR

1

235-255

VVAVYDLGGGTFDISILEIQK

-

47

32

-

-

Acetyl (K)

60

42

1

38-44

FAHKELK

1

56-71

GIDTLAKAVTTTLGPK

1

174-197

DITTTEEIAQVATISANGDTHVG K

1

198-205

LISNAMEK

1

235-249

GYVSPYFITDTKAQK

2

246-264

LEKATPDMLGSTGSITITK

1

388-402

SVISDPATSDYEKEK

1

401-406

EKLQER

1

525-540

GEYVDMIGAGIVDPLK

TE D

24h

EP

gi|295658865

AC C

Heat shock protein

M AN U

Phospho (Y)

49

2

65

SC

Phospho (ST)

RI PT

ACCEPTED MANUSCRIPT

Phospho (ST)

**

**

-

-

Phospho (Y)

48

33

1

383-400

-

52

40

-

-

Acetyl (K)

59

44

1

38-44

FAHKELK

1

56-71

GIDTLAKAVTTTLGPK

2

198-208

LISNAMEKVGK

1

365-376

EDTIILNGEGSK

1

525-540

GEYVDMIGAGIVDPLK

CEQIRSVISDPATSDYEK

ACCEPTED MANUSCRIPT

60

51

SC

RI PT

Phospho (ST)

gi|14538021

24h

4.84/5.05

7.52/5.05

42.67/70.9

24

84

42.67/70.9

82

AC C

EP

5.92/5.05

74.0/70.9

6.09/5.05

Hsp70-like protein

gi|295659116

6h

4.72/5.08

43.17/70.9

76.0/70.92

77

20

53

42

10-20

ASVLSSASSTR

3

63-73

AVTTTLGPKGR

2

156-172

GIQSAVEAVVEYLQTNK

1

198-208

LISNAMEKVGK

1

209-218

EGVITVKDGK

1

219-234

TIDDELEVTEGMRFDR

2

232-246

FDRGYVSPYFITDTK

1

383-400

CEQIRSVISDPATSDYEK

1

477-491

LGISIIKNAITRPAR

2

232-246

FDRGYVSPYFITDTK

1

383-400

CEQIRSVISDPATSDYEK

-

26

13

-

-

-

Acetyl (K)

26

13

-

-

-

Phospho (ST)

26

13

-

-

-

Phospho (Y)

26

13

-

-

-

-

21

11

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

21

11

-

-

-

-

25

16

-

-

-

Acetyl (K)

25

16

-

-

-

Phospho (ST)

32

19

3

386-414

Phospho (Y)

25

16

-

-

-

TE D

Heat shock protein 70

M AN U

Phospho (Y)

2

STNEILLLDVAPLSVGIETAGGV MTPLIK

-

35

21

-

-

Acetyl (K)

37

22

1

347-359

Phospho (ST)

35

21

-

-

-

Phospho (Y)

35

21

-

-

-

-

49

46

-

-

-

Acetyl (K)

52

57

3

96-110

AGKPVISVEFKGEEK

2

107-124

GEEKQFTPEEISSMVLTK

LVSDFFNGKEPNK

52

61

M AN U

SC

Phospho (ST)

RI PT

ACCEPTED MANUSCRIPT

43.83/70.9

AC C

5.79/5.08

36.67/70.9

5.67/5.08

Hsp70-like protein

gi|295673716

24h

4.73/5.39

43.0/70.92

76.0/68.8

109

75

79

21

170-185

IINEPTAAAIAYGLDK

2

416-423

NTTIPTKK

1

500-508

IVITNDKGR

1

509-516

LSKEEIER

1

571-588

TEIDKTVSWLDENQTATK

4

35-54

TTPSFVAFTDTERLIGDAAK

1

55-69

NQVAMNPSNTVFDAK

1

75-86

KFADPEVQSDMK

2

111-126

QFTPEEISSMVLTKMR

3

111-126

QFTPEEISSMVLTKMR

1

250-259

DLSSNARALR

2

310-320

STMDPVERVLR

1

344-355

IQKLVSDFFNGK

2

415-422

RNTTIPTK

2

416-422

NTTIPTK

1

557-566

VDEKLDASDK

Phospho (Y)

49

46

-

-

-

-

**

**

-

-

-

Acetyl (K)

-

-

-

-

-

Phospho (ST)

-

-

-

-

-

Phospho (Y)

-

-

-

-

-

-

28

16

-

-

-

Acetyl (K)

30

17

1

347-359

Phospho (ST)

28

16

-

-

-

Phospho (Y)

28

16

-

23

14

-

-

-

Acetyl (K)

23

14

-

-

-

Phospho (ST)

23

14

-

-

-

Phospho (Y)

23

14

-

-

-

-

36

21

-

-

-

TE D

4.05/5.08

EP

24h

1

LVSDFFNGKEPNK

ACCEPTED MANUSCRIPT

Phospho (ST) Phospho (Y) 7.34/5.39

43.0/68.86

-

80

24h

4.85/4.92

4.69/4.92

95.5/80.7

97.83/80.7

11

10

EP 6h

24h

AC C

gi|295672932

7.06/9.75

6.80/9.75

24.83/28.64

27.0/28.64

120

117

296-302

DLKTMGK

1

342-352

FEELNMDLFKK

2

342-352

FEELNMDLFKK

1

574-588

IDARNTLENYAFSLK

21

-

-

-

36

21

-

-

-

23

12

-

-

-

30

17

1

213-228

VVNEPTAAAIAYGLDK

1

277-278

VISHFVKQYNK

1

574-588

IDARNTLENYAFSLK

-

-

**

**

-

23

12

-

-

-

-

**

**

-

-

-

Acetyl (K)

-

-

-

-

-

Phospho (ST)

-

-

-

-

-

Phospho (Y)

-

-

-

-

-

-

35

25

-

-

-

Acetyl (K)

38

27

1

561-573

LTEKENAMYMEDK

1

582-593

KNELESHIYELR

1

677-688

EEQEAAEEAAKK

1

126-133

TTVSSELK

1

151-160

FNIDIKTNLK

-

-

-

-

Phospho (ST)

30 kDa Heat shock protein

2

Phospho (Y)

TE D

gi|295659787

M AN U

Phospho (ST)

-Heat shock protein HSP88

24

36

SC

Acetyl (K)

40

RI PT

Acetyl (K)

36

26

Phospho (Y)

35

25

-

-

53

17

Acetyl (K)

53

18

1

219-232

Phospho (ST)

**

**

-

-

-

Phospho (Y)

53

17

-

-

-

-

48

19

-

-

-

Acetyl (K)

53

20

1

32-45

1

219-232

TFSFPTRVDQNAVK

LPSQPTSSAYRISK TFSFPTRVDQNAVK

ACCEPTED MANUSCRIPT

26.5/28.64

48

19

-

-

-

Phospho (Y)

48

19

-

-

-

-

57

20

-

-

-

Acetyl (K)

57

22

1

157-172

EYTSSSNGEPGDKGQK

1

219-232

TFSFPTRVDQNAVK

118

Phospho (ST) Phospho (Y) Y20 protein

gi|17980998

6h

7.19/6.09

22.83/21.64

RI PT

7.28/9.75

Phospho (ST)

**

**

-

-

-

-

-

57

20

33

4

-

-

-

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

**

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

124

gi|295675019

24h

7.08/6.46

23.0/21.6

39.17/92.6

123

-

16

12

-

-

Acetyl (K)

21

14

1

152-169

1

571-587

Phospho (ST)

**

**

-

-

Phospho (Y)

17

13

1

170-184

-

**

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

27

8

-

-

-

100

Enolase

gi|146762537

24h

37.83/92.7

AC C

7.85/6.46

EP

TE D

Elongation factor 2

6.90/6.09

M AN U

SC

Acetyl (K)

24h

5.82/5.67

5.66/5.67

49.5/47.4

50.17/47.41

104

62

60

-

ALLELQVTKEDLYQSFSR HNRLYVTAEPLNEEVSK TIESVNVIIATYFDK

Acetyl (K)

27

8

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

27

8

-

-

-

-

64

25

-

-

-

Acetyl (K)

70

32

2

57-67

WLGKGVLNAVK

73

36

gi|295671120

6h

7.08/6.09

38.67/39.72

AC C

Fructose-1,6bisphosphate aldolase

EP

TE D

M AN U

SC

Phospho (ST)

RI PT

ACCEPTED MANUSCRIPT

101

Phospho (Y)

65

29

-

69

30

Acetyl (K)

76

34

Phospho (ST)

69

33

2

243-258

IALDIASSEFYKADEK

1

274-285

WLTYEQLADLYK

1

340-347

SCNALLLK

1

208-216

LAKLNQILR

1

417-431

IEEELGSNAVYAGDK

1

417-433

IEEELGSNAVYAGDKFR

1

1-9

1

51-60

DGDQSKWLGK

1

61-79

GVLNAVKNVNSVIGPAIIK

1

97-105

LDGTPNKSK

1

127-141

GVPLYAHVSDLAGTK

1

185-194

QGSEVYHKLK

1

243-258

IALDIASSEFYKADEK

1

272-285

SKWLTYEQLADLYK

1

340-347

SCNALLLK

1

377-393

SGETEDVTIADIVVGLR

1

399-407

TGAPARSER

1

227-241

GVPLYAHVSDLAGTK

1

185-194

QGSEVYHKLK

1

143-158

IALDIASSEFYKADEK

1

272-285

SKWLTYEQLADLYK

-

-

1

2-9

1

96-115

SIAPSYGIPVVLHTDHCAKK

2

237-250

LHPELLSKHQAYVK

1

253-256

TGSSKNKPVYLVFHGGSGSTK

1

30-52

VFAIPAINVTSSSTVVAALEAAR

1

80-95

QEASVAGAIAAAHYIR

1

278-302

MAITKIHAR

GVKDILSR

EAISYGVVKVNLDTDLQYAYLS GVR

ACCEPTED MANUSCRIPT

24h

7.48/6.09

41.67/39.72

-

89

Acetyl (K) Phospho (ST)

42.67/39.72

9.13/8.26

36.83/36.62

1

278-302

EAISYGVVKVNLDTDLQYAYLS GVR

1

310-328

DYLMSAVGNPEGEDKPNKK

QEASVAGAIAAAHYIR

10

-

-

-

43

10

-

-

-

43

10

-

-

-

10

-

-

-

21

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

66

29

1

5-10

DILSRK

1

10-21

KTGVIVGDDVLR

2

55-73

NSPIILQVSQGGAAFFAGK

1

96-114

SIAPSYGIPVVLHTDHCAK

1

258-273

NKPVYLVFHGGSGSTK

1

278-286

EAISYGVVK

1

347-360

VQEAFDDFNTSNQL

1

80-95

QEASVAGAIAAAHYIR

1

258-273

NKPVYLVFHGGSGSTK

1

278-286

EAISYGVVK

1

328-333

KYFDPR

-

-

1

48-72

YDSTHGQFKGDIQHSSSNNLTV NNK

1

139-162

SYRPDISVLSNASCTTNCLAPLA K

2

260-271

AAVKAASEGELK

2

216-227

AVGKVIPALNGK

83

108

80-95

43

TE D 6h

EP

gi|295658119

AC C

Glyceraldehyde-3phosphate dehydrogenase

1

DYLMSAVGNPEGEDKPNKK

58

-

M AN U

6.33/6.09

35

310-328

43

SC

Phospho (Y)

78

RI PT

Phospho (Y)

1

Phospho (Y)

60

26

-

85

30

Acetyl (K)

90

35

-

ACCEPTED MANUSCRIPT

Phospho (Y)

24h

8.47/8.26

39.67/36.62

-

99

40.17/36.6

2-oxoglutarate dehydrogenase E1

gi|295669416

6h

24h

AC C

EP

8.28/8.26

7.11/6.68

7.30/6.68

96

M AN U

40.17/36.6

103.5/121.6

108.67/121.6

95

1

119-138

VIISAPSADAPMFVMGVNEK

1

260-271

AAVKAASEGELK

2

292-309

SSIFDASAGIALNDRFVK

1

310-323

LISWYDNEWGYSRR

1

272-291

GILGYSEDALVSTDLNGDPR

1

310-323

LISWYDNEWGYSRR

1

323-333

RVLDLIAYIAK

17

-

-

-

67

17

1

41-47

YAAYMLK

1

187-194

TVDGPSHK

2

249-259

TEKPVTYDQIK

Phospho (ST)

**

**

-

-

Phospho (Y)

65

14

1

324-333

-

42

12

-

-

-

Acetyl (K)

42

12

-

-

-

Phospho (ST)

46

15

3

139-162

SYRPDISVLSNASCTTNCLAPLA K

1

110-117

AHLSGGAK

Phospho (Y)

42

12

-

-

-

-

69

17

-

-

-

Acetyl (K)

73

18

2

116-127

AVGKVIPALNGK

Phospho (ST)

73

19

1

110-117

AHLSGGAK

Phospho (Y)

69

17

-

-

-

-

20

17

-

-

-

Acetyl (K)

20

17

-

-

-

Phospho (ST)

21

18

1

4-18

Phospho (Y)

20

17

-

-

-

-

27

19

-

-

-

Acetyl (K)

27

19

-

-

-

Phospho (ST)

27

19

-

-

-

TE D

8.95/8.26

36

63

SC

Acetyl (K)

86

RI PT

Phospho (ST)

VLDLIAYIAK

TSTVKASSTTAVFLR

ACCEPTED MANUSCRIPT

gi|295673931

6h

5.46/6.45

53.67/52.71

27

19

-

-

-

-

33

16

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

47

Phospho (Y) 5.31/6.45

51.83/52.71

-

51

**

**

-

-

-

55

27

-

-

-

63

42

1

82-100

KVGDALAPGDVLVEIETDK

1

157-167

SSALKEPEQPK

2

169-178

ELKVAPAAPK

1

172-195

VAPAAPKEESTPAAEEEPVSTG ER

1

196-211

LQPSLDRESFIAPAVK

1

203-211

ESFIAPAVK

1

203-217

ESFIAPAVKALALER

2

218-225

GVPLKDIK

1

223-232

DIKGTGPGGR

1

226-235

GTGPGGRVTK

2

233-240

VTKNDVEK

1

297-308

EALNNSADGKYK

2

297-308

EALNNSADGKYK

1

367-381

Aconitase

gi|295664721

24h

AC C

EP

TE D

M AN U

SC

Acetyl (K)

RI PT

Pyruvate dehydrogenase protein X component

Phospho (Y)

7.64/6.49

86.5/79.20

17

NAHTLGLSSISSQVK

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

47

40

-

-

-

Acetyl (K)

51

48

1

24-30

1

176-192

VIGVRLSGELSGWTSPK

1

250-258

MYDYLKATK

1

489-498

DTYQAPPKDR

1

535-552

AQGKTTTDHISMAGPWLK

1

601-618

GIKWVVIGDWNYGEGSSR

1

637-647

SFARIHETNLK

EPRYCPK

ACCEPTED MANUSCRIPT

88.17/79.20

Phospho (Y)

48

44

1

250-255

MYDYLK

1

471-488

LKEPTGAGLPANGYDPGR

2

584-598

TGEYGPVPATARDYK

1

648-664

KQGMLPLTFTDPADYDR

-

8.05/6.79

63.67/60.17

39

-

24

19

-

-

-

24

17

-

-

-

Phospho (ST)

24

17

-

-

-

Phospho (Y)

26

20

1

10-20

M AN U

SC

6h

DGSALNTMAKQR

-

VGALGNYINYK

-

50

39

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

52

50

1

24-30

EPRYCPK

1

250-255

MYDYLK

1

260-274

QAIGDFARSYAHSLR

1

473-496

EPTGAGLPANGYDPGRDTYQA PPK

1

489-496

DTYQAPPK

1

601-618

GIKWVVIGDWNYGEGSSR

1

648-664

KQGMLPLTFTDPADYDR

1

649-664

QGMLPLTFTDPADYDR

1

1-10

MDSIEQEDQK

1

48-57

IEYPSNVQSK

1

58-66

KLWGILEER

1

127-140

VNQLWMAQLFHDRK

1

182-189

LTKLFIER

2

190-205

GAAGIHIEDQAPGTKK

1

205-212

KCGHMAGK

EP gi|295660969

AC C

Isocitrate lyase

714-725

-

16

19

1 **

TE D

85.5/79.20

KQGMLPLTFTDPADYDR

**

Acetyl (K)

7.75/6.49

648-664

Phospho (ST)

RI PT

7.51/6.49

1

-

74

51

Acetyl (K)

76

64

ACCEPTED MANUSCRIPT

74

67

M AN U

SC

RI PT

Phospho (ST)

59.67/60.17

38

74

54

AC C 24h

8.10/6.79

57.83/60.2

LFSEAVVDQIKASSIPNK

1

318-333

ASSIPNKQAVIDGFLR

1

516-529

MVSGGISSTSAMGK

1

29-42

YTKRPFTAEQIVAK

1

32-42

RPFTAEQIVAK

1

32-43

RPFTAEQIVAKR

2

44-57

GNLKIEYPSNVQSK

1

349-363

KITGSDIYFDWDAAR

2

350-365

ITGSDIYFDWDAARTR

3

350-365

ITGSDIYFDWDAARTR

1

382-503

AYGELVQEPEMENGVDVVTH QK

1

516-529

MVSGGISSTSAMGK

3

516-537

MVSGGISSTSAMGKGVTEDQF K

1

29-42

1

349-363

KITGSDIYFDWDAAR

1

482-503

AYGELVQEPEMENGVDVVTH QK

YTKRPFTAEQIVAK

70

47

-

-

Acetyl (K)

71

57

1

1-10

MDSIEQEDQK

1

11-19

YWAEVQAVK

2

44-57

GNLKIEYPSNVQSK

1

182-189

LTKLFIER

2

205-212

1

288-298

KCGHMAGK NGADLQAIEDK

1

244-259

TDSEAATLITSTIDPR

2

350-369

ITGSDIYFDWDAAR

1

420-431

LAYNLSPSFNWK

Phospho (ST)

39

307-324

-

EP

8.01/6.79

TE D

Phospho (Y)

1

72

52

-

Phospho (Y)

**

**

-

-

-

-

27

16

-

-

-

ACCEPTED MANUSCRIPT

27

26

-

-

Phospho (ST)

34

17

1

190-204

GAAGIHIEDQAPGTK

2

350-363

ITGSDIYFDWDAAR

2

449-473

LGYAWQFITLAGLHTTALISDQF AK

-

-

-

-

1

87-108

LADLMEQHVDTLAAIEALDNG K

1

126-133

YYGGWADK

1

126-137

YYGGWADKIHGK

1

134-150

IHGKVIDTDSDSFNYTR

1

222-246

VAGAAISSHMDIDKVAFTGSTL VGR

2

247-258

QILQAAAKSNLK

1

390-404

IVKEEIFGPVCCVQK

1

348-356

IMGYIREGK

Phospho (Y) 6h

6.51/5.87

51.5/54.56

-

53

27

16

55

25

57

33

24h

6.19/5.87

TE D

M AN U

SC

Acetyl (K)

52.33/54.5

50

EP

gi|295665123

AC C

Aldehyde dehydrogenase

RI PT

Acetyl (K)

6.46/5.87

5.08/5.87

51.17/54.5

12.0/54.5

54

135

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

57

26

-

-

-

-

41

14

-

-

-

Acetyl (K)

41

14

1

319-325

Phospho (ST)

41

14

-

-

Phospho (Y)

42

15

1

348-353

-

38

10

-

-

Acetyl (K)

38

11

1

222-246

Phospho (ST)

38

10

-

-

-

Phospho (Y)

38

10

-

-

-

-

**

**

-

-

-

Acetyl (K)

-

-

-

-

-

Phospho (ST)

-

-

-

-

-

ARALQNK IMGYIR VAGAAISSHMDIDKVAFTGSTL VGR

ACCEPTED MANUSCRIPT

gi|295674635

6h

8.72/7.55

38.33/38.00

103

-

-

-

84

29

Acetyl (K)

85

35

85

M AN U

SC

Phospho (ST)

RI PT

Alcohol dehydrogenase

Phospho (Y)

Phospho (Y)

8.5/7.55

39.83/38.00

98

EP 6h

24h

AC C

gi|295662426

8.46/8.39

8.83/8.39

42.5/50.91

42.0/50.91

85

88

-

-

-

1

11-18

2

213-228

ELCMKMGATAFVDFSK

2

218-230

MGATAFVDFSKSK

2

229-237

SKDLVADVK

1

248-254

YVLKMPE

1

197-211

AMGLQTIAVDAGNEK

2

213-228

ELCMKMGATAFVDFSK

2

283-298

LQAPVFDTVVRMITIK

1

299-309

GSYVGNRLDAK

2

326-343

TVPLQELGNVFSLMDQGK

1

299-309

GSYVGNRLDAK

1

348-354

YVLKMPE

QWAQVADK

61

23

-

-

Acetyl (K)

66

27

1

1-18

1

218-230

MGATAFVDFSKSK

2

218-230

MGATAFVDFSKSK

1

197-211

AMGLQTIAVDAGNEK

2

218-230

MGATAFVDFSKSK

1

299-309

GSYVGNRLDAK

1

299-309

GSYVGNRLDAK

Phospho (ST)

Aspartate aminotransferase

31

-

-

TE D

24h

86

35

-

57

26

MAPQIPIPEKQWAQVADK

Phospho (Y)

61

24

-

30

9

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phospho (Y)

**

**

-

-

-

-

26

9

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

ACCEPTED MANUSCRIPT

gi|295672652

24h

8.15/6.70

62.83/67.2

**

**

-

-

-

-

20

10

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

36

Phospho (Y) 7.26/6.70

56.17/67.2

-

42

Acetyl (K)

gi|295665468

24h

6.70/9.40

30.0/30.41

6h

24h

40S ribosomal protein S19

gi|295663887

6h

9.59/8.98

EP

gi|295657225

86.33/97.15

AC C

Peroxisomal multifunctional enzyme

10.45/9.6

10.45/9.6 9

17.67/16.41

17.67/16.41

-

29

21

-

-

-

32

23

1

1-13

1

538-548

MADDMTLAQALKK VEFEKVFEEGR

22

-

-

-

21

-

-

-

-

26

13

-

-

-

Acetyl (K)

30

13

1

190-196

Phospho (ST)

**

**

-

-

-

Phopho (Y)

33

13

-

-

-

-

41

18

-

-

-

Acetyl (K)

39

19

1

190-196

ELNELFK

1

190-199

ELNELFKDIK

116

9

-

29

115

18

-

31

TE D

7.18/9.40

30.0/30.41

**

Phospho (Y)

M AN U

Nucleic acid-binding protein

**

SC

Phospho (ST)

RI PT

Bifunctional purine biosynthesis protein ADE17

Phospho (Y)

ELNELFK

Phospho (ST)

**

**

-

-

-

Phospho (Y)

44

21

1

114-123

IVYDNRGMSR

1

153-160

VVVNYSSR

2

140-155

APYGRPLRLDYSLSAR

-

**

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phopho (Y)

**

**

-

-

-

-

38

9

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

38

9

-

-

Phopho (Y)

38

9

-

-

-

80

28

-

-

-

ACCEPTED MANUSCRIPT

10.32/9.6 9

82

29

1

1-18

Phospho (ST)

80

28

-

-

-

Phopho (Y)

78

26

-

-

-

-

77

26

-

-

17.33/16.41

Acetyl (K) Phospho (ST)

gi|295672504

6h

24h

4.84/5.13

4.82/5.13

37.5/36.15

-

36.5/33.55

42.0/33.55

-

-

-

79

27

-

-

-

81

28

87-108

LADLMEQHVDTLAAIEALDNG K

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phopho (Y)

**

**

-

-

-

-

**

**

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

**

**

-

-

-

Phopho (Y)

**

**

-

-

-

-

63

24

-

-

-

Acetyl (K)

63

24

-

-

-

Phospho (ST)

66

26

1

1-13

1

185-198

110

86

26

**

21

12

-

M AN U

Inorganic pyrophosphatase

7.13/5.87

40.5/36.15

TE D

24h

6.87/5.87

EP

6h

AC C

- Arp2/3 complex subunit Arc16

MAPQIPIPEKQWAQVADK

77

SC

Phopho (Y)

RI PT

24h

Acetyl (K)

MSYAKSPSEYTVR HLPGLLRATNEWFR

Phopho (Y)

63

25

1

1-13

-

61

25

-

-

Acetyl (K)

73

43

1

14-24

KVGQPQTLDFR

1

15-29

VGQPQTLDFRAYIEK

2

60-69

WTNAKLEISK

1

65-77

LEISKEEFLNPIK

1

70-77

EEFLNPIK

1

70-81

EEFLNPIKQDVK

1

87-95

YVRNCFPHK

MSYAKSPSEYTVR

80

41

Phospho (Y)

72

34

AC C

EP

TE D

M AN U

SC

Phospho (ST)

RI PT

ACCEPTED MANUSCRIPT

7.0/5.13

115.67/33.55

1

2

107-121

TWEDPNVVHPETKAK

1

120-143

AKGDNDPLDVCEIGELVGYTGQ VK

1

122-143

GDNDPLDVCEIGELVGYTGQVK

1

122-146

GDNDPLDVCEIGELVGYTGQVK QVK

2

144-162

QVKVLGVMALLDEEETDWK

2

102-120

IPDGKPENQFAFSGECKNK

1

1-13

MSYAKSPSEYTVR

3

1-13

MSYAKSPSEYTVR

2

2-13

SYAKSPSEYTVR

3

2-13

SYAKSPSEYTVR

1

65-77

LEISKEEFLNPIK

2

107-119

TWEDPNVVHPETK

2

107-121

TWEDPNVVHPETKAK

1

144-162

QVKVLGVMALLDEEETDWK

1

222-241

YAMEVVHECADAWEKLMSGK

1

237-244

LMSGKSNR

5

245-264

GDISLANTSSEQSPDWVDSK

1

265-285

QVNLPDGQNLPPAPIDGSVDK

1

1-13

MSYAKSPSEYTVR

2

2-13

SYAKSPSEYTVR

1

87-95

YVRNCFPHK

1

90-106

NCFPHKGYLWNYGAFPR

2

90-106

NCFPHKGYLWNYGAFPR

1

222-236

YAMEVVHECADAWEK

1

222-241

YAMEVVHECADAWEKLMSGK

-

59

22

-

-

-

Acetyl (K)

**

**

-

-

-

Phospho (ST)

66

36

1

1-13

MSYAKSPSEYTVR

2

1-13

MSYAKSPSEYTVR

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

EP

AC C

**No PMF results; a GenBank general information identifier; b Time of exposure to zinc starvation or zinc replete conditions; c Experimental/theoretical isoelectric point; d Experimental/theoretical molecular weight; e Spot numbers as depicted in Fig 2; f Post-translational modifications g Amino acid sequence coverage for the identified protein; h Number of peptides identified by using Mascot software analysis; i Peptide position

TE D

Phospho (Y)

63

29

3

1-13

SYAKSPSEYTVR

1

60-69

WTNAKLEISK

1

65-77

LEISKEEFLNPIK

1

107-119

TWEDPNVVHPETK

2

107-119

TWEDPNVVHPETK

1

TWEDPNVVHPETKAK 107-121

2

107-121

TWEDPNVVHPETKAK

1

147-162

VLGVMALLDEEETDWK

1

185-198

HLPGLLRATNEWFR

1

192-201

ATNEWFRIYK

1

202-218

IPDGKPENQFAFSGECK

1

1-13

MSYAKSPSEYTVR

2

1-13

MSYAKSPSEYTVR

1

90-106

NCFPHKGYLWNYGAFPR

1

96-106

GYLWNYGAFPR

1

192-201

ATNEWFRIYK

1

222-236

YAMEVVHECADAWEK

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT

AC C

EP

TE D

M AN U

SC

RI PT

ACCEPTED MANUSCRIPT