Identification of Membrane Proteome of Paracoccidioides Lutzii and its Regulation by Zinc

Figures & data

Figure 1.  Transmission electron microscopy of Paracoccidioides lutzii, whole membranes system fraction.

(A) Intact cells of P. lutzii. (B) Membranes extract. (C) Fragment of membrane, evidencing the lipid bilayer (increase of 40,000 times).

Table 1.  The most abundant membrane proteins.

Accession number^†	Protein identification^‡	Score protein^§	fmol^¶	TMH^#	PTM^††	SignalP score ≥ 0.5^‡‡
PAAG_08620	ADP ATP carrier protein 310 aa	28,165	1955.942	3	–	–
PAAG_08082	Plasma membrane ATPase 930 aa	27,274.75	1292.34	9	–	–
PAAG_07564	Outer mitochondrial membrane protein porin 285 aa^§§	17,746.9	601.808	–	–	–
PAAG_00850	Glucosamine fructose 6 phosphate aminotransferase 489 aa^§§	16,121.64	527.8133	–	–	–
PAAG_00481	Membrane biogenesis protein Yop1 171 aa	11,930.6	449.3233	3	–	–
PAAG_04838	ATP synthase subunit 4 245 aa^§§	13,824.06	372.026	–	–	–
PAAG_04570	ATP synthase D chain mitochondrial 175 aa^§§	3393.7	388.8589	–	–	–
PAAG_05350	Mitochondrial phosphate carrier protein 422 aa^§§	5905.8	335.2175	–	–	–
PAAG_08028	GTP-binding protein ypt1 202 aa	25,168.0	448.6622	–	C:20	–
PAAG_02265	Mitochondrial F1F0 ATP synthase subunit 102 aa	14,358.8	295.1655	1	–	–

†,‡ Accession number and description of protein according to database of Paracoccidioides spp. (www.broadinstitute.org/annotation/genome/paracoccidioides_brasiliensis/MultiHome.html).

§ Protein score obtained from MS data using the PLGS.

¶ Quantification of membrane proteins according to internal standard.

# Represents the amount of transmembrane domains identified in proteins using the program TMHMM version 2.0 (www.cbs.dtu.dk/services/TMHMM/).

†† Indicates the presence of post-translational modification. The program TermiNator (www.isv.cnrs-gif.fr/terminator3/test.php) and Myristoylator (http://web.expasy.org/myristoylator/) were employed in search for myristoylated proteins. To search for palmitoylated proteins was performed with the program TermiNator (www.isv.cnrs-gif.fr/terminator3/test.php). For identification of prenylated proteins, the program was the PrePS (http://mendel.imp.ac.at/sat/PrePS/index.html). The prediction for GPI anchor was performed with the program big-PI fungi predictor (http://mendel.imp.ac.at/gpi/fungi_server.html). C:14 and C:16 indicate the presence of a myristoyl or palmitoyl group in the protein, respectively. C:15 indicates the presence of a recognition site to the addition of a farnesyl group by the enzyme FT and C:20 indicates the presence of a recognition site to addition of a geranyl group by the enzyme GGT1 or a site recognition for addition of a geranyl group by the enzyme GGT2. GPI represents the presence of a GPI anchor in protein.

‡‡ The program SignalP version 4.0 (www.cbs.dtu.dk/services/SignalP/) was employed in the search for signal peptide. The values demonstrate the score protein with signal peptide. The D-Score must be higher or equal to the value of (0.05).

– In all cases, the symbol represents absence in the analyzed category.

The functional classification was performed using the database of FunCat2 (http://pedant.gsf.de/pedant3htmlview/pedant3view?Method=analysis&Db=p3_r48325_Par_brasi_Pb01).

The database GO using the information relative to cellular component was employed to evaluate the possible localization of proteins in cell membranes of Paracoccidioides sp.

§§ Some proteins did not show any form of classical association with the lipid bilayer, but they presented the subcellular localization in GO as belonging to the membrane with score ≥ 50.

FT: Farnesyltransferase; GGT1: Geranylgeranyltransferase; GGT2: Rab Geranylgeranyltransferase; GO: Gene ontology; GPI: Lipid anchor modified; GTP: Guanosine triphosphate; PI: Big-PI fungi database; PLGS: ProteinLynx Global Server; PrePS: Prenylation Prediction Suite; PTM: Post-translational modification; TMH: Transmembrane helices.

Figure 2.  Functional classification of membrane proteins regulated by zinc availability.

The database FunCat2 was used to perform this classification. Light gray bars represent downregulated proteins and dark gray bars represent upregulated proteins. Proteins upregulated: Transport – 56% (10 proteins), Cell cycle and DNA processing – 0% (0 protein), Metabolism – 0% (0 protein), Cellular communication – 0% (0 protein), Protein synthesis – 6% (1 protein), Protein fate – 6% (1 protein), Biogenesis of cellular components – 0% (0 protein), Cell rescue – 6% (1 protein), Energy – 6% (1 protein), Unclassified – 17% (3 proteins), Cell-type differentiation – 0% (0 protein), Cell fate – 6% (1 protein). Proteins downregulated: Transport – 31% (36 proteins), Cell cycle and DNA processing – 2% (2 proteins), Metabolism – 14% (16 proteins), Cellular communication – 5% (6 proteins), Protein synthesis – 1% (1 protein), Protein fate – 10% (11 proteins), Biogenesis of cellular components – 5% (6 proteins), Cell rescue – 4% (5 proteins), Energy – 14% (16 proteins), Unclassified – 13% (15 proteins), Cell-type differentiation – 1% (1 protein), Cell fate – 0% (0 protein).

Figure 3.  Effect of zinc deprivation in Paracoccidioides lutzii yeast cells wall.

Yeast cells were cultivated in MMcM medium depleted or supplemented with zinc for 24 h. (A) The cells were fixed and stained with CFW (increase of 40 times). (B) Fluorescence intensity (in pixels) of the cells under zinc deprivation. The AxioVision Software (Carl Zeiss) was used to obtain the values of fluorescence intensity. The values of fluorescence intensity and the standard error of each analysis were used to plot the graph. Data are expressed as mean ± standard error (represented using error bars).

*p ≤ 0.05.

CFW: Calcofluor white; MMcM: McVeigh/Morton’ liquid medium.

Figure 4.  Zinc deprivation alters proteins glycosylation.

(A) Protein extracts of membranes were fractionated by electrophoresis and stained with periodic acid Schiff. The same extracts were stained with silver. (B) Fluorescence microscopy of P. lutzii cells that were cultured in the presence or absence of zinc for 24 h and subsequently incubated with ConA conjugated to FITC (increase of 40 times). (C) Fluorescence intensity graph. The data for fluorescence intensity evaluation were obtained through the AxioVision Software (Carl Zeiss). The values of fluorescence intensity (in pixels) and the standard error of each analysis were used to plot the graph. Data are expressed as mean ± standard error (represented using error bars), (*) represents p ≤ 0.05.

ConA: Concanavalin A; FITC: Fluorescein isothiocyanate.

Figure 5.  Evaluation of β-1,3 glucan quantities in the cell wall of Paracoccidioides lutzii.

(A) Aniline blue was used to evaluate, by fluorescence microscopy, the presence of β-1,3 glucan in the cell wall of P. lutzii after growth in the presence and absence of zinc (increase of 40 times). (B) Fluorescence intensity graph. The values of fluorescence intensity (in pixels) and the standard error of each analysis were used to plot the graph. Data are expressed as mean ± standard error (represented using error bars).