Figure 5: C. neoformans Cu transporters have distinct properties. | Nature Communications

Figure 5: C. neoformans Cu transporters have distinct properties.

From: Reciprocal functions of Cryptococcus neoformans copper homeostasis machinery during pulmonary infection and meningoencephalitis

Figure 5

(a) A DNA sequence encoding the HA tag was integrated before the stop codon of CTR1 and CTR4. Sequences upstream and downstream of CTR1, CTR4 or CMT1 were cloned into a plasmid containing HA tag construct. The resulting plasmid was amplified and integrated into the genome of the H99 strain to generate strains CTR1HA, CTR4HA or CMT1HA. (b) Cells were grown in SC medium supplemented with 200 μM BCS or 200 μM Cu for 6 h. Proteins were isolated and probed with the anti-HA and anti-histone H3 antibodies (see Supplementary Fig. 5A for full blots). (c) Cells were grown in SC medium supplemented with 1 mM BCS for 6 h and washed three times with H2O. The cells were then treated with 100 μM Cu. Proteins were isolated at different time points and probed with the anti-HA and anti-histone H3 antibodies (see Supplementary Fig. 5B for full blots). (d) C57BL/6 mice were infected with C. neoformans cells expressing CTR1HA, CTR4HA and CMT1HA. Two days post infection, lung tissues were removed and subjected to immunohistochemical anlaysis (scale bar=50 μm). Red circles indicate C. neoformans cells, which are shown in the right panels (scale bar=25 μm). (e) The expression of the Ctr1 and Ctr4 Cu+ transporters was induced by culturing cells in SC medium supplemented with 1 mM BCS for 6 h. Proteins were then isolated and mixed with protein deglycosylation mix (NEB) for 4 h. Proteins were separated on a 15% SDS–PAGE gel and an anti-HA antibody was used to detect Ctr4HA and Ctr1HA (see Supplementary Fig. 5C for full blots). (f) The transmembrane domains of Ctr4 were predicted using TMHMM 2.0 (http://www.cbs.dtu.dk/services/TMHMM-2.0) (left panel). Ctr4 Asn19 was mutated to Gln using a PCR-based approach and the mutated CTR4 genomic DNA sequence was integrated into the CTR4-native allele. The protein was isolated and probed with an anti-HA antibody (see Supplementary Fig. 5D for full blots). (g) ctr1Δctr4Δ cells expressing wild-type CTR4HA and the N19QHA mutation were spotted on SC agar supplemented with 125 μM BCS. Images were acquired after a 2-day incubation at 30 °C.

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