Cell functions and molecular mechanisms of zinc transporters in Neurospora crassa

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"Thesis Title: Cell functions and molecular mechanisms of zinc transporters in Neurospora crassa In this thesis work, I studied the cellular functions and molecular mechanisms of zinc resistance-conferring 1 (ZRC-1), meiotic sister chromatid 2 (MSC-2), and zinc-regulated gene 17 (ZRG-17) that are members of the cation diffusion facilitator (CDF) family of zinc transporters in Neurospora crassa. The Δzrc-1 mutant was unable to grow under high zinc conditions (≥ 0.5 mM). However, the expression of zrc-1 was elevated ~3-fold under low zinc conditions in comparison to normal and high zinc concentrations. The Δmsc-2 mutant showed colony growth and aerial hyphae similar to wild type and the expression of msc-2 was independent of zinc. Furthermore, the double mutant Δzrc-1; Δmsc-2 and Δzrc-1; Δzrg-17 showed additive phenotypes of both the parental single mutants. However, the phenotypic defects such as slow growth rate, defective in asexual sporulation, and inability to degrade cellulose of the Δzrg-17 single mutant were restored in the Δmsc-2; Δzrg-17 double mutant, which showed phenotypes similar to the wild type. The double mutant Δzrc-1; Δzrg-17 showed severe growth defects, stunted aerial hyphae, short septa, and defects in conidiation. In addition, the Δzrc-1; Δmsc-2 and Δzrc-1; Δzrg-17 double mutants showed sensitivity to DTT-induced ER stress and were unable to grow in the medium containing cellulose. Furthermore, zinc-responsive activator protein 1 (ZAP-1) was also studied to understand the molecular mechanism and the interaction of the CDF zinc transporters with the transcription factor. The zap-1 of N. crassa was found to be crucial for survival under low zinc conditions and ZAP-1 was localized in nucleus under all zinc conditions tested. The double mutants Δzap-1; Δzrc-1, Δzap-1; Δmsc-2, and Δzap-1; Δzrg-17 showed slow growth under low zinc like Δzap-1, indicating that ZAP-1 might be functioning upstream of zrc-1, msc-2, and zrg-17. Furthermore, expression analysis of the CDF family of zinc transporter, zrc-1, msc-2, zrg-17, and zrt-3 in Δzap-1 mutant showed very low-level expressions compared to expression in wild type, indicating that the ZAP-1 transcription factor regulates the CDF zinc transporters under low zinc conditions."
Supervisor: Tamuli, Ranjan