Msn5p Is Involved in Formaldehyde Resistance but Not in Oxidative Stress Response in the Methylotrophic YeastCandida boidinii

Abstract

Methylotrophic yeasts, which can utilize methanol as sole carbon and energy source, are exposed to two toxic metabolic intermediates, formaldehyde and hydrogen peroxide, during growth on methanol. Here we report that Msn5p, an importin-β family nuclear exporter, participated in the formaldehyde resistance mechanism but not in the hydrogen peroxide resistance mechanism in Candida boidinii. Disruption of the MSN5 gene in this yeast caused retardation of growth on formaldehyde-generating growth substrates such as methanol and methylamine, but the expression levels of the methanol-metabolizing enzymes did not fall. The Msn5p-depleted strain was sensitive to formaldehyde but not to hydrogen peroxide. Furthermore, a yellow fluorescent protein-tagged Msn5p was diffuse in the cytoplasm of C. boidinii when the cells were treated with high concentrations of formaldehyde or ethanol, but was predominantly associated with the nuclei following treatment with hydrogen peroxide.

Molecular characterization of Candida boidinii MIG1 and its role in the regulation of methanol‐inducible gene expression

“…In the Cbmsn5Δ strain, CbMig1p–YFP was constitutively localized to the nuclei, even in the presence of methanol or glycerol (Figure f, g). However, in the Cbmsn5Δ strain, the AOD1 gene seems to be normally expressed (Zhai et al , ). These results suggest that, if CbMig1p is phosphorylated under non‐fermentable carbon source conditions, as in the case with ScMig1p, phosphorylated CbMig1p does not function as a repressor even though it was localized to the nucleus in the Cbmsn5Δ strain.…”

“…Then, PCR was performed with the primers Pst I–YFP‐Fw and Nhe I–YFP‐Rv, using pTYT as the template (Sasano et al , ). The 5.4 kb Sac I– Spe I fragment of pAPU, the 3.4 kb Sac I– Pst I fragment of the promoter and coding regions of CbMIG1 and the 0.7 kb Pst I– Nhe I fragment of the amplified YFP coding region were ligated to yield pCbMig1–YFP, which was linearized with Aor51HI and used to transform the Cbmig1Δ ura 3 strain and Cbmsn5Δ ura 3 (Zhai et al , ). The resulting strains were named CbMig1p–YFP/ mig1Δ and CbMig1p–YFP/ msn5Δ .…”

“…YFP coding region were ligated to yield pCbMig1-YFP, which was linearized with Aor51HI and used to transform the Cbmig1Δ ura3 strain and Cbmsn5Δ ura3 (Zhai et al, 2012). The resulting strains were named CbMig1p-YFP/mig1Δ and CbMig1p-YFP/msn5Δ.…”

Biological Significance of the Importin‐β Family‐Dependent Nucleocytoplasmic Transport Pathways

“…Candida boidinii is a methylotrophic yeast and, therefore, is exposed to toxic metabolic intermediates, such as formaldehyde and hydrogen peroxide, during growth on methanol. When C. boidinii is treated with formaldehyde or hydrogen peroxide, Msn5p diffuses into the cytoplasm or associates with the nuclei, respectively . This indicates that Msn5p responds to formaldehyde, but not to hydrogen peroxide.…”