A PLAC8-containing protein from an endomycorrhizal fungus confers cadmium resistance to yeast cells by interacting with Mlh3p

Abstract

Cadmium is a genotoxic pollutant known to target proteins that are involved in DNA repair and in antioxidant defence, altering their functions and ultimately causing mutagenic and carcinogenic effects. We have identified a PLAC8 domain-containing protein, named OmFCR, by a yeast functional screen aimed at identifying genes involved in cadmium resistance in the endomycorrhizal fungus Oidiodendron maius. OmFCR shows a remarkable specificity in mediating cadmium resistance. Both its function and its nuclear localization in yeast strictly depend on the interaction with Mlh3p, a subunit of the mismatch repair (MMR) system. Although proteins belonging to the PLAC8 family are widespread in eukaryotes, they are poorly characterized and their biological role still remains elusive. Our work represents the first report about the potential role of a PLAC8 protein in physically coupling DNA lesion recognition by the MMR system to appropriate effectors that affect cell cycle checkpoint pathways. On the basis of cell survival assays and yeast growth curves, we hypothesize that, upon cadmium exposure, OmFCR might promote a higher rate of cell division as compared to control cells.

Figure 1.

Sensitivity of S. cerevisiae strains to cadmium on solid medium. Each yeast strain was transformed with the empty vector (pFL61) and the OmFCR construct (OmFCR) and plated in three serial dilutions onto SD medium with (right column) or without (control, left column) CdSO₄. The concentrations of cadmium used in spot assays are indicated above each row. Plates were incubated at 30°C for 6 days. (A) DY wild-type strain and its isogenic yap1 mutant, BY4741 wild-type strain and three isogenic mutants (hog1, skn7, cup1) on control and cadmium-amended medium. (B) EY39 wild-type strain and the isogenic mlh3 mutant on control and 50 µM cadmium-amended medium. (C) W303 wild-type strain, the isogenic mec1 sml1 double mutant, rad9 mutant and dun1 mutant on control and 10 µM cadmium-amended medium.

Figure 2.

Sequence alignment of four PLAC8 proteins with OmFCR. The Pongo abelii onzin (NP_001124833.1), the Zea mays CNR10 (NM_001157656.1), the Arabidopsis thaliana AtPCR3 (NM_112731) and the A. thaliana AtMCA1 (AB196960.1) were aligned with OmFCR by T-Coffee and visualized by Multiple Align Show. AtMCA1, being a much longer protein in comparison to the other four, was aligned including only its PLAC8 domain. Black-shaded amino acids are identical, gray-shaded amino acids are functionally equivalent. Arrows point to the conserved amino acids which were primarily used as targets for oligonucleotide-directed site-specific mutagenesis. The box shows the PLAC8 domain.

Figure 3.

Localization of OmFCR in cells expressing the OmFCR-EGFP construct. Panels A–F: yap1 mutant cells. (A) Cells are shown in transmitted light. (B) OmFCR-EGFP localization is seen as a single green spot within the cell. (C) DNA is stained in blue with DAPI. (D) Both probes label the nucleus, as seen in the superimposition of the signals. (E) OmFCR-EGFP nuclear localization after 24 h of cadmium exposure. (F) EGFP cytoplasmic localization. (G) In EY39 wild-type cells, the OmFCR-EGFP fluorescence is similar to the one observed in panel B. (H) In mlh3 cells, OmFCR-EGFP localization is observed as a diffused cytoplasmic staining. Scale bars: 10 µm.

Figure 4.

Yeast growth curves and cell survival percentages of unexposed and Cd-exposed cell cultures. (A) The optical densities of OmFCR- and pFL61-ycf1 cell cultures was measured after 2, 3, 7, 24, 48 and 120 h of incubation at 30°C, either with or without 50 µM CdSO₄. Only treated cell cultures were also sampled at 120 h, because untreated cultures reached saturation within 48 h. After 7 h of incubation, the OD₆₀₀ difference between Cd-treated OmFCR-ycf1 cells (OmFCR_Cd) and Cd-treated pFL61-ycf1 cells (pFL61_Cd) began to be highly significant (P < 0.01) (inset). The first points of Cd-treated OmFCR- and pFL61-growth curves were shown in details (inset). (B) From 7 h of incubation on, cell survival of Cd-treated cultures was measured and expressed as a percentage of the cell survival of the corresponding untreated cell cultures, which was demonstrated to be 100%. Different letters above each bar indicate statistically significant differences (P < 0.05).

Figure 5.

Working model for OmFCR. The genotoxic stress caused by cadmium might recruit the MMR system, which, in its turn, might promote the firing of OmFCR through protein–protein interactions with Mlh3p. The signaling pathway promoted by OmFCR appears to merge with the final part of the Mec1p-dependent phosphorylation cascade, at the Rad53p/Dun1p level. In pFL61-transformed cells (left) Dun1p is likely to recruit effector proteins that cause cell cycle arrest, while the presence of OmFCR (right) might enlist alternative effector proteins that ultimately allow the progression of cell division.