Figure 1.
Different pathways of sulfur incorporation into carbon chains and transsulfuration in yeast and filamentous fungal species.
Inorganic sulfide can be combined with O-acetylhomoserine or O-acetylserine to produce homocysteine and cysteine, respectively. The budding yeasts S. cerevisiae (A) has only O-acetylhomoserin pathway for sulfide incorporation, whereas the fission yeast S. pombe (B) and the filamentous fungus A. nidulans (C) possess both pathways. The present study proposes that H. polymorpha (D) has only O-acetylserine pathway.
Table 1.
Yeast strains used in this study.
Figure 2.
Schematic representation of a reconstructed sulfur pathway of H. polymorpha.
The putative sulfur metabolism pathway involved in sulfate assimilation, sulfur amino acid biosynthesis, and the methionine salvage pathway was constructed based on in-house genome information on H. polymorpha DL-1 strain (KRIBB in Korea). The nucleotide sequences of other H. polymorpha genes involved in sulfur assimilation and sulfur amino acid biosynthetic pathway were deposited under accession numbers JN676924-JN676946 (Table S2).
Figure 3.
Sequence alignment of HpCys1p (A) and HpSat1p (B) with other yeast and filamentous fungal homologs.
AnCysBp, A. nidulans cysB protein; SpCys1ap, S. pombe CYSla protein. AnCysAp, A. nidulans cysA protein; ScMet2p, S. cerevisiae MET2 protein; HpMet2p, H. polymorpha MET2 protein.
Figure 4.
Validation of the presence of the complete transsulfuration pathway by gene deletion analysis.
A set of H. polymorpha mutant strains in the sulfur assimilation and transsulfuration pathway were analyzed for their growth on B-medium supplemented with various sulfur sources. (A) Growth analysis of Hpcys1Δ on B-medium with inorganic sulfur source, NH4SO4. (B) Growth analysis of Hpcys1Δ, Hpcys1ΔHpstr2Δ, Hpcys1ΔHpstr3Δ, Hpcys1ΔHpcys3Δ, and Hpcys1ΔHpcys4abcΔ on B medium with organic sulfur compounds as sole sulfur source. (C) Growth analysis of Hpmet2Δ and Hpsat1Δ on B-medium with organic sulfur compounds as sole sulfur source.
Figure 5.
Increased sensitivity of Hpsat1Δ mutant strain to Cd, oxidative, and heat stresses.
H. polymorpha wild-type (WT), Hpmet3Δ, and Hpsat1Δ strains were spotted on YPD medium containing 0.75 µg/ml tunicamycin (TM), 1 mM CdSO4 (Cd), or 30 mM DTT and cultivated at 37°C. For heat stress, yeast cells were cultivated at 45°C.
Figure 6.
Quantitative real-time PCR analysis of transcriptional regulation of H. polymorpha genes in the sulfur pathway.
Expression of a selected set of genes was analyzed by qRT-PCR. H. polymorpha cells were grown in YPD to the exponential phase and then transferred to B-medium supplemented with the indicated sulfur compounds. After 2 hr cultivation, yeast cells were harvested and total RNA was extracted for analysis. The transcript levels of all genes were corrected according to actin levels, and the induced expression levels are shown as relative ratios to the levels of wild-type cultivated in YPD, respectively. Error bars represent standard deviation of triplicate measurements.
Figure 7.
Analysis of GSH biosynthesis by 35S-Met and 35S-Cys labeling.
S. cerevisiae (left panel) and H. polymorpha (right panel) cells were labeled with 35S-Cys (A) or 35S-Met (B) during 2 hr incubation in the presence of Cd at the concentration of 0 mM (lanes 1 and 4), 0.6 mM (lanes 2 and 5), and 2 mM (lanes 3 and 6). Intracellular 35S-labeled metabolites were extracted and separated by TLC. WT, wild-type strain; gsh1Δ, GSH1 null mutant strain. Some 35S-labeled metabolites (GSH, cystathionine, X) are indicated.