Mutation of high-affinity methionine permease contributes to selenomethionyl protein production in Saccharomyces cerevisiae

Abstract

The production of selenomethionine (SeMet) derivatives of recombinant proteins allows phase determination by single-wavelength or multiwavelength anomalous dispersion phasing in X-ray crystallography, and this popular approach has permitted the crystal structures of numerous proteins to be determined. Although yeast is an ideal host for the production of large amounts of eukaryotic proteins that require posttranslational modification, the toxic effects of SeMet often interfere with the preparation of protein derivatives containing this compound. We previously isolated a mutant strain (SMR-94) of the methylotrophic yeast Pichia pastoris that is resistant to both SeMet and selenate and demonstrated its applicability for the production of proteins suitable for X-ray crystallographic analysis. However, the molecular basis for resistance to SeMet by the SMR-94 strain remains unclear. Here, we report the characterization of SeMet-resistant mutants of Saccharomyces cerevisiae and the identification of a mutant allele of the MUP1 gene encoding high-affinity methionine permease, which confers SeMet resistance. Although the total methionine uptake by the mup1 mutant (the SRY5-7 strain) decreased to 47% of the wild-type level, it was able to incorporate SeMet into the overexpressed epidermal growth factor peptide with 73% occupancy, indicating the importance of the moderate uptake of SeMet by amino acid permeases other than Mup1p for the alleviation of SeMet toxicity. In addition, under standard culture conditions, the mup1 mutant showed higher productivity of the SeMet derivative relative to other SeMet-resistant mutants. Based on these results, we conclude that the mup1 mutant would be useful for the preparation of selenomethionyl proteins for X-ray crystallography.

FIG. 1.

Metabolic pathways of sulfur compounds in S. cerevisiae. The main sulfur compounds are methionine, S-adenosylmethionine, and cysteine, which are involved in protein synthesis and sulfur metabolism regulation. The S-adenosylmethionine also participates in the methylation of nucleic acids, proteins, and lipids as a methyl group donor and in the biosynthesis of biotin and polyamines. Glutathione plays a pivotal role in redox homeostasis.

FIG. 2.

Selection and growth phenotypes of SeMet-resistant mutants. The haploid and backcrossed mutants were incubated on SC-Met medium supplemented with or without 0.05 mM SeMet for 3 days at 30°C (A) and on YPAD medium supplemented with or without 10 mM selenate for 2 days at 30°C (B). Twelve tetrads obtained from SRY5-3d (C) and SRY5-7d (D) were incubated on SC-Met medium containing 0.05 mM SeMet for 3 days at 30°C, indicating a 2:2 segregation of viable to nonviable spores in the presence of SeMet.

FIG. 3.

Identification of mutant alleles responsible for SeMet resistance. (A) The SRY5-3 strain was transformed with a centromeric plasmid (URA3 marker) expressing the SAM1 gene under the control of an endogenous promoter. The wild-type and SRY5-3 strains were transformed with empty vector as a control. The transformants were incubated on SC-Met/Ura plates supplemented with or without 0.05 mM SeMet for 3 days at 30°C or on YPAD plates with or without 10 mM selenate for 2day at 30°C. (B) Schematic representation of the mutant protein encoded by sam1-224. The position of the mutation changing Gly-267 to Cys is indicated. (C) Effect of the null mutation of MET6 or MET17 on selenate resistance in the SRY5-7 mutant. Each strain was incubated on SC medium limited in the concentration of Met (5 μg/ml) and supplemented with or without 10 mM selenate for 2 days at 30°C. (D) SRY5-7 was transformed with a centromeric plasmid expressing the MUP1 gene from an endogenous promoter and with the empty vector as a control. The transformants were incubated under the conditions identical to those shown in panel A. (E) Schematic representation of the mutant protein encoded by mup1-100. The position of the mutation changing Met-74 to Ile is indicated.

FIG. 4.

Repression of mRNAs for proteins involved in sulfate assimilation in the presence of Met and AdoMet. The wild-type (left), SRY5-7 (middle), and SRY5-3 (right) strains were grown in SC-Met medium until mid-log phase and then exposed to 0.25 mM Met or AdoMet for 30 min. The expression of MET3, MET14, and MET17 was determined by Northern blot analysis. An ACT1 probe was used as a loading control.

FIG. 5.

Total uptake of Met by the wild-type and SRY5-7 (mup1-100) strains. After cells were cultured in SC-Met medium until mid-log phase, 0.25 mM radiolabeled Met (30.2 kBq) was added, and uptake was measured at the indicated time points. The mean values of Met uptakes obtained from two independent experiments are plotted.

FIG. 6.

Evaluation of recombinant protein yield and SeMet incorporation by wild-type and SRY5-7 cells cultured with SeMet. Yeast strains harboring the plasmid pGAL-L123M were cultured with various concentration of SeMet. The L123M EGF peptides in the culture media were analyzed by reverse-phase chromatography after purification by using an Ni-NTA column, as described in Materials and Methods. Peaks A and B were assigned as the L123M peptides containing Met and SeMet, respectively, by mass spectrometry (see Fig. S1 in the supplemental material). Peaks with asterisks show unidentified compounds. SeMet concentration and OD₆₀₀ values at induction and at harvest are indicated below each chromatogram.

FIG. 7.

(A) The double mutation of mup1-100 and sam1-224 increased SeMet resistance of S. cerevisiae. A serial dilution of TKY371-4A (mup1-100 sam1-224) cells harboring various combinations of pRS315-MUP1, pRS316-SAM1, and empty vectors was spotted on SC-Met/Leu/Ura medium with or without 0.05 mM SeMet for 1 day at 30°C. (B) Tricine-SDS-PAGE analysis of the L123M peptide expressed in the presence of 0.25 mM SeMet by SeMet-resistant mutants. The samples from Ni-NTA affinity chromatography were separated on a Tricine gel (15 to 20%; Wako), and protein bands were visualized by silver staining. The OD₆₀₀ values at induction and harvest are indicated.