Homoserine toxicity in Saccharomyces cerevisiae and Candida albicans homoserine kinase (thr1Delta) mutants

Abstract

In addition to threonine auxotrophy, mutation of the Saccharomyces cerevisiae threonine biosynthetic genes THR1 (encoding homoserine kinase) and THR4 (encoding threonine synthase) results in a plethora of other phenotypes. We investigated the basis for these other phenotypes and found that they are dependent on the toxic biosynthetic intermediate homoserine. Moreover, homoserine is also toxic for Candida albicans thr1Delta mutants. Since increasing levels of threonine, but not other amino acids, overcome the homoserine toxicity of thr1Delta mutants, homoserine may act as a toxic threonine analog. Homoserine-mediated lethality of thr1Delta mutants is blocked by cycloheximide, consistent with a role for protein synthesis in this lethality. We identified various proteasome and ubiquitin pathway components that either when mutated or present in high copy numbers suppressed the thr1Delta mutant homoserine toxicity. Since the doa4Delta and proteasome mutants identified have reduced ubiquitin- and/or proteasome-mediated proteolysis, the degradation of a particular protein or subset of proteins likely contributes to homoserine toxicity.

Fig. 1.

Threonine biosynthetic pathway.

Fig. 2.

Exogenous homoserine (1 mg ml⁻¹) is toxic to S. cerevisiae thr1Δ and thr4Δ and C. albicans thr1Δ mutants.

Fig. 3.

Increased flux through the threonine biosynthetic pathway is deleterious or lethal to thr1Δ mutants. (A) Deletion of THR1 is lethal in combination with a HOM3-feedback-resistant allele (HOM3^FBR). Viable colonies resulting from sporulation and tetrad dissection of strain YJK1595 (HOM3^FBR/hom3::kanMX4 THR1/thr1Δ::natMX4) were replica plated to YPD + Nat and YPD + G418 media to determine genotypes. (B) thr1Δ is synthetically lethal with fpr1Δ. Viable colonies resulting from sporulation and dissection of YJK1792 (FPR1/fpr1Δ::kanMX4 THR1/thr1ΔhphMX4) were replica plated to YPD + G418 and YPD + Hyg media. (C) P_GAL1-induced overexpression of HOM3 in thr1Δ backgrounds. Strains included YJK1012 (P_GAL1-HOM3 thr1), YJK1010 (P_GAL1-HOM3 thr1 met2), YJK1007 (P_GAL1-HOM3^FBR thr1), and YJK1004 (P_GAL1-HOM3^FBR thr1 met2).

Fig. 4.

Serine suppresses, while threonine enhances growth in homoserine-overproducing strains. Strains were applied to YPGal plates, threonine or serine (50 μl of 60 mg ml⁻¹) was added to a central filter disc, and plates were incubated at 37°C for 2 days. Strains included YAG129 (HOM3 THR1), YJK1012 (P_GAL1-HOM3 thr1), YJK1010 (P_GAL1-HOM3 thr1 met2), YJK1007 (P_GAL1-HOM3^FBR thr1), and YJK1004 (P_GAL1-HOM3^FBR thr1 met2).

Fig. 5.

Cycloheximide (CHX) blocks homoserine and threonine starvation-dependent lethality in thr1Δ mutants.

Fig. 6.

(A) Suppression of the thr1Δ mutant homoserine toxicity by doa4Δ and overexpression of ALY1 (pJO335; strain YJK1386), HUA1 (pJO338; strain YJK1388), SSH4 (pJO339; strain YJK1389), and TAT1 (pJO340; strain YJK1517). Other strains included YJK498a (thr1Δ), YJK1074 (doa4Δ), and YJK1077 (thr1Δ doa4Δ). (B) Viability following starvation in SD for thr1Δ and thr1Δ doa4Δ strains.

Fig. 7.

Suppression of the thr1Δ starvation-cidal phenotype by reduced proteasome function. Strains included YJK1766 (thr1Δ), YJK2591 (hom3Δ), YJK2596 (thr1Δ crl3-3), YJK2599 (hom3Δ crl3-3), YJK2620 (thr1Δ crl13-1), YJK2590 (hom3Δ crl13-1), YJK2613 (thr1Δ crl21-1), and YJK2614 (hom3Δ crl21-1). Starvation assays were performed with two isogenic strains for each thr1Δ crl genotype, the results of which were virtually identical, and only one strain is shown.