Studies on the Properties of the Sporulation Specific Protein Dit1 and its Product Formyl Tyrosine

Abstract

The dityrosine layer is a unique structure present in the spore wall of the budding yeast Saccharomyces cerevisiae. The primary constituent of this layer is bisformyl dityrosine. A sporulation-specific protein, Dit1 is localized in the spore cytosol and produces a precursor of bisformyl dityrosine. Although Dit1 is similar to isocyanide synthases, the loss of Dit1 is not rescued by heterologous expression of the Pseudomonas aeruginosa isocyanide synthase, PvcA, indicating that Dit1 does not mediate isocyanidation. The product of Dit1 is most likely formyl tyrosine. Dit1 can produce its product when it is expressed in vegetative cells; however, formyl tyrosine was not detected in the crude cell lysate. We reasoned that formyl tyrosine is unstable and reacts with some molecule to form formyl tyrosine-containing molecules in the cell lysate. In support of this hypothesis, formyl tyrosine was detected when the lysate was hydrolyzed with a mild acid. The same property was also found for bisformyl dityrosine. Bisformyl dityrosine molecules assemble to form the dityrosine layer by an unknown mechanism. Given that bisformyl dityrosine can be released from the spore wall by mild hydrolysis, the process of formyl tyrosine-containing molecule formation may resemble the assembly of the dityrosine layer.

Keywords: Dit1; Saccharomyces cerevisiae; dityrosine layer; formyl tyrosine; spore wall.

Figure 1

Heterologous expression of P. aeruginosa PvcA in yeast cells. (A) Wild-type (WT) or dit1Δ spores harboring pRS316TEF-PvcA (PvcA) or pRS316TEF (Vector), were stained with CFW and observed by bright field (BF) or florescence microscopy (CFW). Bar, 5 µm. (B) Lysates of dit1Δ spores harboring pRS316TEF-PvcA (PvcA) and pRS316TEF (Vector) were hydrolyzed with 6 N HCl and subjected to HPLC analysis. Dityrosine (di-Tyr) is shown as a control. (C) Lysates of wild-type spores harboring pRS316TEF-PvcA (PvcA) or pRS316TEF (Vector) were hydrolyzed with 6 N HCl and subjected to HPLC analysis. Dityrosine (di-Tyr) was assayed as a control. (D) Lysates of wild-type spores harboring pRS316TEF-PvcA (PvcA) or pRS316TEF (Vector) were hydrolyzed with 6 N HCl and subjected to HPLC analysis. The relative amount of dityrosine detected by HPLC is shown. The data presented are the mean ± SE of six independent samples. * p < 0.05.

Figure 2

Analysis of Dit1 localization. (A) A diploid strain in which the chromosomal DIT1 genes were fused to GFP. Vegetative growing cells (Vege) and spores of the DIT1-GFP harboring cells were lysed and subjected to Western blotting analysis using anti-GFP antibodies. (B) Vegetative growing cells (Vege) and spores of the DIT1-GFP harboring cells were observed by bright field (BF) and florescence microscopy (GFP). Bar, 5 µm.

Figure 3

Ectopic expression of Dit1 in vegetative cells and analysis of formyl tyrosine production. (A) Wild-type vegetative cells harboring pRS424GAL1 (Vector) or pRS424GAL1-DIT1-GFP (+Dit1-GFP) were cultured in galactose-containing media and observed by bright field (BF) or florescence microscopy (GFP). Bar, 5 µm. (B) Lysates from wild-type vegetative cells harboring pRS424GAL1-DIT1-GFP cultured in glucose (Glc) or galactose (Gal) containing media were subjected to Western blotting analysis using anti-GFP antibodies. (C) Lysates of vegetative cells harboring pRS424GAL1 (Vector) or pRS424GAL1-DIT1-GFP (Dit1) were analyzed by HPLC. Stars indicate tyrosine peaks. Tyrosine (Tyr) and formyl tyrosine (f-Tyr) are shown as controls.

Figure 4

Dit1-GFP is active in vegetative cells. (A) Lysates from wild-type vegetative cells harboring pRS424GAL1-DIT1-GFP (Dit1-GFP) and/or pRS426GAL1-DIT2-FlAG (Dit2-FLAG) were subjected to Western blotting analysis using anti-GFP antibodies and anti-FLAG antibodies. (B) Lysates of vegetative cells harboring pRS424GAL1-DIT1-GFP (Dit1) and/or pRS426GAL1-DIT2-FlAG (Dit2) were treated with or without (no treatment) 6 N HCl and subjected to HPLC analysis. Dityrosine (di-Tyr) and formyl dityrosine (f-di-Tyr) are shown as controls.

Figure 5

In vitro Dit2 assay to assess the presence of formyl tyrosine in Dit1-expressing cells. (A) Vegetative cells harboring pRS426GAL1-DIT2-FlAG (Dit2) or pRS426GAL1 (Vector) were permeabilized or lysed and incubated with formyl tyrosine (f-Tyr). The reaction mixtures were hydrolyzed with 6 N HCl and analyzed by HPLC. Di-tyrosine (di-Tyr) is shown as a control. Arrows indicate dityrosine peaks. (B) Lysates of vegetative cells harboring pRS424GAL1-DIT1-GFP (Dit1) were permeabilized or lysed and mixed with lysates of vegetative cells harboring pRS426GAL1-DIT2-FlAG (Dit2). The mixtures were incubated with tyrosine. The reaction mixtures were hydrolyzed with 6 N HCl and analyzed by HPLC. Dityrosine (di-Tyr) is shown as a control. Arrows indicate dityrosine peaks.

Figure 6

Trisodium citrate treatment of yeast lysates and formyl tyrosine detection. (A) The lysate of vegetative cells harboring pRS424GAL1-DIT1-GFP (Dit1) or pRS424GAL1 (Vector) were treated with trisodium citrate and subjected to HPLC analysis. Formyl tyrosine (f-Tyr) is shown as a control. Arrows indicate formyl tyrosine peaks. (B) The formyl tyrosine peak detected in the lysates of vegetative cells harboring pRS424GAL1-DIT1-GFP was collected, hydrolyzed with 6 N HCl and subjected to HPLC analysis. Tyrosine (Tyr) and formyl tyrosine (f-Tyr) are shown as controls.

Figure 7

Bisformyl dityrosine detection in vegetative cells coexpressing Dit1 and Dit2. (A) The lysate of vegetative cells harboring pRS424GAL1-DIT1-GFP and pRS426GAL1-DIT2-FlAG (Dit1 and Dit2), or pRS424GAL1 and pRS426GAL1 (Vector) were treated with or without (no treatment) trisodium citrate and subjected to HPLC analysis. Formyl dityrosine (f-di-Tyr) is shown as a control. Arrows indicate formyl dityrosine peaks. (B) The formyl dityrosine peak detected in the lysates of vegetative cells harboring pRS424GAL1-DIT1-GFP and pRS426GAL1-DIT2-FlAG was collected, hydrolyzed with 6 N HCl and subjected to HPLC analysis. Dityrosine (di-Tyr) and formyl dityrosine (f-di-Tyr) are shown as controls.