Zymolyase Treatment of Saccharomyces cerevisiae Affects Cellular Proteins and Degrades Tyrosyl-DNA Phosphodiesterase I

Abstract

Saccharomyces cerevisiae is a genetically tractable, affordable, and extensively documented eukaryotic single-cell model organism. This budding yeast is amenable for the development of genetic and biochemical experiments and is frequently used to investigate the function, activity, and mechanism of mammalian proteins. However, yeast contains a cell wall that hinders select assays including organelle isolation. Lytic enzymes, with Zymolyase as the most effective and frequently used tool, are utilized to weaken the yeast cell wall resulting in yeast spheroplasts. Spheroplasts are easily lysed by, for example, osmotic-shock conditions to isolate yeast nuclei or mitochondria. However, during our studies of the DNA repair enzyme tyrosyl-DNA phosphodiesterase I (Tdp1), we encountered a negative effect of Zymolyase. We observed that Zymolyase treatment affected the steady-state protein levels of Tdp1. This was revealed by inconsistencies in technical and biological replicate lysates of plasmid-born galactose-induced expression of Tdp1. This off-target effect of Zymolyase is rarely discussed in articles and affects a select number of intracellular proteins, including transcription factors and assays such as chromatin immunoprecipitations. Following extensive troubleshooting, we concluded that the culprit is the Ser-protease, Zymolyase B, component of the Zymolyase enzyme mixture that causes the degradation of Tdp1. In this study, we report the protocols we have used, and our final protocol with an easy, affordable adaptation to any assay/protocol involving Zymolyase.

Keywords: TDP1; Zymolyase; budding yeast; nuclei; protein degradation; spheroplasts.

FIG. 1.

Tdp1 degradation following Zymolyase treatment to isolate nuclei. Galactose-induced exponentially growing cultures of top1Δ, tdp1Δ (KWY4) cells cotransformed with vector control YCpGAL1•U and control vector YCpGAL1•L (tdp1Δ) or the indicated YCpGAL1-TDP1•L plasmid were split in two for total protein isolation (lysed by glass-bead method) and isolation of nuclear extracts following generation of spheroplasts. The spheroplast-cells underwent nondenaturing lysis to isolate nuclear extracts. Furthermore, 40 μg/lane of each lysate was resolved with 10% Bis-Tris SDS-PAGE, immunoblotted, stained for yeast Tdp1, and then stripped and restained for histone H3 as loading control. FL: Full-length Tdp1 protein. The boxes represent the degraded Tdp1 protein products. The tdp1Δ lanes show the nonspecific immunostaining by the yeast Tdp1 antibody.

FIG. 2.

Zymolyase treatment causes loss of Tdp1 immuno-signal. Two technical replicates of total cell extract from galactose-induced exponentially growing cultures of top1Δ, tdp1Δ (KWY4) cells cotransformed with vector control YCpGAL1•U and YCpGAL1-TDP1•L plasmid were treated with and without Zymolyase. Furthermore, 40 μg/lane of each lysate was resolved following 10% Bis-Tris SDS-PAGE, immunoblotted, stained for yeast Tdp1, and stripped and stained for histone H3 as loading control.

FIG. 3.

Ser-protease inhibitor prevents Zymolyase-facilitated degradation of intracellular Tdp1. Galactose-induced exponentially growing top1Δ, tdp1Δ (KWY4) cells cotransformed with vector control YCpGAL1•U and YCpGAL1-TDP1•L plasmid cultures were aliquoted followed by total protein lysate isolation from cells treated without Zymolyase (Pre-Zym), and from cells that were treated with (+) and without (−) PMSF pretreated Zymolyase, incubated at room temperature (RT) or 37°C for 10 or 30 min. Furthermore, 40 μg/lane of each lysate was resolved on 10% Bis-Tris SDS-PAGE, immunoblotted, stained for Tdp1, and subsequently stripped and stained for GAPDH as loading control.

FIG. 4.

Effect of Zymolyase treatment on total cellular protein. Wild-type cells (MGY250) grown in galactose for 6 h were divided into 5 aliquots. Total protein lysates were isolated from cells pretreatment (Pre), cells treated without Zymolyase (−) incubated at 30°C and 37°C for 60 min, and from cell aliquots that were treated with Zymolyase (+) and Zymolyase pretreated with PMSF (+/PMSF), incubated at 37°C for 60 min as indicated. Furthermore, 40 μg/lane of each sample was resolved on 10% Bis-Tris SDS-PAGE and stained with SYPRO Ruby.

FIG. 5.

Effect of Zymolyase treatment on Tdp1, Top1, histone H3, and GAPDH. Galactose-induced exponentially growing top1Δ, tdp1Δ (KWY4) cells transformed with vector control YCpGAL1•U (vector) or YCpGAL1-TDP1•U (Tdp1) or YCpGAL1-TOP1•U (Topo1) plasmid. The cultures were aliquoted, and spheroplasts were generated with (+) and without (−) PMSF pretreated Zymolyase for 60 min at 37°C. Furthermore, 40 μg/lane of each spheroplast lysate was resolved on 10% Bis-Tris SDS-PAGE, immunoblotted, stained with anti-yTDP1 or anti-yTOPO1, and subsequently stripped and stained with anti-GAPDH or anti-histone H3, respectively. Tdp1-stained blot (α-TDP1): FL-Arrow: points at the full-length Tdp1 protein band and the nonspecific reactive bands caused by the anti-yTdp1 antibody are shown in the vector and Topo1 lanes.