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.

• PMID: 38682313
• DOI full text
• PMC full text
• PubMed
• PubTator

Reference Type

Journal Article

Authors

Brettrager EJ, Frederick AJ, van Waardenburg RCAM

Primary Lit For

Additional Lit For

Review For