Development of fluorescent localization reporter strains of the budding yeast

Abstract:

[Objective] To facilitate the localization of proteins in budding yeast, we developed yeast reporter strains expressing fusion proteins that localize to specific subcellular structures. [Methods] The yeast expression vectors expressed organelle-specific proteins tagged with much brighter variant red fluorescent protein (RedStar) at their carboxy terminal were constructed and transferred into yeast cells to generate the reporter strain collection. To this end, the yeast codon that optimized coding sequence of RedStar were in-frame inserted immediately preceding the stop codon of each ORF that controlled by their endogenous promoters. Each strain was verified by PCR and sequencing, and then analyzed by fluorescence microscopy. Co-localization of DNA-binding dyes (DAPI), Mitotracker (mitochondria) and SipA from Salmonella enteritidis in the corresponding reporter strain were also performed. [Results] The yeast strain collection containing indicators for the actin, endoplasmic reticulum, nuclei, mitochondria, peroxisomes, lipids, endosomes, and the Golgi apparatus were constructed successfully. [Conclusion] Our studies provide a fundamental tool for observing the dynamic changes of organelles and identifying the localization of unknown proteins in yeast.