A Validated Set of Ascorbate Peroxidase-Based Organelle Markers for Electron Microscopy of Saccharomyces cerevisiae

Abstract

ABSTRACT Genetically encoded tags, such as engineered ascorbate peroxidase APEX2, offer unique advantages for the specific labeling of subcellular structures in electron microscopy (EM). However, the use of APEX2 in EM investigation of yeast has been limited. Here we describe the development of APEX2-based organelle markers for Saccharomyces cerevisiae. We found that with regard to APEX2 -catalyzed formation of diaminobenzidine precipitation, cell wall removal was not essential during sample preparation, yet the presence of fluorescent proteins in APEX2 chimeras had a negative impact. We showed that major organelles including endoplasmic reticulum, early Golgi, late Golgi/early endosomes, late endosomes, mitochondria, peroxisomes, and lipid droplets could be labeled by appropriate APEX2 chimeras. The subcellular localization of our APEX2 chimeras was verified by EM visualization and supplemented with immunofluorescence colocalization analysis when necessary, validating their feasibility as organelle markers. IMPORTANCE Yeast is an excellent single cellular model system for studying basic cellular processes. However, yeast cells are much smaller than most animal and plant cells, making the observation and recognition of yeast subcellular structures challenging. Here we developed a set of yeast organelle markers for use in electron microscopy and documented our technical approach for using this method.

Keywords

• organelle
• ascorbate peroxidase
• fluorescent protein
• electron microscopy
• immunofluorescence
• yeast