A Validated Set of Fluorescent-Protein-Based Markers for Major Organelles in Yeast (Saccharomyces cerevisiae)

Abstract

Eukaryotic cells share a basic scheme of internal organization featuring membrane-based organelles. The use of fluorescent proteins (FPs) greatly facilitated live-cell imaging of organelle dynamics and protein trafficking. One major limitation of this approach is that the fusion of an FP to a target protein can and often does compromise the function of the target protein and alter its subcellular localization. The optimization process to obtain a desirable fusion construct can be time-consuming or even unsuccessful. In this work, we set out to provide a validated set of FP-based markers for major organelles in the budding yeast (Saccharomyces cerevisiae). Out of over 160 plasmids constructed, we present a final set of 42 plasmids, the recommendations for which are backed up by meticulous evaluations. The tool set includes three colors (green, red, and blue) and covers the endoplasmic reticulum (ER), nucleus, Golgi apparatus, endosomes, vacuoles, mitochondria, peroxisomes, and lipid droplets. The fidelity of the markers was established by systematic cross-comparison and quantification. Functional assays were performed to examine the impact of marker expression on the secretory pathway, endocytic pathway, and metabolic activities of mitochondria and peroxisomes. Concomitantly, our work constitutes a reassessment of organelle identities in this model organism. Our data support the recognition that “late Golgi” and “early endosomes,” two seemingly distinct terms, denote the same compartment in yeast. Conversely, all other organelles can be visually separated from each other at the resolution of conventional light microscopy, and quantification results justify their classification as distinct entities. IMPORTANCE Cells contain elaborate internal structures. For eukaryotic cells, like those in our bodies, the internal space is compartmentalized into membrane-bound organelles, each tasked with specialized functions. Oftentimes, one needs to visualize organelles to understand a complex cellular process. Here, we provide a validated set of fluorescent protein-based markers for major organelles in budding yeast. Yeast is a commonly used model when investigating basic mechanisms shared among eukaryotes. Fluorescent proteins are produced by cells themselves, avoiding the need for expensive chemical dyes. Through extensive cross-comparison, we make sure that each of our markers labels and only labels the intended organelle. We also carefully examined if the presence of our markers has any negative impact on the functionality of the cells and found none. Our work also helps answer a related question: are the structures we see really what we think they are?

New Perspectives on SNARE Function in the Yeast Minimal Endomembrane System

“…Similarly, both FM4-64 and fluorescently labeled α-factor, a yeast mating pheromone that trafficks to the vacuole for degradation, colocalize with the Golgi marker peaking at 3 min after endocytosis, before moving to the PVE at 10 min, indicating that the Golgi serves as the early endosome in yeast [23]. Improved fluorescent markers capable of labeling key compartments continue to emerge [35], facilitating more detailed insights into the yeast endomembrane system. Most recently, a study investigating the yeast v-SNARE Snc1 has indicated that one of its interacting proteins, Rcy1, may be responsible for the delivery of endocytic plasma membrane to the TGN [1].…”

Excess diacylglycerol at the endoplasmic reticulum disrupts endomembrane homeostasis and autophagy

“…Strains and plasmids constructed in this study are listed in Additional file 2: Tables S1 & S2. Plasmids for additional organelle markers were described previously [75,76]. Restriction sites and PCR primers utilized in plasmid construction are listed in Additional file 2: Table S3.…”

“…Yeast cells in liquid culture were incubated at 30°C with shaking. Media recipes for YPD, SD-N, and SMD amino acid dropouts were described previously [76,79]. Unless otherwise noted, YPD was used to grow cells to mid-log phase and SD-N was used for nitrogen starvation.…”

“…Fluorescence microscopy, pho8Δ60 assay, and GFP-Atg8 processing assay were performed as described previously [75,76,79]. For quantification of microscopy images, the experiments were repeated at least 3 times, and a total of at least 150 cells were analyzed for each sample.…”

Genetically controlled mtDNA editing prevents ROS damage by arresting oxidative phosphorylation

“…Cox4 localises to the mitochondrial inner membrane (J. Zhu et al, 2019). We used Cox4-EGFP as a mitochondrial signal in Figure 2B and Figure S3A.…”