Visualizing Yeast Organelles with Fluorescent Protein Markers

Subcellular compartmentalization into membrane-bound organelles is a common principle in the organization of eukaryotic cells. Each organelle fulfills specific functions. Like in many other aspects of eukaryotic biology, the budding yeast, Saccharomyces cerevisiae, has been a classic model system in elucidating the basic principles of organelle organization and dynamics. Examples include the seminal discoveries in the protein secretion pathway, the peroxisomal protein import pathway, and the autophagy pathway^1,2,3.

In typical nutrient-rich conditions, fast-growing yeast cells contain endoplasmic reticulum (ER), early Golgi, late Golgi/early endosomes, late endosomes, vacuoles, and mitochondria. Some peroxisomes, lipid droplets, and autophagosomes (even fewer than the first two, mainly of the Cvt vesicle type, which are present in nutrient-rich conditions⁴) are also present, but not as prominent as it would be under specific culturing conditions (lipid-rich media, starvation media, etc.). Compared to other common eukaryotic models, yeast cells are quite small; the diameter of a typical yeast cell is around 5 µm, compared to tens of micrometers for most animal and plant cells. As a result, in the same imaging field that normally contains a single adherent animal cell, one normally sees tens of yeast cells at various cell cycle stages. Besides the size difference, yeast organelle morphology also has some peculiar features. At the ultrastructural level, yeast ER is composed of sheets and tubules, like in other systems. Under fluorescent microscopy, yeast ER manifests as two rings with some interconnecting structures in between. The inner ring is the nuclear ER, which is continuous with the nuclear envelope, and the outer ring is the peripheral ER, which is a tubular network lying beneath the plasma membrane⁵. Similar to plant cells but different from animal cells, a hybrid organelle, the late Golgi/early endosome, sits at the intersection between the secretory pathway and endocytic pathway^6,7. Morphologically, yeast Golgi apparatuses are dispersed in the cytoplasm. Vacuoles are functionally analogous to lysosomes in animal cells. They often occupy large portions of the cytoplasm and undergo frequent fission and fusion. Besides the use of fluorescent colocalization markers, the vacuolar membrane can be distinguished from the nuclear ER by at least two criteria: The vacuolar membrane is generally more rounded than the nuclear ER, and the concaving appearance of the vacuole in DIC is also more pronounced than that of the nucleus.

Routinely, we use a set of fluorescent protein-based markers to visualize the aforementioned organelles in live yeast cells (Table 1). The fidelity and functionality of these organelle markers have been experimentally verified^7,8. These marker constructs are intended to introduce fluorescent protein chimera cassettes into the yeast genome. As outlined below, in preparation for yeast transformation, linear DNA fragments are generated either by enzymatic digestion or PCR amplification^7,8. The linear DNA fragments get integrated into the genome via homologous recombination. For plasmids described in this protocol, three types of design are employed. In the first type, covering the majority of the plasmids, it is often possible to obtain transformants carrying multiple copies of the construct. This is usually undesired because it introduces expressional and possibly functional variations across transformants. Single-copy transformants need to be identified through imaging as described in this protocol, by immunoblotting or by carefully designed PCR tests. In the second type, covering GFP-Sed5, GFP-Pep12, and GFP-Atg8, only single-copy integration is produced in haploid yeast cells. Both the first type and the second type keep the endogenous copy of the marker gene intact in the genome. A third type of plasmid design, covering Sec7-2GFP and Vph1-2GFP, is intended to introduce C-terminal knock-ins, leading to the chimeras being the sole copy of the corresponding marker gene.

Here we describe the procedure to utilize these organelle markers, provide exemplary microscopy images, and discuss precautions geared toward researchers new to yeast organelle imaging.