A high-throughput method to globally study the organelle morphology in S. cerevisiae

High-throughput methods to examine protein localization or organelle morphology is an effective tool for studying protein interactions and can help achieve an comprehensive understanding of molecular pathways. In Saccharomyces cerevisiae, with the development of the non-essential gene deletion array, we can globally study the morphology of different organelles like the endoplasmic reticulum (ER) and the mitochondria using GFP (or variant)-markers in different gene backgrounds. However, incorporating GFP markers in each single mutant individually is a labor-intensive process. Here, we describe a procedure that is routinely used in our laboratory. By using a robotic system to handle high-density yeast arrays and drug selection techniques, we can significantly shorten the time required and improve reproducibility. In brief, we cross a GFP-tagged mitochondrial marker (Apc1-GFP) to a high-density array of 4,672 nonessential gene deletion mutants by robotic replica pinning. Through diploid selection, sporulation, germination and dual marker selection, we recover both alleles. As a result, each haploid single mutant contains Apc1-GFP incorporated at its genomic locus. Now, we can study the morphology of mitochondria in all non-essential mutant background. Using this high-throughput approach, we can conveniently study and delineate the pathways and genes involved in the inheritance and the formation of organelles in a genome-wide setting.

2. Incorporating Acp1-GFP in the DMA: The following protocol for making a GFP array is adapted from the Synthetic Genetic Analysis (SGA) technology (Tong and Boone, 2006) with modifications. All of the replica pinning steps are done by using a Singer ROTOR high-density arraying robotic system. Alternatively, manual pinning or a liquid-based high-density arraying robot may be used. Acronyms: YPD, yeast extract-peptone-dextrose; SD, synthetic dropout; LRK, Lue/Arg/Lys dropout media; LHRK, Lue/His/Arg/Lys dropout media Preparation 1. Grow a lawn of Acp1-GFP cells by pouring a 5 ml of overnight Acp1-GFP culture onto a YPD plate. Allow the plate to dry sufficiently by a flame or in a biological fume hood. 2. Incubate the plate at 30˚C overnight. 3. Using a robot, array Acp1-GFP strain into 1536 colonies per plate. At the same time, prepare a fresh copy of the DMA by replica pinning to YPD + G418 plates. 4. Incubate the plates at 30˚C overnight.
Mating and diploid selection 5. Mate the Acp1-GFP strain with the DMA by replica pinning and over-laying the colonies on YPD plates. 6. Incubate the plates at 30˚C overnight. 7. Replica-plate the colonies onto SD -His + G418 plates to select diploid cells (MATa/MATalpha). 8. Incubate the plates at 30˚C overnight.
Sporulationg and germination 9. Replica-plate the diploid colonies on enhanced sporulation media, which contains reduced levels of carbon and nitrogen sources to induce the formation of meiotic spores. 10. Incubate the plates at 25˚C for a minimum of 5 days. 11. Germinate the MATa meiotic progenies by replica-plating the colonies onto LRK media and incubate the plates at 30˚C for two days. 12. This media will induce the germination of haploid cells from spores. Since a LUE2 opening reading frame (ORF) is integrated at the MATa-specific STE2 promoter, all the haploids germinated will be MATa. Selection of both alleles 13. It is now necessary to select both the GFP allele and the single mutant allele. To accomplish this, MATa cells are replica-plated onto LRK + G418 media, which selects for haploid cells that carry the gene deletion (xxx::kanR). 14. Incubate the plates at 30˚C overnight.
The following protocol for making a GFP array is adapted from the Synthetic Genetic Analysis (SGA) technology (Tong and Boone, 2006) with modifications. All of the replica pinning steps are done by using a Singer ROTOR high-density arraying robotic system. Alternatively, manual pinning or a liquid-based high-density arraying robot may be used. Acronyms: YPD, yeast extract-peptone-dextrose; SD, synthetic dropout; LRK, Lue/Arg/Lys dropout media; LHRK, Lue/His/Arg/Lys dropout media Preparation 3. Microscopy: 1. Pick the desired mutant(s) expressing Acp1-GFP directly from plate. 2. Grow cells at 30°C in YPD or SD -His media to early log phase. 3. Visualize by confocal microscopy using the GFP filterset. An example of expected result is show in Figure 1.

Add in order Amount
Yeast nitrogenous base with ammonium sulfate 2.7 g Amino acid mix 0.

Discussion
This method can help efficiently incorporate a mitochondrial marker, Acp1-GFP into various mutant backgrounds. It relies on the use of a robotic system, and can easily adopted for use with any robotic system. This procedure can be also used for incorporating other types of markers. For example, to visualize ER, we routinely use the marker Erg11-GFP. In our representative images, a mutant and a wild type with the Acp1-GFP maker were visualiz ed by confocal microscopy to study the mitochondria morphology. The mutant showed disrupted mitochondrial phenotype, and is therefore a good candidate for further investigation.