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

Summary

GFP-fusion proteins are widely used to visualize organelles by confocal microscopy. However, screening for mutations that affect the morphology of organelles generally requires individual mutagenesis and is time consuming. Here, we demonstrate a method to simultaneously incorporate organelle-GFP markers in almost 5,000 non-essential genes in yeast.

Abstract

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.

Protocol

Materials and methods:

1. Yeast strains:
   • Acp1-GFP (GFP::His): C-terminal GFP-tagging of Acp1 was generated by a PCR-mediated homologous recombination using plasmid pKT128 (contains GFP and HIS5). Positive transformants were confirmed by confocal microscopy and colony PCR. The strain background was BY7043 (MAT alpha can1∆::STE2pr-lue2 lyp1∆ his3∆1 leu2∆0 ura3∆0 met15∆0) from Boone lab (Tong and Boone, 2006).
   • Deletion Mutant Array (DMA) (xxx::KanR): it is an array of about 5,000 deletion mutants of non-essential genes. All of these nonessential genes were knocked out on G418. The stra....

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 s.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
SGA media			The growth media (YPD) and synthetic dropout media (SD) including LRK and LHRK, and enriched sporulation media used in this protocol is routinely used in yeast molecular biology. Please refer to Methods in Yeast (Amberg et al., 2005) for detailed descriptions.