Imaging-based live cell yeast screen identifies novel factors involved in peroxisome assembly.
We describe an imaging-based method in intact cells to systematically screen yeast mutant libraries for abnormal morphology and distribution of fluorescently labeled subcellular structures. In this study, chromosomally expressed green fluorescent protein (GFP) fused to the peroxisomal targeting sequence 1, consisting of serine-lysine-leucine, was introduced into 4740 viable yeast deletion mutants using a modified synthetic genetic array (SGA) technology. A benchtop robot was used to create ordered high-density arrays of GFP-expressing yeast mutants on solid media plates. Immobilized live yeast colonies were subjected to high-resolution, multidimensional confocal imaging. A software tool was designed for automated processing and quantitative analysis of acquired multichannel three-dimensional image data. The study resulted in the identification of two novel proteins, as well as of all previously known proteins required for import of proteins bearing peroxisomal targeting signal PTS1, into yeast peroxisomes. The modular method enables reliable microscopic analysis of live yeast mutant libraries in a universally applicable format on standard microscope slides, and provides a step toward fully automated high-resolution imaging of intact yeast cells.
Wolinski
Heimo
H
Institute of Molecular Biosciences, University of Graz, Austria.
Petrovic
Uros
U
Mattiazzi
Mojca
M
Petschnigg
Julia
J
Heise
Bettina
B
Natter
Klaus
K
Kohlwein
Sepp D
SD
D005656:Fungal Proteins / Q000378:metabolism*; D017353:Gene Deletion; D049452:Green Fluorescent Proteins / Q000378:metabolism; D050356:Lipid Metabolism; D018613:Microscopy, Confocal / Q000379:methods; D008856:Microscopy, Fluorescence / Q000379:methods*; D008954:Models, Biological; D009154:Mutation; D020411:Oligonucleotide Array Sequence Analysis; D000074435:Peroxisome-Targeting Signal 1 Receptor; D020675:Peroxisomes / Q000378:metabolism*; D040901:Proteomics / Q000379:methods*; D018160:Receptors, Cytoplasmic and Nuclear / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism
D005656:Fungal Proteins; D000074435:Peroxisome-Targeting Signal 1 Receptor; D018160:Receptors, Cytoplasmic and Nuclear; D029701:Saccharomyces cerevisiae Proteins; D049452:Green Fluorescent Proteins