Mitochondria-targeted green fluorescent proteins: convenient tools for the study of organelle biogenesis in Saccharomyces cerevisiae.
We describe the construction and characterization of a novel set of plasmids for expression of mitochondria-targeted green fluorescent protein (GFP) in Saccharomyces cerevisiae. The vectors include constructs with strong regulatable and constitutive promoters, four different auxotrophic markers for yeast transformation, and a green (S65T) and a blue-shifted (P4-3) variant of GFP. Mitochondria are brightly fluorescent in living yeast cells grown on different carbon sources and at different temperatures, with virtually no background staining. Specific staining of mitochondria is also shown for a respiratory-deficient mutant with abnormal mitochondrial morphology. The plasmids facilitate convenient analysis of mutants defective in mitochondrial morphology or inheritance and, at the same time, are suitable vectors for easy construction of different kinds of GFP fusion proteins to study various aspects of organelle biogenesis in living yeast cells.
Westermann
B
B
Institut für Physiologische Chemie, Ludwig-Maximilians-Universität München, 80336 München, Germany. benedikt.westermann@bio.med.uni-muenchen.de
Neupert
W
W
D003001:Cloning, Molecular; D017931:DNA Primers / Q000737:chemistry; D004271:DNA, Fungal / Q000737:chemistry; D015966:Gene Expression Regulation, Fungal; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted; D007202:Indicators and Reagents / Q000737:chemistry; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics*; D008856:Microscopy, Fluorescence; D008858:Microscopy, Phase-Contrast; D008928:Mitochondria / Q000737:chemistry / Q000235:genetics*; D010957:Plasmids; D016133:Polymerase Chain Reaction; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics* / Q000648:ultrastructure
D017931:DNA Primers; D004271:DNA, Fungal; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C409504:blue fluorescent protein, Aequorea victoria; D049452:Green Fluorescent Proteins