1. Ras membrane trap: overview and screen.A, principle of the method.
Ras membrane trap: overview and screen.A, principle of the method. Only inserts encoding membrane-targeted proteins will bring the fused Ras to the vicinity of the plasma membrane, thus allowing yeast growth. B, map of the pMet-Ras vector, indicating the library cloning sites. The Met25 promoter shuts down expression from the plasmid in the presence of methionine. C, schematic of the main stages of an RMT screen. Transformation and incubation at 24 °C is followed by replica-plating and selection at the restrictive temperature of 36 °C. Positive clones are then picked and replated for elimination of false positives by methionine selection. D, an example of methionine selection at the final step of a screen. Positive colonies are those that grow in the absence of methionine and are repressed by addition of 50 μg/ml methionine. E, categories of clones obtained from the Lymnaea CNS library. Left, membrane association of the selected clones as predicted by on-line software tools. Right, subcellular localization of close homologs for those clones with informative homologies in the data bases. PM, plasma membrane; ER, endoplasmic reticulum; mito, mitochondria; cytoskel, cytoskeleton; sec, secreted.
Hanna Jaaro et al. Mol Cell Proteomics 2005;4:
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