Journal: Molecular Biology of the Cell
Article Title: Identification of the endocytic sorting signal recognized by the Art1-Rsp5 ubiquitin ligase complex
Figure Lengend Snippet: Two regions in the Mup1 N-terminal cytosolic tail are critical for endocytic down-regulation. (A) Top, sequence of the cytosolic N-tail (residues 1–60) of the yeast methionine transporter Mup1. The two regions critical for endocytosis are underlined. Bottom, a collection of GFP-tagged Mup1 mutants was stably integrated into yeast cells expressing Vph1-mCherry as vacuolar marker. In each mutant, five consecutive residues of the N-tail were mutated to alanines. Cells were grown to mid log phase in synthetic medium at 30°C, stimulated with 20 μg/ml methionine for 90 min, and analyzed by fluorescence microscopy. Average Mup1 sorting was calculated as the ratio of vacuolar GFP to total cell GFP and normalized to wild type before (0% sorting) and after methionine stimulation (100% sorting). The numbers below each bar of the graph correspond to the position of the substituted residues within Mup1. Error bars indicate SD between fields. (B) Representative images from cells quantified in A. Cells expressing GFP-tagged Mup1 or the indicated Mup1 alanine substitutions were grown to mid log phase in synthetic medium at 30°C and subjected to fluorescence microscopy after treatment with methionine for 90 min. A complete set of images is shown in Supplemental Figure S2. Dashed lines indicate cell outlines. Scale bar, 2.5 μm; vacuolar marker: Vph1-mCherry. (C) Yeast cells expressing GFP-tagged Mup1 or the indicated Mup1 alanine substitutions were grown to mid log phase in synthetic medium at 30°C and stimulated with 20 μg/ml methionine. Equal volumes of culture were harvested at the indicated time points after stimulation, and total cell lysates were prepared and analyzed by SDS–PAGE and immunoblot. G6PDH serves as a control for equal loading.
Article Snippet: The frozen pellet was resuspended in 450 μl RIPA buffer (50 mM Tris HCl, pH 7.6, 150 mM NaCl, 20 mM NaF, 1 mM EDTA, 0.5 mM ethylene glycol tetraacetic acid) supplemented with 1 mM DTT, 1 mM PMSF, and cOmplete protease inhibitor (Sigma-Aldrich), and 20 mM N -ethylmaleimide.
Techniques: Sequencing, Stable Transfection, Expressing, Marker, Mutagenesis, Fluorescence, Microscopy, SDS Page