Journal: Scientific Reports
Article Title: Heterologous mammalian Akt disrupts plasma membrane homeostasis by taking over TORC2 signaling in Saccharomyces cerevisiae
Figure Lengend Snippet: Akt phosphorylates Slm1 but its effects are independent of TORC2-Slm-Ypk signaling. ( a ) Immunoprecipitation of Slm1-6×His expressed from plasmid BG1805- SLM1 in wild type YPH499 cells co-expressing the empty vectors YCpLG and pYES3 and/or YCpLG-p110α and pYES3-GFP-Akt1. Arrows indicate the two electrophoretic mobility bands of Slm1-6×His observed with anti-polyHis antibody. ( b ) Input and immunoprecipitation (IP) of Slm1-6×His from cell lysates co-expressing BG1805- SLM1 , YCpLG-p110α and pYES3-GFP-Akt1. Immunoprecipitates were treated with alkaline phosphatase (AP) and additionally with ortovanadate (OV) to inhibit phosphatase activity. ( c ) Immunoprecipitation experiments as in ( a ) but using the non-phosphorylatable Slm1 S659A mutant. ( d ) Immunoprecipitation of Slm1-6×Myc, endogenously expressed from strain IRE12 ( SLM1 -6× myc slm2 Δ), co-transformed with the empty vectors and/or plasmids YCpLG-p110α and pYES3-GFP-Akt1 and immunodetected with anti-Myc antibody. ( e ) As in ( d ), but Slm1-6×Myc was expressed from strain IRE11 and immunoprecipitates were blotted with anti-(R/K)X(R/K)XX(pT/pS) motif (anti-p-Akt-substrate) antibodies. ( f ) Growth assays of wild type (SEY6210) and AAY1663 ( slm1 ∆ slm2 ∆ sac7 ∆) cells transformed with YCpLG-p110α and pYES2-GFP-Akt1 K179M or pYES2-GFP-Akt1 cultured in SD (Glucose) and SG (Galactose). ( g ) Western-blotting of cells (YPH499) co-expressing the PLB187 (empty plasmid), PLB215 (Ypk1-HA) and PLB534 (Ypk1 S644A, S662A -HA) and YCpLG/YCpLG-p110α and/or pYES3/pYES3-HA-Akt1. Cells were induced in SR-Gal for 3 h, and, where indicated, treated with 1.5 µM myriocin for 2 additional hours. Extracts were probed with anti-phospho-T662 for Ypk1 phosphorylation, anti-HA for Ypk1 immunodetection, and anti-G6PDH as loading control. The ‘M’ lane denotes the Mw marker. ( h ) Western-blotting of lysates from cells (YDB146) co-transformed with the catalytically-inactive mutant YCpLG-p110α K802R (p110α−) or YCpLG-p110α (p110α+) and pYES2-GFP (GFP-Akt1−), pYES2-GFP-Akt1 (WT) or kinase-dead pYES2-GFP-Akt K179M (KD), immunodetected with anti-FLAG or anti-G6PDH as loading control. As in ( g ), cultures were treated, when indicated, with 1.5 µM myriocin. ( i ) Representative fluorescence images and quantitative analyses of PM invaginations in wild type (YPH499) or YPT40 ( ypk1-1ts ypk2 ∆) at permissive (24 °C) or restrictive (34 °C) temperatures, co-transformed with YCpLG-p110α and pYES2-GFP-Akt1, after 24 h incubation in SG medium. Data are the average from 3 different fields (n > 30 cells/field). Error bars correspond to standard deviation. Images in ( a - e , g , h .
Article Snippet: Immunodetection was carried out with anti-phospho-Ypk1 (T662) (1:20000, kindly provided by Dr. T. Powers, UC Davis, CA, USA), anti-HA 12CA5 (1:1000, Roche), anti-FLAG clone M2 (1:1000, Sigma), anti-cMyc 9E10 (1:1000, Santa Cruz), anti Phospho-Akt Substrate [(R/K)X(R/K)XX(pT/pS)] (1:1000, cs 9611 s, Cell Signaling) and anti-polyHistidine (1:1000, Clone HIS1, Sigma).
Techniques: Immunoprecipitation, Plasmid Preparation, Expressing, Activity Assay, Mutagenesis, Transformation Assay, Cell Culture, Western Blot, Immunodetection, Marker, Fluorescence, Incubation, Standard Deviation