ptdins3p grip  (Echelon Biosciences)

Journal: Autophagy

Article Title: The ULK1 complex mediates MTORC1 signaling to the autophagy initiation machinery via binding and phosphorylating ATG14

doi: 10.1080/15548627.2016.1140293

Figure Lengend Snippet: Figure 6 ATG14 Ser29 phosphorylation is important for starvation-induced activation of ATG14-associated PIK3C3. (A) Validation of our assay to measure the kinase activity of ATG14-associated PIK3C3. WT ATG14 and ATG14 KO HCT116 cells were incubated in DMEM or EBSS for 1 h. ATG14 immunoprecipitates were incubated with PI and ATP for 30 min. The production of PtdIns3P was assayed by dot blot analysis (see Materials and Methods). (B) Quantitative analysis of PtdIns3P production in (A). Values are mean ± SD (**, P

Article Snippet: The sources of antibodies and chemicals used in the experiments are: antibodies for ULK1 (sc-10900 for IP and sc-33182 for WB of human protein), ATG14 (sc-164767 for IP), UVRAG (sc-82115), RUBCN (sc-99955), BECN1 (sc-11427 for WB and sc-10086 for IP), SQSTM1 (sc-28359), GSK3A (sc-1844) and GAPDH (sc-25778) from Santa Cruz Biotechnology; anti-ULK1 antibody (A7481 for WB of mouse protein), anti-WIPI2 antibody (SAB4200400 for immunostaining), EBSS (2888), bafilomycin A1 (B1793–10UG) from Sigma-Aldrich; antibodies for PIK3C3 (3358), ATG9A (13509), ATG14 (rabbit monoclonal clone D3H2Z for immunostaining and WB), MTOR (2972 and 2983 for WB), phospho-AKT Ser473 (4051), AKT (9272), MAP1LC3B/LC3B (2775 for WB), phospho-RPS6KB1 Thr389 (9205), and RPS6KB1 (9202) from Cell Signaling Technology; anti-MYC 9E10 monoclonal antibody (OP10), WIPI2 antibody (MABC91 for immunostaining), and Akt inhibitor VIII (124018) from EMD-Millipore; anti-HA antibody HA.11 from Covance (AFC-101P); anti-GST antibody (27457701), glutathione-sepharose 4B beads (17–0756–01), and PreScission protease (27–0843–01) from GE Healthcare Life Sciences; LC3B antibody (PM036 for immunostaining) from MBL (Woburn, MA); phospho-ATG13 Ser318 (600–401-C49) from Rockland Immunochemicals Inc.; active ULK1 kinase (U01–11G-10) from SignalChem; DMEM (11995), RPMI 1640 (11875093), HBSS (24020), and Lipofectamine 2000 (11668–019) from Life Technologies; Torin1 (4247) and rapamycin(1292/1) from R & D; anti-phosphatidylinositol (840042P) from Avanti Polar Lipids; PtdIns3P Grip (G-0302) from Echelon Biosciences; Protein G-agarose beads (GenDEPOT, P9202); FuGENE™ 6 (PRE2692) and DSP (dithiobis[succinimidylpropionate]) (22586) from (Thermo Fisher Scientific, Inc.).

Techniques: Activation Assay, Activity Assay, Incubation, Dot Blot

Journal: Nature Communications

Article Title: Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

doi: 10.1038/ncomms10470

Figure Lengend Snippet: PI(3)P is not localized in the ER of P. falciparum . ( a ) Chimeric proteins expressed in P. falciparum . p40PX-GFP, no signal sequence; STEVORss-p40PX-GFP, STEVOR signal sequence; STEVORss-p40PX-GFP SDEL , STEVOR signal sequence and ER retention signal; Hrs-GFP, no signal sequence; STEVORss-Hrs-GFP, STEVOR signal sequence; STEVORss-Hrs SDEL -GFP, signal sequence and ER retention signal; STEVORss-Hrs C215S -GFP, STEVOR signal sequence and mutation of lipid-binding residue C215S in both FYVE fingers. ( b ) p40PX and Hrs reporters are expressed in P. falciparum -infected erythrocytes. Blots probed with anti-GFP antibodies. First panel: p40PX-GFP, STEVORss-p40PX-GFP and STEVORss-p40PX-GFP SDEL. A ‘GFP core' band was observed for STEVORss-p40PX-GFP. This is observed for all GFP chimeras secreted to the PV and does not indicate degradation specific to this protein. Note the absence of ‘GFP core' when SDEL ER-retention signal was attached, which prevented secretion to PV (lane 3). Second panel: Hrs-GFP, STEVORss-Hrs-GFP, STEVORss-Hrs-GFP SDEL and STEVORss-HrsC 215S -GFP. Note the lack of ‘GFP core' for STEVORss-Hrs fusions, confirming they were not secreted. ( c ) p40PX-GFP localizes to the cytoplasm and to membranes of the food vacuole (labelled with anti-CRT) and apicoplast (labelled with anti-ACP). STEVORss-p40PX-GFP localizes to the PV in P. falciparum -infected erythrocytes, as shown by co-localization with anti-EXP2, and not in the ER, which was labelled with anti-PMV. If PI(3)P was located within the ER, this chimera would be expected to remain within the ER via p40PX binding. Fusion to an ER-retention signal (STEVORss-p40PX-GFP SDEL ) retains the protein in ER, co-localization with anti-ERC. Scale bar, 5 μm. ( d ) Localization of Hrs-GFP to the cytoplasm and membranes of food vacuole (labelled anti-CRT) and apicoplast (labelled anti-ACP). STEVORss-Hrs-GFP localizes to the ER as shown by co-localization with anti-ERC; however, mutation of residues necessary for lipid binding in both Hrs FYVE fingers (STEVORss-Hrs C215S -GFP) does not abrogate ER localization, demonstrating PI(3)P-independent retention in ER. Addition of ER retention signal (STEVORss-Hrs-GFP SDEL ) also causes ER localization. Scale bar, 5 μm. ( e ) Quantification of GFP localization in the PV (black), ER (grey), or mix of both (white) determined using 40 P . falciparum -infected erythrocytes per construct (20 cells per experiment, performed twice).

Article Snippet: Recombinant GST-p40PX was also purchased (Echelon Biosciences; G-0302).

Techniques: Sequencing, Mutagenesis, Binding Assay, Infection, Construct

Journal: Nature Communications

Article Title: Export of malaria proteins requires co-translational processing of the PEXEL motif independent of phosphatidylinositol-3-phosphate binding

doi: 10.1038/ncomms10470

Figure Lengend Snippet: Exported P. falciparum proteins do not bind PI(3)P. ( a ) Recombinant proteins expressed in this study as fusions to GST or GFP/6His. ( b ) Recombinant proteins stained with Coomassie blue stain. Molecular weights (kDa) are shown. ( c ) Binding of p40PX and HRPII with native PEXEL (WT) or mutant PEXEL (RLE > A) to PI(3)P liposomes measured by SPR. Binding to control PC/PE liposomes was subtracted to give the sensorgrams 24 . BSA was used to measure nonspecific binding to the liposome-coated chip (red). Inset: a similar level of low binding of HRPII WT and HRPII RLE > A to PI(3)P liposomes. Experiments were performed in triplicate. ( d ) Binding of proteins to PI(3)P liposomes or control PC/PE liposomes, determined by ultracentrifugation. Pellet and supernatant fractions were resolved by SDS–PAGE and Coomassie blue stain. Proteins were ultracentrifuged in buffer to remove potential aggregates before incubation with liposomes 39 , explaining why input and sum of pellet and supernatant sometimes differ. Experiments were performed in triplicate. Full-length gels shown in Supplementary Fig. 6 . ( e ) Binding of p40PX and P. falciparum proteins with native PEXEL (WT) or mutant PEXEL (M) to PI(3)P-coated beads (+) or control beads lacking lipid (−). Bound protein was eluted in SDS–PAGE sample buffer and detected by immunoblot with anti-GST (p40PX) or anti-GFP ( P. falciparum proteins). Ten per cent of unbound fraction volume was loaded to visualize protein inputs. Densitometry (histogram shown) of the p40PX bands shows that 68% of p40PX input bound to PI(3)P-coated beads (PI3P+) compared with 18% of input to control beads (PI3P−). No binding was detected for P. falciparum exported proteins. Experiments were performed in triplicate. Full-length gels shown in Supplementary Fig. 7 . ( f ) Binding of recombinant proteins to phospholipid head group of PI(3)P (inositol 1,3 bisphosphate; I1,3P2) in solution, measured by isothermal calorimetry. The titration did not differ between I1,3P2 (2.4 mM) into dialysis buffer (150 mM NaCl, 20 mM HEPES pH 7.4) (negative control) and the same buffer containing either GBP130 66–88 , HRPII WT or HRPII RLE > A . As controls, binding of Htp1 from S. parasitica to Fmoc-Tyr(SO 3 )-OH and also p40PX binding to I1,3P2 are shown. Experiments were performed in duplicate.

Article Snippet: Recombinant GST-p40PX was also purchased (Echelon Biosciences; G-0302).

Techniques: Recombinant, Staining, Binding Assay, Mutagenesis, SPR Assay, Chromatin Immunoprecipitation, SDS Page, Incubation, Titration, Negative Control