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. 2007 Jul;18(7):2646–2655. doi: 10.1091/mbc.E06-10-0897

Figure 5.

Figure 5.

PI4P promotes GAT binding to Ub. (A) Ub-agarose binding. Mammalian GST-GAT or yeast His-VHS-GAT, 0.5 μM, was incubated with increasing amounts of PI4P in the presence of either protein A-agarose (Ctrl) or Ub-agarose. Bound proteins were detected with antibodies by Western blotting. (B) GAT binding to Ub-agarose was preferentially increased by PI4P. GST GGA1 GAT, 0.5 μM, was incubated with 20 μM PI4P or PI3P (left and middle panels) or with PC/PE liposomes containing either PS or PI4P (right panel). PI4P increased GGA1 GAT binding to Ub by 3.73 ± 0.8-fold and PI3P 1.5 ± 0.3-fold (n = 3). *p = 0.024. NS, p = 0.775. (C) His-Ub did not bind PI4P on lipid dot blots. His-Ub (at 8 or 40 nM) and His-VHS-GAT from yeast Gga1p (at 8 nM) were incubated with lipid dots made in the laboratory (lipids: 100, 50, and 25 pmol), and bound proteins were detected with anti-His antibody. (D) PI4P did not promote mutant GAT binding to Ub-agarose. GGA1 GAT wt, R265A, and R260A (0.25 μM) were incubated with Ub-agarose in the presence of 15 μM PI4P. The proteins used for the binding studies are shown (input). (E) GGA1 ubiquitination in vivo. HeLa cells were cotransfected with myc-tagged wt or mutant GGA1 and HA-Ub cDNAs. Myc-GGA1 was immunoprecipitated and blotted with anti-myc antibody to detect GGA1 and anti-HA to detect Ub associated with GGA1. The extent of ubiquitination for each sample was expressed as a ratio of the intensity of the HA-Ub to myc-GGA1 signals in the myc-GGA1 immunoprecipitates, and the value for the wt GGA1 is defined as 1.0. Data shown are mean ± SE (n = 3). (F) ITC analysis. GGA1 GST-GAT and bovine Ub were each preincubated with 50 μM water-soluble diC8PI4P before mixing. The kcal/mol of Ub as a function of the molar ratio of Ub to GGA1 GAT is shown, and the curves were fitted by using a one-site model. The Kd values shown are mean ± SE of five experiments using several different preparations of GGA1 GAT. The insets show the heat change elicited by successive injections of Ub into a chamber containing the GAT1 solution.