YopE associates with intracellular membrane compartments. (A) YopE colocalizes with markers of intracellular membrane compartments. Cells expressing GFP-YopE were fixed in cold methanol and were incubated with monoclonal antibodies that recognize the Golgi marker comitin and the ER marker protein disulfide isomerase (PDI) followed by incubation with Cy3-labeled anti-mouse IgG. GFP is visualized directly. Images are confocal sections. Scale bar, 10 μm. (B) Fractionation of Dictyostelium cells expressing GFP-YopE. Cells were lysed by sonication and cytosolic and membrane fractions were separated by ultracentrifugation. Samples were resolved in 12% polyacrylamide gels, blotted onto nitrocellulose membranes and probed with antibodies against GFP, PDI (marker for the membrane fraction) and RhoGDI (marker for the cytososlic fraction). (C) Sucrose gradient fractionation of cells expressing GFP-YopE. Fractions were collected from the top and analyzed in Western blots using antibodies for the indicated proteins or in enzymatic reactions. Interaptin is a protein of the nuclear envelope and ER. RhoGDI is a predominantly cytosolic protein but a small amount appears associated to membrane compartments. Alkaline phosphatase is a marker for plasma membrane and the contractile vacuole and acid phosphatase is a marker for lysosomes.