Figure 1. Preparation and pH indicator loading of p7-containing membrane vesicles.

HEK-293FT cells were transiently transfected with either FLAG-p7 or FLAG-p7KR, homogenized 24 h later, and subjected to an initial centrifugation at 3,000× g followed by ultracentrifugation of the supernatant at 120,000× g. (A) Western blot of the cytosolic extract (3k Sup), heavy membrane fraction (3k Pellet) and light membrane fraction (120k pellet) showing that FLAG-p7 (left panel) and FLAG-p7KR (right panel) were present in whole cell extracts as well as in the heavy membrane and light membrane fractions. Marker proteins were LAMP-2 (lysosomal/late endosomal protein), PDI (ER chaperone) and GRP75 (mitochondrial chaperone). (B) HPTS (100 nM solution) was titrated to varying pH values from 4.5 to 10.5 and fluorescence emission (F) at 520 nm was determined using excitation wavelengths of 450 and 405 nm. The ratio of F₄₅₀/₄₀₅ is presented as a function of pH. Dotted lines represent the linear range. (C) Frozen 120k vesicle pellet was thawed in the presence of HPTS and re-homogenized as described in Materials and Methods. The mixture was applied to a Bio-Gel P-10 size exclusion column and eluted with constant flow. The column void volume eluted at approximately fraction 28. The right panel shows both fluorescence and protein content of fractions over the whole column profile while the left panel shows results around the void volume only with a magnified ordinate. Solid symbols (•) represent fluorescence; bars indicate protein concentration. (D) Protocol was similar to that shown in C except that the vesicle prep was first exposed to Triton X-100 prior to gel filtration. In this experiment the void volume eluted approximately at fraction 15. Note the absence of a fluorescence peak associated with the eluted protein.