Mint3/X11 Is an ADP-Ribosylation Factor-dependent Adaptor that Regulates the Traffic of the Alzheimer's Precursor Protein from the Trans-Golgi Network

Supplemental Materials

This article contains the following supporting material:

• Supplementary Figure 1 - Resolution of light vesicles from larger organelles and endosomes was achieved by sucrose velocity sedimentation S2 (1 mL; 8.5 mg/ml) was loaded on top of a 15-45% linear sucrose gradient prior to centrifugation for 1 hr at 100,000xg. A total of 16 fractions of 700 µl each were collected from the top, with only the top eight fractions shown here. A portion of each fraction (25 µl) was analyzed by immunoblot, using the antibodies indicated next to each panel. Note that recycling endosomes (TfR) migrate into the gradient while soluble proteins and light vesicles remain near the top.
• Supplementary Figure 2 - Histogram of diameters of APP/Mint3-positive vesicles Light vesicles (fractions 4-6) from an Optiprep gradient were immunopurified on magnetic beads using antibodies specific to either Mint3 or the cytoplasmic tail of APP before being analyzed by transmission electron microscopy, as described under Materials and Methods. The size distribution of the vesicles was determined by measuring the diameters of immunomagnetic isolated vesicles attached to magnetic beads in electron micrographs. Vesicles isolated with APP antibodies had a median diameter of 93.24 nm (N = 12; range 80.0-133.2 nm) and those obtained with Mint3 antibodies were 99.9 nm (N = 23; range 66.6-136.5 nm). Data shown are for all vesicles.
• Supplementary Figure 3 - Decreased expression of Mint3 does not alter syntaxin 6 or EEA1 staining Cells were depleted of Mint3 by siRNA and over-expressing human APP₆₉₅, before imposition of a 4 hr temperature block, as described in the legend to figure 6. Cells were then fixed and stained for APP and syntaxin 6 (A) or EEA1 (B). No differences were observed in the size or distribution of syntaxin6- or EEA1-positive puncta in controls vs Mint3 depleted cells. This is in contrast to what is seen shortly after release from the temperature block (Figure 7). Bar = 10 µm.