Phosphatidylinositol-4-Kinase Type II Is a Component of Adaptor Protein-3-derived Vesicles

Supplemental Materials

This article contains the following supporting material:

• Supplemental Table 1 - AP-3-derived Vesicle Protein Identification.

  Table depicts proteins identified in the proteome of the fraction enriched in AP-3 derived vesicles. Peptide # corresponds to the cumulative peptide count in three proteomic experiments for any given protein. AP-3 corresponds to proteins confirmed to be present in AP-3 vesicles; asterisks denote proteins confirmed in other papers. For dynamin see Faundez et al 1998, for ATM see Lim et al. 1998 and for vimentin see Styers et al. 2005. CCV denotes those proteins also found in the brain clathrin-coated vesicle proteome (Blondeau et al., 2004; Ritter et al., 2004). Proteins were confirmed to be present in these vesicles by immunomagnetic isolation of glycerol purified microvesicle fractions using beads coated with AP-3 δ antibodies followed by immunoblot with antibodies against the specific antigen and/or changes in the subcellular distribution in mocha brain small membranes. Asterisks denote proteins exclusively identified by MALDI-TOF, double asterisk correspond to proteins identified by MALDI-TOF and MS/MS. All other proteins were identified by MS/MS.

• Supplemental Figure 1 - AP-3-derived vesicle isolation scheme.

  A) PC12 cells expressing ZnT3-HA were treated in the absence or presence of MβCD to interfere with SLMV biogenesis from plasma membrane. Plasma membrane-derived SLMV were monitored with synaptophysin and AP-3-derived vesicles with ZnT3. Homogenate was fractionated by differential centrifugation to generate S2 supernatants. S2 fractions were sequentially fractionated by velocity and isopicnic sedimentation. Alternatively, S2 sedimentation at high speed generates a P3 pellet (not shown). B) The protein profile of an S2 supernatant resolved using glycerol velocity sedimentation. S2 represents ~55% of the total homogenate protein (see insert). Only 0.3% of the homogenate protein is recovered in the microvesicle peak fractions (see Insert, fraction 8-11). C) Protein profile of the isopycnic sucrose flotation shows that the majority of the protein present in glycerol fractions 8-11 remains in the soluble pool (fractions 12-16). Closed circles represent protein concentration, open circles sucrose %. The y axis represents the % of the gradient total protein present on any given fraction. D) Silver staining of microvesicle fractions depicted in C. E) Higher magnification view of fraction 8 from C (marked with asterisks in C-D) where the highest content of ZnT3 is present. Note the reduction of the protein content following treatment with MβCD.

• Supplemental Figure 2 - AP-3-is enriched in microvesicles comigrating with SLMV fractions.

  A) PC12 cells were fractionated by differential and glycerol velocity gradient sedimentation. Glycerol gradient fractions were probed with antibodies against AP-3 β3, AP-2 α, and AP-1 ƒ× adaptins. Most of the adaptor complexes are present in the fractions containing cytosolic proteins (fractions 14-17). A significant portion of AP-3 β3 is readily detectable as a second peak in fraction 8 that contains synaptic vesicle markers. B) 160 ng of P1, P2 and glycerol fraction 8 protein were resolved by SDS-PAGE and analyzed by immunoblot with antibodies against AP-3 β3 adaptin and ZnT3. C) Graph depicts the quantification of the ZnT3 and AP-3 blots presented in (B). Crude P1 membranes were assigned an arbitrary value of 1. AP-3 is enriched ~16-fold over crude P1 membranes.

• Supplemental Figure 3 - AP-3-derived vesicles are present in brain.

  A) Rat brains were fractionated by sequential differential, glycerol velocity and isopycnic sucrose flotation as described in Fig.1. Sucrose gradient fractions were probed for AP-3 σ3 and AP-1 γ adaptins, and the synaptic vesicle markers ZnT3 and VAMP II. Inserts on the right depict positive controls with brain homogenate. Synaptic vesicle markers co-migrating with AP-3 are devoid of AP-1 coated membranes.