Specific and Nonspecific Membrane-binding Determinants Cooperate in Targeting Phosphatidylinositol Transfer Protein -Isoform to the Mammalian Trans-Golgi Network

Supplemental Material

This article contains the following supporting material:

• Figure 1 - PITPβ localizes to the MEF trans-Golgi network. An animated 3-dimensional reconstruction of a PITPα^-/- MEF imaged for PITP antigen (green channel), the medial-Golgi marker giantin (blue channel), and the trans-Golgi marker TGN38 (red channel). Rotation of the reconstructed image clearly shows the coincidence of PITPβ and TGN38 localization and also the segregation of that localization from that of giantin.

• Figure 2 - C-terminal PITPβ localization elements necessary for TGN association. Alignment of the C-terminal 28 residues of PITPβ with the corresponding region of PITPα is given. The BOX motifs are shown at top. The most divergent residues within each are highlighted (•). Schematic illustrations of PITPα, PITPβ and each of the reciprocal C-terminal swaps are depicted at bottom. At right, for each corresponding PITP version, is given the number of imaged cells that exhibited a Golgi (G) or non-Golgi (N) immunofluorescence profile when that construct was expressed in COS-7 cells as a PITP-GFP chimera and visualized along with the GM130 marker. The percentage of imaged cells with Golgi profiles is also given.

• Figure 3 - C-terminal PITPβ localization elements sufficient for redirecting PITPα to TGN membranes. Swap series of divergent BOX motifs from PITPβ into the context of PITPα is shown. The most divergent residues within each are highlighted (•). The series of hybrid PITPs is illustrated and each swap is further defined at left by identification of which PITPβ residues were introduced to generate the swap. Quantification of COS-7 cells expressing each individual hybrid with respect to number of cells displaying Golgi (G) or non-Golgi (N) localization profile, along with percentages of cells displaying Golgi localization, is also given.

• Figure 4 - Comparison of PITPβ and PITPβ^QGQR localization in MEFs. PITPα nullizygous MEFs expressing PITPβ-GFP or PITPβ ^QGQR-GFP were fixed and decorated with primary antibodies directed against either the trans-Golgi marker TGN38 (A), the medial-Golgi marker mannosidase II (B) , or the cis-Golgi marker GM130 (C) . GFP-chimera localization was followed by intrinsic GFP fluorescence. The individual and merged profiles are identified.

• Figure 5 - PITPβ localization in PKCδ^-/- MEFs. (A) Endogenous PITPβ profile of E16.5 PITPα^-/- PKCε^-/-, and PKCδ^-/- MEFs, as indicated. The NT-PITP serum was employed in these experiments, and the PITPα^-/- MEFs represent the positive control. In all cases essentially all cells exhibited a robust Golgi profile for endogenous PITPβ species. (B) PMA-induced phosphorylation of PITPβ is ablated in PKCδ^-/- MEFs. Cell-free extracts from MEFs of indicated PKCδ genotype were generated, resolved by SDS-PAGE, transferred to nitrocellulose and developed with PITPβ-specific antibodies (PITPβ ^QGQR is not detected) and an ECL scheme. Instances where cells were treated with PMA (100nM; 15 min), or extracts were incubated with calf-intestine phosphatase (CIP), are identified at bottom. MEF PKCδ genotypes for each condition are also identified. PITPβ is a phosphoprotein as indicated by the collapse of the PITPβ doublet to the lower apparent molecular mass form upon treatment of lysates with CIP, and by the complete conversion of the PITPβ doublet to the higher apparent molecular mass form by treatment of cells with PMA. This PMA effect is not observed in PKCδ^-/- MEFs, although the PITPβ doublet is maintained.

• Table 1 - Summary of effect of missense substitutions on PITP localization in COS-7 cells. Quantification of COS-7 cells expressing each indicated mutant form of PITPβ-GFP and PITPα-GFP with respect to number of cells displaying Golgi (G) or non-Golgi (N) localization profile. Corresponding percentages of cells displaying Golgi localization is also given.

• Figure 1 movie