Scrib can target LPP to an ectopic location in vivo through its PDZ domains
Evidence for an in vivo interaction between Scrib and LPP was obtained by performing mitochondrial targeting experiments. We tested if Scrib was sufficient to recruit LPP to an ectopic location in living cells. The membrane anchor of the ActA sequence has been shown previously to be sufficient to target proteins expressed in mammalian cells to the surface of mitochondria [27,28]. This ectopic localization allows testing ligand recruitment in vivo. For this purpose, we generated a chimera named Xpress-hScrib-mito made up by an Xpress-epitope tag fused to the amino-terminus of human full length Scrib and linked in frame to the membrane anchor of the Listeria monocytogenes protein ActA (mito). Expression of this construct was confirmed by Western blotting with the use of an anti-Xpress antibody (results not shown). CV-1 cells were transiently transfected with Xpress-hScrib-mito and full length wild-type or carboxy-terminally mutated LPP green fluorescent protein fusions. Cells were stained with an anti-Xpress antibody and examined by fluorescence microscopy. In all transfected cells, the Xpress-hScrib-mito chimera localized to mitochondria, as shown in Fig. 7, left upper and middle panels. As shown in Fig. 7, upper right panels, wild-type LPP can be recruited to Xpress-hScrib-mito on mitochondria. This recruitment of LPP to Xpress-Scrib-mito-coated mitochondria was completely abolished when the carboxy-terminus of LPP was mutated (Fig. 7, middle panels).
Figure 7  Scrib can recruit LPP to an ectopic location in vivo through its PDZ domains. CV-1 cells were transiently co-transfected with Xpress-hScrib-mito or Xpress-hScribdPDZ-mito, and GFP-fusions of wild-type full length human LPP, or LPP with a mutated carboxy-terminus (T610A). Xpress-hScrib-mito and Xpress-hScribdPDZ-mito are composed of the human full length Scrib protein with or without its PDZ domains, respectively, which is fused to an Xpress-epitope-tag at its amino-terminus, and to an ActA-derived mitochondrial membrane anchor at its carboxy-terminus, Cells were stained with an anti-Xpress antibody to detect Xpress-Scrib(dPDZ)-mito. Immunofluorescence and GFP were visualized by epifluorescence microscopy. The focal adhesion localization of the GFP-LPP proteins is not visible in these pictures because a focal plane corresponding to mitochondrial staining is shown.   To investigate the importance of the PDZ domains of Scrib in this recruitment of LPP, we deleted all four PDZ domains (amino acids 724–1192) from Xpress-hScrib-mito (=Xpress-hScribdPDZ-mito) and tested whether this PDZ-less protein still was able to recruit LPP to mitochondria. As shown in Fig. 7, lower panels, Xpress-hScribdPDZ-mito lost its ability to recruit LPP to mitochondria. These results indicate that Scrib can recruit LPP to an ectopic location in vivo, and that the PDZ domains of Scrib are an absolute requirement for this activity.
As mentioned above, the Xpress-hScrib-mito chimera localized to mitochondria in all cells that expressed this protein. However, LPP, which was co-expressed, was only recruited to mitochondria in a small fraction of these cells. This issue will be further addressed in the Discussion section.