Retention of p63 in an ER-Golgi intermediate compartment depends on the presence of all three of its domains and on its ability to form oligomers

When we carefully inspected the critical p63 sequence, a repetitive feature consisting of a positively charged amino acid followed by a glycine residue became apparent. The sequence contains an arginine-glycine combination at amino acid positions 7 and 8, and two pairs of lysine-glycine at positions 10 and 11, and 21 and 22, respectively. In addition, there is a single lysine residue present at amino acid position 5. The distribution of these residues relative to the amino acids altered in the overlapping alanine substitution experiments was consistent with these residues serving as components of a redundant retention signal. To test this possibility, K ~, R 7, G s, K 1°, G H, K 21, and G 22 in the ~24-101 sequence were simultaneously changed to aianines (Z~.4-101,KSRTGSKI°GnK2tGa2-A [MP1]; Fig. 7 ). Immunofluorescence analysis of this mutant expressed in COS cells revealed that it was highly expressed at the plasma membrane of non-permeab~ transfected cells (Fig. 8 b) . Furthermore, in permeabilized ceils transfected with MP1 the staining was also characteristic for a surface protein (Fig. 8 a) . The phenotype of MP1 is therefore very similar to that of the tailminus mutant A2-101AA. We next tested whether alunine substitutions of only the positively charged amino acids (K s, W, K 1°, and K29 (A24-101,KSRTK'°Ke~-A [MF2]; Fig. 7 ) or only the glycine residues (C_J, G n, and Cr n) (A94-101,CJG"G22-A [MP3]; Fig. 7 ) present in the K-RG-KG-KG element would also disrupt p63 localization. Both constructs behaved similarly to A24-101 and p63wt as indicated by the lack of cell surface staining (Fig. 8 , d and f) and the typical internal pattern (Fig. 8, c and e). This demonstrated that changing positive charges or glycines alone does not affect retention of p63.