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

To further define the sorting determinant that is present within amino acids 1-23 of p63, this region was subjected to extensive site-directed mutagenesis. First, a series of overlapping alanine stretches was created in the A24-101 mutant. As indicated in Fig. 5 A, amino acids 2-8 (A24-101, 2-8A), amino acids 6-12 (A24-101,6-12A), amino acids 9-15 (A24-101,9-15A), amino acids 13-19 (A24-101, 13-19A), or amino acids 17-23 (A24-101, 17-23A), respectively, were simultaneously changed to alanines using PCR mutagenesis. Surprisingly, these substitutions had no detectable effect on the localization of p63. Immunofluorescence of transfected nonpermeabilized cells showed that none of the mutants were transported to the plasma membrane (for an example see Fig. 6 b) . The internal distribution of the constructs was also indistinguishable from that of/,24-101 or p63wt (for an example see Fig. 6 a) . When a similar type of alanine scan was carried out in the m6-101 background (Fig. 5 B) , a different result was obtained. All of the mutant proteins exhibited some degree of increased transport to the cell surface, with the zk16-101,6-12A mutant showing the strongest effect (Fig. 6 d) . When COS cells transfected with this mutant were permeabilized, the predominant staining pattern was characteristic for the ER and the Golgi apparatus indicating that/,16-101,6-12A had lost p63wt localization (Fig. 6 c) .