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

We next tested whether the ability of p63 to oligomerize correlated with retention and proper localization. To address this point, a selected set of p63 cytoplasmic tall mutants were expressed in COS cells and the transfected ceils were then solubilized with Triton X-100 at pH 6.5 and analyzed as described above. The results of a typical experiment are shown in Fig. 12 A and the quantitation of multiple experiments is given in Fig. 12 B. P63wt which served as a control was exclusively found in the pellet fraction (97 ± 5 %). A24-101, MP2, and MP3 which have intraceilular distributions COS ceils were trmsfeoed with wt or mutant p63, solubRized with Triton X-100 at pH 6.5 and further analyzed as described in Fig. 11 . (P) pellet, (S) supematant. The upper band seen in the pellet lanes of p63 mutants represents endogenous p63 that is exclusively found in the pellet fractions. (B) The immtmoblots shown in A and those from additional experiments were quantitated by densitometric scanning. For each sample (x-axis) the amount of wt or mutant 1063 detected in the supernatant (dotted light bars) and pellet (striated dark bars) fraction, respectively, is plotted as the percentage of p63 present in both fractions (y-axis). Each value is the average of at least four independent experiments. The standard deviations were ,g8.5 %. similar to p63wt were also recovered predominantly in the 100,000 g pellet (81 + 9%, 78 + 4%, and77 ± 5%, respectively). In contrast, A2-101AA and MP1 which are not localized properly, were found mainly in the supernatant fraction (75 ± 2 %, and 83 ± 2 %, respectively). The upper band detected in the pellet lane of each of the p63 mutants represents endogenous p63 from COS ceils. Similar to transfected p63wt, endogenous 1363 was exclusively found in the pellet fraction regardless of the behavior of the transfected p63 mutants and therefore served as a useful internal standard for the distribution of the various mutants. Affinity-purified A2-101AA gave similar results to those obtained with homogenates of COS ceils transfected with A2-101AA (Fig. 11 B, lanes 7-10) .