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

The retention behavior of p63 described so far suggested that the underlying molecular mechanism might be related to physical properties within the p63 protein itself. Two findings in particular supported this idea. First, overexpression of p63 did not saturate the retention mechanism. And second, the p63 localization could not be transferred to another protein. We therefore speculated that individual 1363 molecules might interact with each other to form higher order structures. To test this hypothesis p63wt was analyzed by centrifugation. In this experiment COS cells transfected with p63wt were solubilized with Triton X-100 at different pHs (5.8, 6.3, 6.8, 7.4, and 8.0) and then subjected to centrifugation at 100,000 g for 1 h. The resulting supernatant and pellet fractions were then assayed for the presence of p63 by SDS-PAGE and immunoblotting with an anti-p63 mAb. As shown in Fig. 11 A there was a pH dependent shift of 1363 from the soluble fraction to the pellet. At pH 8.0, p63 was equally distributed between the pellet and supernatant fractions, whereas increasing amounts of p63 were recovered in the COS cells transfected with p63wt were solubilized with Triton X-100 at the indicated pHs and then separated by centrifugation at 100,000 g into supernatant (S) and pellet (P). Proteins of the two fractions were subjected to SDS-PAGE (8% gels) and immunoblotring with an anti-p63 mAb. The numbers at the left margin of the blot indicate known molecular mass in kilodalton. (B) Samples of affinity purified p63wt protein (lanes 1-6) or A2-101AA (lanes 7-10) in MNT pH 8.0-Triton X-100 were incubated for 40 rain (I), adjusted to pH 5.8 and then incubated for 40 rain (II), or shifted to pH 5.8 for 40 mill, then returned to pH 8.0 and incubated for another 40 rain (III). Separation into supernatant (S) and pellet (P) and further analysis was as described in A.