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

What is the role of the cytoplasmic tail in conferring proper intracellular retention of p63? A potential clue emerged when we analyzed the solubility of the various constructs in the nonionic detergent Triton X-100. When transfected cells were solubilized with Triton X-100 at pH 6.5 and then subjected to ultracentrifugation at 100,000 g, the proteins that were properly retained (p63wt, A24-101, MP2, and MP3) were predominantly found in the pellet fraction whereas the nonretained proteins (A2-101AA and MP1) were mostly recovered in the supernatant. Endogenous p63 from COS cells was exclusively found in the 100,000 g pellet, demonstrating that the property is not a function of transfection and/or overexpression. While the extent of Triton X-100-insolubility of p63wt increased with decreasing pH, most of the protein was insoluble at pH 7.4, which is similar to the pH of the cytoplasm. Since highly purified p63wt exhibited the same pHdependent insolubility in Triton X-100, we conclude that this behavior is an intrinsic property of the protein. This does not exclude the possibility that p63 also interacts with other resi-dent proteins of the compartment or with cytoplasmic elements, such as members of the cytoskeleton.