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

For the generation of the PPD and A24-101PPD chimeras (complete or A24-101 cytoplasmic tail of 1)63, transmembrane domain of p63, and lumenal domain of DPPIV), the DPPIV cDNA was subcloned into a Bluescript SK-vector that has the KpnI site in its polylinker deleted. A KpnI site was then introduced between nucleotides 166 and 171 of the DPPIV sequence without altering the amino acid sequence. The resulting plasmid was digested with SalI and KpnI to give a fragment including the complete lumenal domain of DPPIV except for the first 11 nucleotides (bp 160-170). The pECE-p63 or pECE-A94-101 plasmid, respectively, were digested with SalI and NotI, and the corresponding fragments that encode the complete or mutant (A24-101) I)63 cytoplasmic tail plus the first 12 amino acids of the p63 transmembrane domain, were isolated. Two complementary oligonucleotides corresponding to nucleotides 441-465 of the p63 sequence followed by nucleotides 160-165 of the DPPIV sequence and a KpnI site were sequenced and annealed. These components were assembled in a three-part ligation to give plasmids pECE-PPD or pECE-A24-101 PPD, respectively.