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 define more precisely the location of the retention information in the p63 cytoplasmic tail we made a construct (A16-101, Fig. 3 ) in which amino acids 2-15 of p63wt were added back to the A2-101 deletion mutant. When cells expressing this construct were analyzed by immunofluorescence, an internal staining pattern close to that of p63wt was observed except that no cells with the tubular network type pattern were found (Fig. 4 a) . Labeling of nonpermeabilized COS cells with anti-p63 mAbs revealed that A16-101 is predomi- For detection an mAb to p63 followed by goat anti-mouse FITC was used. In contrast to p63wt, constructs A2-101 and A2-101AA were observed at the plasma membrane. Bar, (a and b) 28/~m; (c-f) 42/~m. nantly localized intracellularly (Fig. 4 b) ; an occasional cell showed weak staining of the plasma membrane (Fig. 4 b, inset) . When the NH~-terminal portion of p63 was extended to include the 23 NH2-terminal residues of the p63wt sequence (A24-101, Fig. 3) , the resultant protein behaved like p63wt when analyzed by indirect immunofluorescence after transfection (Fig. 4, c and d) . The mutant protein was completely retained inside the cell (Fig. 4 d) . In addition, the A24-101 staining in permeabilized ceils was indistinguishable from that ofp63wt (Fig. 4 c) , including cells that exhibit the striking tubular network that is typically found in p63wttransfected ceils (Fig. 4 c, inset) . We conclude from these results that the NH~-terminal 23 amino acid residues of the p63 cytoplasmic domain are required for proper intracellular localization. Deletion of amino acids 24-101 of the p63 tail, on the other hand, had no detectable effect on the distribution of p63 in the cell.