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

In our attempt to determine the features of p63 responsible for retention we initially concentrated on the cytoplasmic domain of the protein. The first construct to be prepared was a truncated form of p63 in which the amino-terminal residues 2-101 of the 106-amino acid-long p63 tail were deleted (A2-101, Fig. 3) . Since p63 has a type II membrane orientation, the first NH2-terminal amino acid representing the initiator methionine could not be removed. When A2-101 was expressed in COS cells, it was clearly present on the plasma membrane in about half of the transfected cells (Fig. 2 d) . Upon permeabilization, A2-101 was detected primarily in the rough ER as indicated by the reticular staining pattern along with labeling of the outer nuclear membrane (Fig. 2 c) . Occasionally it was found in the Golgi region or at the cell surface (see insert of Fig. 2 c for an example of surface staining). In the construction of A2-101, two arginine residues became positioned near the extreme NI-I2-terminus due to the deletion of 100 amino acids within the cytoplasmic tail (Fig. 3) . We therefore considered the possibility that these positively charged amino acids might serve as an ERtargeting signal, thereby impairing the normal transport of the protein (Jackson et al., 1990) . These arginine residues (located in position -2 and -3 from the NH2 terminus of A2-101) were therefore changed to alanines (A2-101AA, Fig. 3 ) and the effect monitored by immunofluorescence as outlined above. A2-101AA showed strong cell surface expression over the entire transfected cell population (Fig. 2 f) . Further, the staining pattern observed in permeabilized cells (Fig. 2 e) was typical for a cell surface protein including juxtanuclear Golgi and plasma membrane staining. Taken together, these data demonstrated that the cytoplasmic domain of p63 is necessary for correct intracellular localization.