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

We first analyzed a chimeric protein (DPP; DPPIV cytoplasmic, p63 transmembrane, p63 lumenal; Fig. 9 ) in which the cytoplasmic tall of DPPIV was fused to the transmembrane and lumenal domains of p63. COS cells were transfected with DPPIVwt or the chimeric construct and the localization of the proteins was examined by indirect immunofluorescence of permeabilized and nonpermeabilized cells using mAbs to a lumenal epitope of each protein. Nontransfected control cells did not react with the anti-DPPIV mAb (data not shown) consistent with evidence that COS cells have tittle or no endogenous activity of DPPIV (Ogata et ai., 1992) . As shown in Fig. 10 , a and b DPPIVwt was located on the cell surface as expected. Bright cell surface staining was also observed for DPP (Fig. 10 d) . However, unlike DPPIVwt a portion of DPP was also found in the ER (Fig. 10 c) , indicating that the export of this chimeric protein out of the ER is impaired to some extent: Very rarely a cell showed a p63wt-related staining pattern. A similar result was obtained with a construct that only contained the lumenal domain of p63 (DDP; DPPIV cytoplasmic; DPPIV transmembrane, p63 lumenal; Fig. 9 ) except that most of the ER staining was now replaced by cell surface expression (data not shown). These results confirm that the p63 cytoplasmic tail is necessary for proper localization. In the absence of the p63 cytoplasmic tail, the lumenal p63 domain alone or in combination with its transmembrane domain is not sufficient to achieve retention.