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

Previous studies have established that p63 is stably retained between the rough ER and the Golgi apparatus (Schweizer et al., 1993a) . In the present study, we have analyzed the contribution of the individual p63 domains to retention by creating chimeric constructs with the type II plasma membrane protein DPPIV. The role of the cytoplasmic tail was further analyzed by deletions and point mutations within the p63 protein itself. The appearance of mutant p63 proteins at the cell surface and the concomitant loss of the characteristic p63wt internal staining pattern were used as criteria for loss of proper retention. Using these approaches, we have identiffed the domains of p63 that determine retention and obtained data that are consistent with the notion that oligomerization of p63 serves as a major retention mechanism. Table I presents a summary of all the constructs analyzed in this study. The striking characteristic of p63 retention is that no single topological domain is sufficient to specify retention. Rather, all three domains are required to achieve complete intracellular retention. The cytoplasmic and lumenal domains of p63 are most important for proper localization with the transmembrane domain playing a lesser role in this process. These findings with p63 differ from those obtained with most other proteins of the early secretory pathway where one domain has been shown to be sufficient for retention. In the case of resident lumenal ER proteins, the four-amino acid sequence KDEL at the carboxyl terminus serves as retrieval signal (Munro and Pelham, 1987; Pelham, 1989 Pelham, , 1990 . Similarly, KKXX/KXKXX elements at the carboxyl terminus of the cytoplasmic domains of type I transmembrane proteins function as retrieval signals (Nilsson et al., 1989; Jackson et al., 1990 Jackson et al., , 1993 . When transplanted onto marker proteins, these sequences are sufficient to cause retention in the ER. The carboxyl-terminal cytoplasmic domain of TGN38 is also sufficient for localization of chimeric proteins to the TGN (Bos et al., 1993; Humphrey et al., 1993; Wong and Hong, 1993) . Other Golgi proteins are localized by means of their transmembrane domains (N-acetylglucosaminyltransferase I (Burke et al., 1992; Tang et al., 1992) , galactosyltransferase (Nilsson et al., 1991; Aoki et al., 1992; Russo et al., 1992) , and the M glycoprotein of avian coronavirus (Swift and Machamer, 1991; Machamer et al., 1993) or in association with the cytoplasmic and lumenal flanking sequences (or 2,6-sialyltransferase (Munro, 1991; Dahdal and Colley, 1993) . The E1 glycoprotein of the mouse coronavirus is an exception among the proteins localized to the Golgi apparatus (Armstrong and Patel, 1991) . While a short deletion in the cytoplasmic tail destroyed retention, the cytoplasmic segmet~t alone was not sufficient for retention. Similar to the finding with p63, most of the sequence appeared to be required for proper localization.