RETENTION OF P63 IN AN ER-GOLGI INTERMEDIATE COMPARTMENT DEPENDS ON THE PRESENCE OF ALL 3 OF ITS DOMAINS AND ON ITS ABILITY TO FORM OLIGOMERS

The type II membrane protein p63 is a resident protein of a membrane n etwork interposed between rough ER and Golgi apparatus. To study the r etention of p63, mutant forms were expressed in COS cells and the intr acellular distribution determined by immunofluorescence microscopy. In vestigation of chimeric constructs between p63 and the plasma membrane protein dipeptidylpeptidase IV showed that protein sequences from all three domains of the p63 protein are required to achieve complete int racellular retention. Mutational analysis of the 106-amino acid cytopl asmic tail of p63 revealed that the NH2-terminal 23 amino acids are ne cessary for retention. When p63 was solubilized with Triton X-100 and subjected to centrifugation at 100,000 g, it formed large, insoluble o ligomers, particularly at neutral pH and below. A comparison of the be havior of wildtype and mutant p63 proteins in this assay revealed a pe rfect correlation between the formation of large oligomers and correct intracellular retention. These results suggest that self-association may he a major mechanism by which p63 is retained between the rough ER and the Golgi apparatus.