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.
The type II membrane protein p63 is a resident protein of a membrane network interposed between rough ER and Golgi apparatus. To study the retention of p63, mutant forms were expressed in COS cells and the intracellular distribution determined by immunofluorescence microscopy. Investigation 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 intracellular retention. Mutational analysis of the 106-amino acid cytoplasmic tail of p63 revealed that the NH2-terminal 23 amino acids are necessary for retention. When p63 was solubilized with Triton X-100 and subjected to centrifugation at 100,000 g, it formed large, insoluble oligomers, particularly at neutral pH and below. A comparison of the behavior of wildtype and mutant p63 proteins in this assay revealed a perfect correlation between the formation of large oligomers and correct intracellular retention. These results suggest that self-association may be a major mechanism by which p63 is retained between the rough ER and the Golgi apparatus.
Schweizer
A
A
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Rohrer
J
J
Hauri
H P
HP
Kornfeld
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001692:Biological Transport; D002451:Cell Compartmentation; D002478:Cells, Cultured; D004252:DNA Mutational Analysis; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D006056:Golgi Apparatus / Q000378:metabolism*; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
C081876:CKAP4 protein, human; D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins