A 13-amino acid n-terminal domain of a basic proline-rich protein is necessary for storage in secretory granules and facilitates exit from the endoplasmic reticulum.
We have investigated the role of different domains of a salivary basic proline-rich protein in intracellular transport and sorting of proline-rich proteins to the secretory granules. We have cloned a full-length cDNA of a basic proline-rich protein from the rat parotid and expressed it in AtT-20 cells. It was correctly sorted into secretory granules as shown by EM immunolocalization and by its presence in 8-bromocyclic AMP-stimulated secretion. Deletion of the N-terminal thirteen amino acid domain upstream from the proline-rich domain eliminated storage whereas deletion of the C-terminal 20-amino acid domain downstream from the proline-rich domain had no effect. Intracellular transport of full-length and mutant proline-rich proteins was unusually slow due to slow exit from the endoplasmic reticulum. However, the rate of transport increased with increasing level of expression for the full-length protein and the C-terminal deletion mutant. In contrast, the rate of transport of the N-terminal deletion mutant was independent of the level of expression. These results imply that the N-terminal domain is necessary for both storage and efficient intracellular transport. Moreover, interactions (self-aggregation?) that mediate sorting may begin as early as the endoplasmic reticulum.
Castle
A M
AM
Department of Anatomy and Cell Biology, University of Virginia Health Sciences Center, Charlottesville 22908.
Stahl
L E
LE
Castle
J D
JD
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002460:Cell Line; D003594:Cytoplasmic Granules / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism*; D007150:Immunohistochemistry; D007700:Kinetics; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D055232:Proline-Rich Protein Domains; D051381:Rats; D012471:Salivary Proteins and Peptides / Q000737:chemistry / Q000378:metabolism*
D010455:Peptides; D012471:Salivary Proteins and Peptides