Abstract
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.
Highlights
A 13-Amino Acid N-terminal Domain of a Basic Proline-rich Protein Is Necessary for Storage in Secretory Granules andFacilitates 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
Storage of bPRP-We have been using a salivary basic proline-rich protein, bPRP derived from bPRP23 cDNA, as a prototype to identify domains that function in secretory sorting in AtT-20 cells
Summary
Cell Culture and Transfection-AtT-20 D16v cells were cultured under a 5% COn atmosphere in Dulbecco’s modified Eagle’s medium/ nutrient mixture F-12 supplemented with 10% Nuserum (Collaborative Research, Lexington, MA), 5% inactivated horse serum (HyClone, Logan,UT), mM HEPES, and0.1 pg/ml kanamycin sulfate. The labeling was carried out (residues 1-16), the transition region (residues 17-33), a proline-rich region composed of one amino acid segment and seven aminoacidrepeats(residues 34-203), and the Cterminalregion(residues 204-223). The cDNA contains a single consensus site for N-glycosylation at Asn-47 Both the for 15h with 0.4 mCi/ml [3H]proline(Amersham Gorp.) in Dulbecco’s modified Eagle’smedium supplemented with 15% of dialyzed Nuserum and 20mM HEPES. Labeled cells were chased in Dulbecco’s modifed Eagle’s medium containing 15% Nuserum, mM HEPES, 1mM proline.
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