Secretory Granule Content Proteins and the Luminal Domains of Granule Membrane Proteins Aggregate in Vitro at Mildly Acidic pH

Abstract

A major unresolved issue in the field of secretory granule biogenesis is the extent to which the aggregation of granule content proteins is responsible for the sorting of regulated from constitutively secreted proteins. The aggregation process is postulated to take place in the trans-Golgi network and immature secretory granules as the proteins encounter mildly acidic pH and high calcium concentrations. We have developed in vitro assays that reconstitute the precipitation out of solution of secretory granule content proteins of anterior pituitary gland and adrenal medulla. In the assays, all of the major granule content polypeptides form a precipitate as the pH is titrated below 6.5, and this precipitate can be recovered in the pellet fraction after centrifugation. Addition of calcium is required for the aggregation of chromaffin granule content. In contrast to the proteins secreted by the regulated pathway, the constitutively secreted proteins IgG, albumin, and angiotensinogen, when added to the assays, remain predominantly in the supernatant. Among the individual proteins tested, prolactin is found to aggregate homophilically under these conditions and can drive the co-aggregation of other proteins, such as the chromogranins. Soluble forms of granule membrane proteins, including dopamine beta-hydroxylase and peptidyl glycine alpha-amidating enzyme also co-aggregated with granule content proteins. The results are consistent with the idea that spontaneous aggregation of proteins occurring under ionic conditions similar to those at the sites of granule formation is a property restricted to those proteins packaged in secretory granules. In addition, the association of luminal domains of membrane proteins with content proteins in vitro raises the possibility that analogous interactions between membrane-bound and content proteins also occur during granule formation in intact cells.

Response of an Integral Granule Membrane Protein to Changes in pH

“…The lumenal pH in each secretory compartment is postulated to play an important role in peptide sorting and vesicle biogenesis (8,(13)(14)(15).…”

“…The chromogranins (13, 16, 16 -19), furin (20), carboxypeptidase E (21,22), prohormone convertase 2 (23), prolactin (13), and insulin (24) each demonstrate reversible, low pH, high calcium-dependent aggregation. Peptidylglycine ␣-amidating monooxygenase (PAM) has also been found among the group of proteins that form aggregates (13).…”

“…The chromogranins (13, 16, 16 -19), furin (20), carboxypeptidase E (21,22), prohormone convertase 2 (23), prolactin (13), and insulin (24) each demonstrate reversible, low pH, high calcium-dependent aggregation. Peptidylglycine ␣-amidating monooxygenase (PAM) has also been found among the group of proteins that form aggregates (13). This tendency to aggregate appears to be specific to proteins belonging to the regulated secretory pathway, because constitutively secreted proteins, such as immunoglobulin, albumin, and angiotensinogen, remain soluble at low pH, even in the presence of calcium (13).…”

“…Peptidylglycine ␣-amidating monooxygenase (PAM) has also been found among the group of proteins that form aggregates (13). This tendency to aggregate appears to be specific to proteins belonging to the regulated secretory pathway, because constitutively secreted proteins, such as immunoglobulin, albumin, and angiotensinogen, remain soluble at low pH, even in the presence of calcium (13). Although the exact function of protein aggregation is unknown, it is postulated that this process contributes to the sorting of secretory proteins (5,25).…”

Protein Composition and Biogenesis of the Pancreatic Zymogen Granules

“…The membrane association of GP-2 is via a glycosylphosphatidylinositol (GPI) anchor (47,67). Since GP-2 can form stable complexes with zymogens at mildly acidic pH but not at alkaline pH (12,48), it was suggested that GP-2 may act as a sortase for aggregated secretory proteins (39). However, the findings that ZGs in mouse pancreas can form in the absence of GP-2 indicated it is not required for ZG biogenesis (87).…”