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

doi:10.1074/jbc.271.1.48

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

From the Department of Cell Biology and Kaplan Cancer Center, New York University Medical Center, New York, New York 10016

^¶Crawford-5nard Established Investigator of the American Heart Association, NYC affiliate. To whom correspondence should be addressed:
Dept. Cell Biology, NYU Medical Center, 550 First Ave., New York, NY 10016.
Tel: 212-263-5812; Fax: 212-263-8139.

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 β-hydroxylase and peptidyl glycine α-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.

Footnotes

↵^§ Recipient of an Ella Fitzgerald Award from the American Heart Association, NYC affiliate.
↵* This work was supported in part by National Institutes of Health Grant DK44238. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵¹ The abbreviations used are:

TGN

trans-Golgi network

ISG

immature secretory granule

IgG

immunoglobulin G

BSA

bovine serum albumin

GH

growth hormone

Prl

prolactin

Cg

chromo/secretogranin

DBH

dopamine β-hydroxylase

PAM

peptidyl glycine α-amidating monooxygenase

FSH

follicle stimulating hormone

POMC

pro-opiomelanocortin

IGF-1

insulin-like growth factor 1

ACTH

adrenocorticotropic hormone

PAGE

polyacrylamide gel electrophoresis

MES

4-morpholineethanesulfonic acid

LH

luteinizing hormone.
- Received August 14, 1995.