Intracellular Transport of Proinsulin in Pancreatic 
-Cells
STRUCTURAL MATURATION PROBED BY DISULFIDE ACCESSIBILITY (*)
- From the (1) Department of Microbiology, University of Alabama, Birmingham, Alabama 35209 and the
- (2) Program in Biological and Biomedical Sciences, Harvard Medical School and the Division of Endocrinology, Beth Israel Hospital, Boston, Massachusetts 02215
- ¶ To whom correspondence should be addressed: Division of Endocrinology, Beth Israel Hospital Research North, 99 Brookline Ave., Boston, MA 02215. Tel.: 617-667-4280; Fax: 617-667-2927.
Abstract
In pancreatic islets, formation of β-secretory granule cores involves early proinsulin homohexamerization and subsequent insulin
condensation. We examined proinsulin conformational maturation by monitoring accessibility of protein disulfide bonds. Proinsulin
disulfides are intact immediately upon synthesis, but are ≥90% sensitive to in vivo reduction with 2 mM dithiothreitol; wash out of dithiothreitol leads to reoxidation, proinsulin transport, and conversion
to insulin. With t 
10 min, newly synthesized proinsulin becomes resistant to disulfide reduction, correlating with endoplasmic reticulum (ER)
export. However, inhibition of ER export with brefeldin A blocks acquisition of resistance to reduction, and once proinsulin
arrives in the Golgi, it resists reduction despite brefeldin treatment. Moreover, in vivo, resistance of proinsulin disulfides is overcome after increasing [dithiothreitol] > 10-fold, or in vitro, in islets lysed in a zinc-free, but not a zinc-containing, medium. Employing 30 mM dithiothreitol in vivo, a further decrease in disulfide accessibility is observed following proinsulin conversion to insulin. Incubation of islets
with chloroquine or zinc enhances and diminishes accessibility of insulin disulfides, respectively. We hypothesize that two
major conformational changes culminating in granule core formation, proinsulin hexamerization and insulin condensation, are
sensitive to zinc and occur upon ER exit and arrival in immature secretory granules, respectively.
Footnotes
-
↵§ This work represents partial fulfillment of requirements for a Ph.D. degree.
-
↵* This work was supported in part by a Research Grant from the American Diabetes Association and by National Institutes of Health Grant DK48280. 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:
- ER
-
endoplasmic reticulum
- DTT
-
dithiothreitol
- DME
-
Dulbecco's modified Eagle's medium
- IAA
-
iodoacetamide
- PAGE
-
polyacrylamide gel electrophoresis
- Tricine
-
N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine
- BFA
-
brefeldin A
- BSA
-
bovine serum albumin
- Mes
-
4-morpholineethanesulfonic acid.
-
↵2 Calculations from the chase times selected in this paper are based on a model of intercompartmental vesicular transport that assumes first-order kinetics (Pfeffer and Rothman, 1987). Since some of our experiments employed no pharmacological block of transport, no “perfect” chase times could be selected in these experiments (i.e. times in which a radioactive wave of proinsulin labeled one compartment to the exclusion of all others (Farquhar et al., 1978; Salpeter and Farquhar, 1981)). For the purposes of this study, 20 min of chase was selected as a time when a majority of proinsulin is thought to be in Golgi cisternae, although a minority may have reached the trans-Golgi network and another minor fraction may have lingered in the ER; whereas 30 min of chase was selected as a time when a majority of proinsulin is thought to have reached the trans-Golgi network, a minority may have reached immature granules and another minor fraction may have lingered in prior compartments (Howell et al., 1969; Orci, 1982).
-
↵3 Despite attempts to control digestion conditions during islet isolation, the size distribution of islets from different preparations varied. Unlike studies of proinsulin in the ER (which was always reduced with low dose DTT), in experiments attempting to achieve reduction that required high dose DTT, it appeared that preparation-dependent variability in disulfide cleavage related to differences in DTT penetration. This variability was thought to be an acceptable consequence of islet experiments, since such results were thought to have greater physiological significance than comparable experiments performed in cell lines (where penetration of DTT is more uniform). Despite increased reduction observed in experiments where the islets were smaller (e.g. Fig. 10), comparative data between samples within the same preparation were reproducible and thus did not affect our conclusions.
-
↵4 We could not be sure that we achieved complete proinsulin denaturation, even in the presence of 8 M urea. With this in mind, additional thermal denaturation (boiling the sample) led to quantitatively identical and complete reduction of proinsulin at all pH values, including pH 5.2.
-
- Received April 10, 1995.
- Revision received June 14, 1995.
- © 1995 by The American Society for Biochemistry and Molecular Biology, Inc.
