(Received for publication, April 10, 1995; and in revised form, June 14, 1995)
From the
In pancreatic islets, formation of -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.
The distinguishing feature of regulated secretory cells is the
packaging of a distinct subset of proteins into insoluble dense cores
within storage granules (Palade, 1975; Kelly, 1985). Although most
storage granule contents are rapidly solubilized upon exocytosis,
intracellular accumulation of secretory proteins at supersaturating
conditions appears to represent a conserved mechanism by which
glandular tissues ready themselves for an acute response to demand for
protein secretion. In the -cells of pancreatic islets, insulin is
normally stored within a polymerized condensate in the cores of
granules, which are stimulated to undergo exocytotic discharge in
response to glucose. In the secretory pathway of these cells, insulin
is initially made as the single chain precursor, proinsulin.
Endoproteolysis converts the soluble prohormone into insulin that
becomes insoluble (Steiner, 1973; Michael et al., 1987;
Kuliawat and Arvan, 1994), a process that begins after the former
enters immature secretory granules (Orci et al., 1987; Huang
and Arvan, 1994).
While remaining highly soluble in the early
secretory pathway, proinsulin normally progresses from monomers to
homohexamers (Frank and Veros, 1968, 1970; Grant et al., 1972;
Steiner, 1973; Baker et al., 1988). It has been hypothesized
that proinsulin assembly to homohexamers proceeds within the ER ()(Emdin et al., 1980) and indeed for many
secretory proteins, such oligomerization is a prerequisite for ER
export (for review, see Hurtley and Helenius (1989)). Recently, several
remarkable studies have shown that conformational maturation of newly
synthesized proteins in the secretory pathway can be monitored in
vivo, by changes in the accessibility of protein disulfide bonds
(Alberini et al., 1990; Braakman et al., 1992; Kaji
and Lodish, 1993; Lodish and Kong, 1993). Moreover, it has been
established that disulfide bonds in exportable proteins, including
regulated secretory proteins, may be reduced in vivo not only
within the ER but also in Golgi and post-Golgi compartments (Chanat et al., 1993; Tatu et al., 1993; Valetti and Sitia,
1994). If disulfide accessibility represents a useful means to follow
proinsulin structural maturation during its intracellular transport,
then it should be possible to use this approach to examine the stages
of conformational maturation of this regulated secretory protein, which
lead ultimately to granule core condensation.
With this in mind, we
have examined pancreatic -cells of live islets, to explore
dithiothreitol (DTT)-mediated disulfide bond reduction of proinsulin
and insulin while in transit through the secretory pathway. We find
that, in live
-cells, the accessibility of proinsulin disulfides
to reducing agents is lost in two discrete stages. Surprisingly, the
first stage, which is likely to represent proinsulin hexamerization,
does not occur within the ER but occurs just after ER export. The
second stage, which is likely to represent granule core formation,
occurs shortly after proteolytic conversion to insulin. Interestingly,
both of these stages appear to be facilitated by zinc ions.
Chase incubations for batches of islets were performed as described (Huang and Arvan, 1994). When employed, brefeldin A was used at 10 µg/ml. Islets were lysed by sonication in 150 mM NaCl, 20 mM Tris, pH 7.4, containing Triton X-100 ranging from 0.1 to 1.0% in different experiments. An anti-protease mixture of aprotinin (1 milliunit/ml), leupeptin (0.1 mM), pepstatin (10 mM), EDTA (5 mM), and diisopropyl fluorophosphate (1 mM) was added to the islet lysates. The alkylating agent, iodoacetamide (IAA) was also added (described below).
Prior to lysis, islets were either mock-treated or treated with DTT in complete DME for 10 min at 37 °C. At the conclusion of the incubation with reducing agent, the islets were rapidly washed in ice-cold phosphate-buffered saline containing 50 mM IAA and lysed (as described above) in a buffer including 50 mM IAA (except in Fig. 7, where the IAA dose was 150 mM). The islet lysate was spun briefly in a Microfuge to remove debris. The supernatant was further incubated in the presence of the alkylating agent (overnight at 4 °C) before electrophoretic analysis. In Fig. 6, lysed islets were either mock-treated or treated with DTT in lysis buffer, before alkylation overnight at 4 °C in the presence of 100 mM IAA, and in Fig. 8, alkylation employed 150 mM IAA.
Figure 7: In vivo dose-response curve of disulfide reduction by DTT in proinsulin at 20 min of chase. Isolated islets were pulse labeled for 5 min and then divided into five equal aliquots chased for 20 min as shown. At this time, the islets were treated for an additional 10 min at 37 °C in complete chase medium containing DTT at the doses shown. After treatment with DTT, the islets were washed in ice-cold buffer containing 150 mM IAA before lysis and further incubation in lysis buffer plus 150 mM IAA overnight at 4 °C. The lysates were then analyzed by Tricine-urea-SDS-PAGE without prior immunoprecipitation. The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown with arrows on the left. Although there was some variability in the dose-response curves for different preparations of islets chased for 20 min, in all cases proinsulin was largely resistant to low dose DTT and became sensitive to reduction with high dose DTT. An unrelated band which migrated slightly faster than fully reduced proinsulin was unaffected by DTT exposure; variable amounts of this protein were detected in different experiments.
Figure 6:
In vitro, proinsulin re-acquires
sensitivity to disulfide reduction, but its structure is stabilized in
the presence of zinc. A, isolated islets were pulse labeled
for 5 min with S-labeled amino acids and then chased for
the times indicated. At each chase time the islets were lysed at
neutral pH as described under ``Experimental Procedures.'' B, a separate preparation of islets were pulse labeled for 5
min and chased for 20 min before lysis in the presence or absence of
0.5 mM ZnSO
, at neutral pH as described under
``Experimental Procedures.'' Each lysate was then treated
with low dose DTT either at the doses shown (A) or at 2 mM (B) for 10 min at 37 °C. After treatment with DTT,
the lysates were incubated in ice-cold buffer containing 100 mM IAA overnight at 4 °C. All samples were then analyzed by
Tricine-urea-SDS-PAGE without prior immunoprecipitation. The positions
of fully reduced (Red.) and fully oxidized (Ox.)
proinsulin standards (St'd) are shown with arrows on the left of each panel.
Figure 8:
DTT-mediated disulfide reduction is more
sensitive to protein conformation than to changes of pH within the
physiologic range. A, isolated mouse pancreatic islets were
continuously labeled for 10 min with S-labeled amino acids
and then lysed in unbuffered 0.1% Triton X-100, 150 mM NaCl,
and 8 M urea, with the intention to fully denature newly
synthesized proinsulin. The lysate was then divided into five aliquots
containing 0.1% Triton X-100, 150 mM NaCl, 40 mM
Tris-Mes (20 mM Tris, 20 mM Mes) at the pH values
shown and treated with 30 mM DTT for 10 min at 37 °C. B, a constitutive secretory protein, BSA, was dissolved in 150
mM NaCl plus 0.1% Triton X-100 (lanes marked
``TX''). The samples were then divided into five
aliquots (lanes 3-7) and reduced as in A. In
parallel, two additional BSA samples were denatured by exposure to 2%
SDS and then divided into equal portions. One was treated with 5 mM DTT (lane 1) and a second was nonreduced (lane
2). Finally, all samples in A and B were
alkylated with 150 mM IAA overnight at 4 °C. A,
the samples were analyzed by Tricine-urea-SDS-PAGE and fluorography.
The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown
with arrows on the left. B, the samples were
analyzed by nonreducing SDS-PAGE (8% acrylamide) and stained with
Coomassie Blue. 5 µg of BSA was run in each lane. The position of
fully oxidized albumin standard is shown on the left.
Figure 1:
Resolution of proinsulins with
differentially reduced disulfide bonds. Isolated mouse pancreatic
islets were continuously labeled for 10 min with S-labeled
amino acids and then lysed in 1% Triton X-100, 150 mM NaCl in
25 mM Tris, pH 7.4. The lysates were then diluted into a
4-fold concentrated SDS-gel sample buffer. The samples were
subsequently exposed to DTT at the concentrations indicated for 10 min
at 37 °C, alkylated with IAA, and analyzed by Tricine-urea-SDS-PAGE
without prior immunoprecipitation. The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown with arrows on the left
side. A fifth band, which migrates slightly faster than fully
reduced proinsulin, is unrelated to proinsulin and is unaffected by DTT
exposure; variable amounts of this protein were detected in different
experiments.
Figure 2:
Rapid formation of native proinsulin
disulfide bonds. Isolated mouse pancreatic islets were pulse labeled
for 1 min with S-labeled amino acids and chased (in the
presence of 100 µg/ml cycloheximide to inhibit further proinsulin
synthesis) for the times indicated. The islets were then lysed and
analyzed by Tricine-urea-SDS-PAGE without prior immunoprecipitation.
The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown
with arrows on the left side. It is clear that most
labeled proinsulin is already fully oxidized at the zero chase
time.
Figure 3:
In
live islets, proinsulin acquires resistance to disulfide reduction with
low dose DTT. Isolated mouse pancreatic islets were pulse labeled for 5
min with S-labeled amino acids and then chased (in the
presence of 100 µg/ml cycloheximide to inhibit further proinsulin
synthesis) for the times indicated. At each chase time, the islets were
treated for an additional 10 min at 37 °C in complete chase medium
containing 2 mM DTT. After treatment with DTT, the islets were
washed in ice-cold buffer containing 50 mM IAA, lysed in the
presence of 50 mM IAA as described under ``Experimental
Procedures,'' and further incubated at 4 °C in this buffer
overnight. The islets were then analyzed by Tricine-urea-SDS-PAGE
without prior immunoprecipitation. The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown with arrows on the left
side.
Figure 4: Reduction of proinsulin disulfides in the ER is reversible. Isolated islets were pulse-labeled for 5 min (lane 1) and immediately exposed to 5 mM DTT for 10 min with the intention of fully reducing proinsulin in vivo (lane 2). The islets were then washed and chased in normal medium for the recovery times indicated (lanes 3 and 4) before lysis and overnight alkylation as in Fig. 3. The lysates were analyzed by Tricine-urea-SDS-PAGE without prior immunoprecipitation. The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin standards (St'd) are shown with arrows on the left side. Upon further chase, re-oxidized proinsulin was proteolytically converted to insulin (not shown), indicating normal intracellular transport to secretory granules.
Based on the reported t of
10-20 min for proinsulin exit from the ER (Steiner et
al., 1986) and a similar rate through the Golgi stacks (Howell et al., 1969), it is expected that by 20 min of chase, at
least half of labeled proinsulin should have exited the ER and been in
transit through Golgi cisternae. Over the course of a 20-min chase,
labeled proinsulin disulfides changed progressively from being largely
reduced upon low dose DTT exposure (Fig. 3, lane 1) to
predominantly resistant to reduction (Fig. 3, lane 4).
At 5 min of chase, roughly 70% of labeled proinsulin remained sensitive
to low dose DTT, whereas at 10 min this value approached 50%; these
data suggest a t for acquisition of DTT resistance 10
min.
Thus, based on the foregoing information,
conformational maturation of proinsulin in vivo appeared to
correlate with its intracellular transport. Specifically, these data
appeared consistent with a published hypothesis (Emdin et al.,
1980) that proinsulin might hexamerize before its exit from the ER.
However, these kinetic data do not exclude the possibility of initial
oligomerization in the Golgi (Jascur et al., 1991; Musil and
Goodenough, 1993) or the possibility of initial oligomerization in the
ER with larger assembly units forming subsequently (Wagner and Marder,
1984; Colley and Baenziger, 1987).
To more precisely define the
relationship between the initial acquisition of DTT resistance and
proinsulin export from the ER, pulse-chase experiments were performed
in the presence of brefeldin A (BFA, 10 µg/ml). Previous studies
have indicated that in -cells of mouse pancreatic islets (Huang
and Arvan, 1994), like in many other cells (Klausner et al.,
1992), the use of BFA blocks anterograde traffic of proteins out of the
ER. As expected for pulse-labeled islets exposed to DTT without prior
chase, the presence of BFA during the labeling period did not diminish
the susceptibility of
90% of newly synthesized proinsulin to
reduction with low dose DTT (Fig. 5A, lane 1). However,
even after 20 min of chase in the continuous presence of BFA,
proinsulin in live islets failed to acquire resistance to reduction
with low dose DTT (Fig. 5A, lane 2). Because BFA itself
is not known to interfere with protein folding or assembly within the
ER (Doms et al., 1989; Chen et al., 1991; Russ et
al., 1991; Collins and Mottet, 1992), the conformational
maturation revealed by DTT resistance must be a consequence of
intracellular transport of proinsulin, whereupon it encounters some
feature of the Golgi lumenal environment. Thus, the t of
10 min (Fig. 3) appears to reflect the kinetics of a
process that occurs immediately after rather than before proinsulin exit from the ER.
Figure 5:
Proinsulin resistance to DTT in vivo requires export from the ER. A, isolated islets were
preincubated for 10 min, pulse-labeled for 3 min, and chased for either
0 (lane 1) or 20 min (lane 2), all in the presence of
10 µg/ml BFA to block proinsulin transport from the ER. B,
a separate preparation of islets were pulse labeled for 5 min with S-labeled amino acids. In B, groups of islets
were examined either without chase (lane 1), after 20 min
chase (lane 2), or after chase for 20 min followed by an
additional 20-min chase in the presence of BFA (lane 3). In
both A and B, each sample was then exposed to low
dose DTT before lysis/alkylation with IAA as in Fig. 3. All
samples were then analyzed by Tricine-urea-SDS-PAGE without prior
immunoprecipitation. The positions of fully reduced and fully oxidized
proinsulin standards are shown with arrows on the left of each panel.
When pulse-labeled islets (Fig. 5B, lane 1) were chased 20 min so that most proinsulin had reached the Golgi (Fig. 5B, lane 2), further chase in the presence of BFA failed to disassemble or unfold proinsulin back to its previous, DTT-susceptible state (Fig. 5B, lane 3). These data confirm that, separate from general effects on membrane traffic, BFA has no specific effect on proinsulin conformation. However, independent of BFA, when pulse-labeled proinsulin was chased for either 15 or 30 min before islet lysis in a zinc-free medium, most labeled proinsulin was no longer resistant to reduction in the presence of low dose DTT (i.e.in vitro reduction with either 0.5 or 1.5 mM DTT, Fig. 6A). Importantly, significant resistance to reduction by 2 mM DTT was maintained simply by adding 0.5 mM zinc to the islet lysis medium (Fig. 6B). These data (Fig. 3-6) demonstrate that a new state in conformational maturation can be detected in live cells upon proinsulin transport from the ER to the Golgi; this new state is unaffected by subsequent addition of BFA, but is largely reversed in vitro upon dilution of proinsulin into a zinc-free, but not a zinc-containing, buffer.
During proinsulin
trafficking from the Golgi stacks to mature storage granules, there is
progressive acidification of the lumen of the secretory pathway (Orci et al., 1986). Since the ability of DTT to reduce disulfide
bonds is affected by the prevailing pH (Means and Feeney, 1971), we
were concerned that detection of delayed changes in proinsulin
disulfide sensitivity might be obscured by direct effects of pH on the
potency of DTT. To control for this, we initially examined the effect
of pH on the ability of DTT to reduce proinsulin that was largely
denatured by exposure to 8 M urea. Denaturation was intended
to minimize potential effects of pH on protein conformation per se (Bewley and Li, 1969), so that these data could be used largely as
a measure of DTT efficacy in disulfide reduction. As shown in Fig. 8A using a 10-min exposure at 37 °C to high
dose DTT, proinsulin was essentially fully reduced over the entire pH
range from 6.8 to 5.2, although a slight decrease was noted at pH 5.2. ()A similar point was demonstrated by in vitro reduction of bovine serum albumin (BSA), a constitutive secretory
protein that is highly disulfide-bonded and highly soluble (Gerdes et al., 1989; Freedman and Scheele, 1993). When treated with
SDS for denaturation, essentially full reduction of protein disulfides
was accomplished (Fig. 8B, lane 1). By contrast, when
BSA was dissolved in 150 mM NaCl containing only 0.1% Triton
X-100 (comparable with the islet lysis buffer), disulfide reduction was
limited, causing only minor SDS-PAGE mobility shifts for a large
fraction of the protein, although a very small fraction was fully
reduced at the highest pH (Fig. 8B, lane 7). Evidently,
in contrast with the effects of protein conformation per se (compare lanes 1 and 7), changes of pH in the
range from 6.8 to 5.2 (Fig. 8B, lanes 3-7) have
at most a modest effect on DTT-mediated disulfide reduction. Thus, the
ability of high dose (30 mM) DTT to cause full disulfide
reduction is not limiting (Fig. 8A),
except
to the extent that protein structure alters disulfide
reactivity/accessibility (Fig. 8B).
We therefore
proceeded to examine the susceptibility of disulfides to reduction in
pulse-labeled islets in which labeled proinsulin had been chased to the
trans-Golgi network or later compartments. Arrival of labeled
proinsulin at immature secretory granules could be monitored by ongoing
proteolytic conversion to insulin (Huang and Arvan, 1994). In these
experiments, although proinsulin in live islets was largely sensitive
to reduction with high dose DTT prior to the appearance of labeled
insulin, it was clear that a major step-up in resistance to reduction
occurred following proteolytic processing to insulin (Fig. 9A). Moreover, except for a possible small lag
time after initial insulin formation, resistance of newly made insulin
to high dose DTT relatively rapidly approached that found in mature
granules from the same preparation (as measured after overnight chase,
see Fig. 9B). These data suggest strongly that in
addition to initial conformational changes that take place upon
proinsulin transport from ER Golgi, new DTT resistance occurs
within immature granules (at least in part reflecting quaternary
structural maturation that occurs after C-peptide is released from the
proinsulin backbone (Huang and Arvan, 1994)), and this state is
preserved during the maturation of storage granules.
Figure 9:
Decreased accessibility of protein
disulfides follow proinsulin conversion to insulin. A and B represent independent experiments with two different
preparations of mouse islets. Isolated islets were pulse labeled for 5
min with S-labeled amino acids and then chased for the
times indicated. At each chase time, the islets were either
mock-treated or treated for an additional 10 min at 37 °C in
complete chase medium containing 30 mM DTT. After this, the
islets were washed in ice-cold buffer containing 50 mM IAA,
lysed, and further incubated in lysis buffer containing 50 mM IAA overnight at 4 °C. The islets were then analyzed by
Tricine-urea-SDS-PAGE without prior immunoprecipitation. The positions
of fully reduced (Red.) and fully oxidized (Ox.)
proinsulin (Pro) standards, as well as oxidized insulin (Ins), are shown with arrows on the left. A, in this experiment, the rate of proinsulin processing was
comparable to that in the majority of islet preparations. B,
this experiment was unusual in that proinsulin processing appeared
considerably faster than in other islet preparations, with visible
accumulation of proinsulin conversion intermediate (asterisk,
which appeared largely sensitive to DTT-mediated disulfide reduction).
In the 2-h chase sample, roughly half of labeled proinsulin was loaded
on the gel; thus, fluorography of these lanes was carried out for a
longer period. However, this difference in film exposure has no impact
on the evaluation of disulfide
accessibility.
Figure 10: Effects of chloroquine (100 µM) and zinc (100 µM) on stability of the insulin granule core in vivo. Isolated islets were pulse labeled for 5 min and then divided into six aliquots. Control islets (lanes 5 and 6) were chased for 2 h as shown. Another two groups of islets were treated with chloroquine from 70 to 120 min of chase. The last two groups of islets were treated with zinc throughout the chase. In each sample, the islets were either mock-treated or treated for an additional 10 min at 37 °C in complete chase medium containing 30 mM DTT. After this the islets were washed in ice-cold buffer containing 50 mM IAA, lysed, and further incubated in lysis buffer containing 50 mM IAA overnight at 4 °C. The islets were then analyzed by Tricine-urea-SDS-PAGE without prior immunoprecipitation. The positions of fully reduced (Red.) and fully oxidized (Ox.) proinsulin (Pro) standards, as well as oxidized and partially reduced insulin (Ins), are shown with arrows on the left. Note that, in agreement with previous findings (Kuliawat and Arvan, 1994), chloroquine treatment does not reduce the extent of proinsulin processing (compare lanes 1 and 5), although it eliminates that fraction of insulin which is fully resistant to reduction (compare lanes 2 and 6). By contrast, zinc treatment reduces the extent of proinsulin processing (compare lanes 3 and 5), although it augments the insulin fraction which is fully resistant to reduction (compare lanes 4 and 6).
This investigation has concentrated on new methods to explore
the quaternary structural maturation of proinsulin in the secretory
pathway of mouse pancreatic -cells. In selecting in vivo reduction as our primary tool, we were aware that, since it is
based on the spatial positioning of protein disulfide bonds, this
method may not work for all proteins and by itself cannot define the
size of assembled protein complexes. However, we are extremely
fortunate that the tertiary and quaternary structures of proinsulin and
insulin, the location of their three highly conserved disulfide bonds,
and their reduction have all been well characterized (Bewley and Li,
1969; Blundell et al., 1972). Furthermore, hexamerization of
this protein within the secretory pathway of
-cells is an
established fact (see below).
As measured by Tricine-urea-SDS-PAGE, exposure to increasing doses of DTT in vitro led to a stepwise mobility shift, confirming our ability to detect the integrity of the three disulfide bonds (Fig. 1). In vivo, although native disulfide bonds were intact immediately upon synthesis (Fig. 2), nearly all proinsulin disulfides appeared susceptible to low dose DTT ( Fig. 3and Fig. 5A). The reduced proinsulin readily re-oxidized upon washout of DTT (Fig. 4); these features of extensive reduction and efficient reoxidation may be a specific reflection of the ER environment (Braakman et al., 1992; Valetti and Sitia, 1994).
As a
function of chase in pulse labeled pancreatic islets, proinsulin became
nearly fully resistant to low dose DTT with a t (10 min)
that closely approximates the t of proinsulin transport from
the ER (Fig. 3). Thus, it initially seemed quite reasonable to
think that conformational maturation such as hexamerization might be a
rate-limiting step for proinsulin export. However, in islet
-cells
producing abundant quantities of a mutant proinsulin that is unable to
hexamerize (Gruppuso et al., 1984; Carroll et al.,
1988), there are no obvious defects in transport of the mutant through
the ER (nor the rest of the secretory pathway, as measured by
proteolytic conversion to mutant insulin). During intracellular
transport in normal
-cells, proinsulin encounters zinc, which in
most species is required for homohexamerization (Bentley et
al., 1992); furthermore, zinc stabilizes hexamer close-packing
during insulin condensation (Emdin et al., 1980). Thus,
proinsulin differs from most other exportable proteins in that even
after achieving conformational competence, hexamerization does not
proceed until the secretory protein reaches a compartment in which
sufficient zinc is present (Gold and Grodsky, 1984), which has never
been demonstrated in the ER. Thus, despite the kinetics showing
acquisition of resistance to low dose DTT, proinsulin hexamerization in
the ER cannot be assumed.
When BFA was added to mouse islets in
order to prevent protein export from the ER, the ability of proinsulin
to acquire resistance to reduction with low dose DTT was fully
inhibited (Fig. 5A). Since proinsulin receives no
post-translational glycosylation that might influence monomer folding,
this initial decrease in disulfide accessibility almost certainly
signifies oligomeric assembly (see below). Because BFA has no direct
inhibitory effects on protein folding or assembly (Doms et
al., 1989; Chen et al., 1991; Russ et al., 1991;
Collins and Mottet, 1992; and this report, Fig. 5B),
the present data, taken together, indicate that while closely
correlated with intracellular transport, proinsulin hexamer formation
is unlikely to occur within the ER of pancreatic -cells.
It is
believed that oligomeric assembly of proinsulin proceeds from folded
monomers to homodimers to homohexamers (Emdin et al., 1980;
Steiner et al., 1986). It is extremely unlikely that the
conformational maturation reflected by disulfide resistance to low dose
DTT (Fig. 3) represents either proinsulin monomer folding or
dimerization (as opposed to hexamer formation), for several reasons.
First, quality control mechanisms ensure that monomers are folded to an
advanced stage prior to export from the ER (Copeland et al.,
1988; Kim et al., 1992; Tatu et al., 1993), whereas
proinsulin resistance to low dose DTT occurs after ER export (see
above). Second, there is strong reason to suspect that proinsulin
dimers can form within the -cell ER, because in a manner dependent
essentially only on protein concentration, monomers and dimers
interconvert in dynamic equilibrium (Jeffrey and Coates, 1966). Third,
unlike hexamers, proinsulin dimers have no zinc requirement for
assembly and therefore should be able to form independently of
intracellular transport (Blundell et al., 1972; Goldman and
Carpenter, 1974; Emdin et al., 1980; Carroll et al.,
1988).
By contrast, the present data strongly support the view that
in pancreatic -cells, assembly of proinsulin hexamers occurs upon
exit from the ER, whereupon proinsulin encounters zinc ions. If newly
made proinsulin, chased to the Golgi complex, is intentionally diluted
(by cell lysis) into a zinc-deficient medium, the molecules reacquire
their previous sensitivity to low dose DTT (Fig. 6A).
This is analogous to the
10
-fold dilution of
exocytosed granule cores into the hepatic portal bloodstream (which
contains micromolar levels of zinc), causing near-instantaneous
dissolution of condensed insulin into free insulin monomers (Gold and
Grodsky, 1984). More importantly, when Golgi proinsulin is comparably
diluted into a medium containing 0.5 mM zinc ions, resistance
to low dose DTT is largely preserved (Fig. 6B). Based
on our knowledge of proinsulin structure, these findings are very
likely to signify stabilization of proinsulin hexamers as a consequence
of zinc addition (Emdin et al., 1980).
It must be
emphasized that just because exportable proteins normally dimerize in
the ER, it does not necessarily follow that folded monomers must be
restrained from transport (Hoshina and Boime, 1982; Peters et
al., 1984; Singh et al., 1990). Thus, it should not be
surprising that a proinsulin mutant defective for homodimerization can
in fact be transported through the secretory pathway (Quinn et
al., 1991). The significance of this observation with respect to
ER quality control remains unclear, since this finding is not based on
studies of a homozygous mutation in -cells, but rather represents
the results from transfected heterologous cells where possible
heterotypic proinsulin associations with abundant endogenous secretory
proteins have not been excluded (Quinn et al., 1991). Thus, in
pancreatic
-cells where proinsulin is the predominant secretory
protein, previously published studies and the current results lead us
to hypothesize that proinsulin normally forms dimers in the ER, but
does not hexamerize. We propose that upon export from ER to Golgi,
proinsulin encounters zinc and hexamerization proceeds.
In the
-cells of pancreatic islets, most if not all condensation of
regulated secretory protein begins in immature secretory granules,
because proinsulin does not form higher order insoluble aggregates
until after its proteolytic conversion to insulin (which takes place
within this compartment (Huang and Arvan, 1994)). This conclusion is
based upon the biophysical properties of proinsulin and insulin in
vitro (Frank and Veros, 1968; Grant et al., 1972;
Steiner, 1973; Emdin et al., 1980; Baker et al.,
1988; Weiss et al., 1990; Kuliawat and Arvan, 1994). To
complement these findings we have exploited disulfide accessibility in
Golgi and post-Golgi compartments in vivo (Chanat et
al., 1993; Tatu et al., 1993; Chanat et al.,
1994), using high dose DTT to monitor further quaternary structural
maturation of proinsulin during this stage of the secretory pathway (Fig. 7). After acquisition of resistance to low dose DTT, no
further change in sensitivity of proinsulin disulfides occurred until
shortly after the appearance of labeled insulin (Fig. 9). This
was true in spite of experimental variation in proinsulin conversion
rates and degrees of sensitivity to reduction between islet
preparations. In some samples where processing to insulin neared
completion (
90% converted), small amounts of residual proinsulin
also appeared to exhibit increased resistance to DTT; this is
consistent with the reported ability of small quantities of proinsulin
to participate in higher order insulin assemblies (Steiner, 1973).
Moreover, insulin condensation in immature granules was clearly
inhibited or enhanced by the presence of chloroquine or zinc,
respectively (Fig. 10), in agreement with previous studies
suggesting that not only do these agents alter the stability of the
-granule core (Kuliawat and Arvan, 1994), but zinc plays a role in
insulin condensation that is over and above its role in proinsulin
hexamer formation.
In conclusion, the studies in this report support
the notion that storage of insulin by formation of the -granule
core represents the culmination of a stepwise process of quaternary
structural maturation in the secretory pathway (Carroll et
al., 1988). Specifically, proinsulin exhibits at least three
predominant conformational states that are reflected in three discrete
stages of disulfide accessibility. We propose that proinsulin monomers
and probably dimers within the ER are reduced maximally, proinsulin
hexamers within the Golgi complex are reduced at an intermediate level,
and insulin (and a small quantity of co-condensed proinsulin) forms
higher order assemblies within secretory granules that are maximally
resistant to disulfide reduction. Thus, at least in the
-cells of
mouse pancreatic islets, condensation of insulin is predicated upon the
prior formation of proinsulin hexamers, and both processes are
facilitated by the presence of zinc. However, we must point out that at
present, nothing is known about the mechanism by which zinc ions enter
the secretory pathway in pancreatic
-cells; this will be an
important area for future investigation.