FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2044679375> ?p ?o ?g. }

• W2044679375 endingPage "20423" @default.
• W2044679375 startingPage "20417" @default.
• W2044679375 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. 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. 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 β-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 1The abbreviations used are: ERendoplasmic reticulumDTTdithiothreitolDMEDulbecco's modified Eagle's mediumIAAiodoacetamidePAGEpolyacrylamide gel electrophoresisTricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycineBFAbrefeldin ABSAbovine serum albuminMes4-morpholineethanesulfonic acid. (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. endoplasmic reticulum dithiothreitol Dulbecco's modified Eagle's medium iodoacetamide polyacrylamide gel electrophoresis N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine brefeldin A bovine serum albumin 4-morpholineethanesulfonic acid. With this in mind, we have examined pancreatic β-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 β-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. Collagenase was from Worthington (Freehold, NJ); Hypaque, human serum albumin, bovine serum albumin, soybean trypsin inhibitor, chloroquine, zinc sulfate, iodoacetamide, and DTT were from Sigma; calf serum and antibiotic-antimycotic solution were from Life Technologies, Inc.; [35S]methionine/cysteine (Express) were from DuPont NEN; and brefeldin A was from Epicentre Technologies Corp. (Madison, WI). Islets from 30-g CD-1 mice were isolated by pancreatic ductal perfusion with collagenase, flotation on a hypaque gradient, picking of individual islets, and recovery overnight in Dulbecco's modified Eagle's medium (DME) containing 10% calf serum plus 1% penicillin/streptomycin. Islets were washed twice with Met-free, Cys-free DME, prior to pulse labeling for times ranging up to 5 min at 37°C in the same medium containing ∼300 μCi of [35S]methionine and cysteine. At the conclusion of the pulse labeling, the islets were washed and chased in complete DME. All labeling and chase media also contained 0.5 mg/ml human serum albumin and 0.005% soybean trypsin inhibitor. 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 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 35S-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 ZnSO4, 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT)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 35S-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Islet lysates were analyzed by 15% acrylamide SDS-PAGE plus urea using a Tricine buffer system (Schagger and von Jagow, 1987). In this system, mouse proinsulins I and II were not resolved from one another, nor were insulins I and II. Reduced and oxidized mouse proinsulin were run as standards on all gels. Insulin gels were fixed initially in 20% trichloroacetic acid without alcohol, then in 12.5% trichloroacetic acid plus 50% methanol, then incubated briefly with water, and finally incubated with 1 M sodium salicylate for 20 min. Dried gels were exposed to XAR film at −70°C. Proinsulin has three highly conserved disulfide bonds; all are preserved within insulin. Based on the x-ray structure of the 2-Zn/insulin crystal, solvent accessibility is highest for the interchain B7-A7 bond, lower for the interchain B19-A20 disulfide, and most buried in the case of the intrachain A6-A11 bond (Blundell et al., 1971; Baker et al., 1988). In order to resolve different forms of proinsulin exhibiting either a full or partial complement of disulfide bonds, pulse-labeled mouse pancreatic islets were lysed and then treated with low doses of DTT in SDS-sample buffer. The partially reduced lysates were then alkylated with iodoacetamide and analyzed by Tricine-urea-SDS-PAGE and fluorography. A progressively decreasing mobility was observed for proinsulin as a function of the DTT dose used for in vitro exposure. Specifically, four discrete regions of proinsulin were detected (Fig. 1). It is apparent from these data that the four observed bands signify increasing reduction of proinsulin, corresponding to the hydrolysis of none, one, two, or all three proinsulin disulfide bonds. In order to estimate the rate at which native proinsulin disulfide bonds are formed in vivo, isolated mouse islets were pulse-labeled for 1 min and rapidly lysed either without chase or after a brief chase period. At each chase time, the islets were lysed in the presence of iodoacetamide and then analyzed under nonreducing conditions by Tricine-urea-SDS-PAGE and fluorography. As shown in Fig. 2, most labeled proinsulin was already fully oxidized at the zero chase time. Eliminating the possibility that proinsulin disulfides might form artifactually at the time of cell lysis, the same results were obtained when the islets were preincubated with 20 mMN-ethylmaleimide in ice-cold phosphate-buffered saline for 10 min before the lysis step (data not shown). Since the labeling itself was 1 min in duration, it is clear that formation of the three native disulfide bonds in proinsulin is complete within a minute and is likely to occur co-translationally. Nascent secretory proteins within the ER undergo significant monomer folding (Kim and Arvan, 1993) which may allow presentation of a suitable surface for recognition of oligomerization partners (Hurtley and Helenius, 1989). Because these processes are expected to result in decreased accessibility of protein disulfide bonds, we examined the susceptibility of disulfides in newly synthesized proinsulin to reduction by low doses of DTT. First, isolated islets were pulse-labeled for 5 min and chased for times ranging from 0 to 20 min, before exposure of the live cells to reducing agent. At the zero chase time, proinsulin disulfides were ≥90% reduced upon subsequent exposure to low dose (2 mM) DTT (Fig. 3, lane 1). If, after DTT exposure, the live islets were washed free of DTT and the 37°C chase continued in fresh medium, reduced proinsulin was quantitatively reoxidized (Fig. 4). Reoxidation was complete within 5 min after DTT washout and did not impair the ability of previously reduced protein to be transported through the secretory pathway and converted to insulin (data not shown). Thus, like for other exportable proteins (Braakman et al., 1992), proinsulin was reduced in the ER, and this reduction was fully reversible.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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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. 2Calculations 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). 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 β-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. 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. Since proinsulin eventually proceeds along the intracellular transport pathway to immature secretory granules where proteolytic conversion to insulin occurs (Huang and Arvan, 1994), we performed new dose-response curves of proinsulin at later chase times in order to develop an assay of further changes in the accessibility of proinsulin disulfide bonds in live pancreatic β-cells. At 20 min of chase when initial resistance of proinsulin disulfides to reduction with low dose DTT had achieved a maximum (Fig. 3), progressive reduction could still be detected as the DTT dose was incrementally raised by more than an order of magnitude (Fig. 7). Although at delayed chase times, some variability was observed in the degree of sensitivity of proinsulin and processed insulin to in vivo reduction (i.e. in occasional experiments, essentially complete proinsulin reduction and even significant insulin reduction was observed with high dose DTT (see below)), 3Despite 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. we elected to employ 30 mM DTT as our standard means by which to monitor further conformational maturation of proinsulin in the distal secretory pathway. 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. 4We 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. 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),4 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. Previous reports have indicated that in pancreatic β-cells, most of the formation of insoluble aggregates of regulated secretory protein begins within immature granules (Huang and Arvan, 1994) and that 100 μM chloroquine or zinc added to the islet chase medium selectively inhibits or promotes granule core condensation, respectively (Kuliawat and Arvan, 1994). Since these conclusions are based upon in vitro observations, we sought to examine the effect of these perturbations on β-granule core formation as monitored by proinsulin and insulin disulfide accessibility in vivo. In control islets at 2-h chase, roughly 70% of proinsulin had been converted to insulin (Fig. 10, lane 5). Although the remaining proinsulin in the control islets was nearly fully reduced by in vivo exposure to high dose DTT (lane 6), insulin contained within the immature granules of these islets was fairly evenly divided into three portions in which: 1) no disulfide reduction was evident, 2) at least one disulfide bond had been broken (the insulin band shifted up in the gel), or 3) both interchain disulfide bonds were broken (insulin hydrolyzed into separate A and B chains ran off our Tricine-urea-SDS-PAGE system and therefore could only be detected as protein under-recovery). The addition of chloroquine from 70 to 120 min of chase did not inhibit the conversion of proinsulin to insulin (Fig. 10, compare lanes 1 and 5), nor the fraction of proinsulin that was reduced by exposure to DTT (lanes 2 and 6). However, upon chloroquine exposure, none of the insulin contained within immature granules was protected from disulfide reduction: a minority of the insulin was partially reduced (band shift up), whereas most had lost both interchain disulfide bonds (compare lanes 1 and 2). Finally, islet exposure to zinc appeared to modestly slow the rate of proinsulin conversion within immature granules (Fig. 10, lane 3). Nevertheless, this increased fraction of proinsulin remained highly sensitive to high dose DTT, whereas the insulin contained within immature granules showed markedly increased resistance to reduction (lane 4). These data indicate that chloroquine treatment did not block proinsulin conversion to insulin but inhibited granule core formation, whereas zinc treatment modestly slowed proinsulin conversion and yet enhanced granule core formation. This investigation has concentrated on new methods to explore the quaternary structural maturation of proinsulin in the secretory pathway of mouse pancreatic β-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 β-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 (Figure 3:, Figure 4:, Figure 5:A). 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 β-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 β-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 β-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 β-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 β-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 ∼107-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 β-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 β-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 β-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 β-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 β-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 β-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 β-cells; this will be an important area for future investigation. We thank members of the Arvan laboratory for help during the progress of these studies." @default.
• W2044679375 created "2016-06-24" @default.
• W2044679375 creator A5072170846 @default.
• W2044679375 creator A5080668619 @default.
• W2044679375 date "1995-09-01" @default.
• W2044679375 modified "2023-10-13" @default.
• W2044679375 title "Intracellular Transport of Proinsulin in Pancreatic β-Cells" @default.
• W2044679375 cites W1429534966 @default.
• W2044679375 cites W1487304679 @default.
• W2044679375 cites W1493572921 @default.
• W2044679375 cites W1494127461 @default.
• W2044679375 cites W1530972565 @default.
• W2044679375 cites W1537255763 @default.
• W2044679375 cites W1562863707 @default.
• W2044679375 cites W1576764274 @default.
• W2044679375 cites W1580586970 @default.
• W2044679375 cites W1597334704 @default.
• W2044679375 cites W1606310877 @default.
• W2044679375 cites W1931642121 @default.
• W2044679375 cites W1963661748 @default.
• W2044679375 cites W1965686966 @default.
• W2044679375 cites W1974175370 @default.
• W2044679375 cites W1975073093 @default.
• W2044679375 cites W1975085160 @default.
• W2044679375 cites W1981862236 @default.
• W2044679375 cites W1985959092 @default.
• W2044679375 cites W1996562607 @default.
• W2044679375 cites W200246087 @default.
• W2044679375 cites W2006943139 @default.
• W2044679375 cites W2006989237 @default.
• W2044679375 cites W2009468057 @default.
• W2044679375 cites W2010127283 @default.
• W2044679375 cites W2017134019 @default.
• W2044679375 cites W2018546389 @default.
• W2044679375 cites W2027186246 @default.
• W2044679375 cites W2030093057 @default.
• W2044679375 cites W2041331557 @default.
• W2044679375 cites W2045039384 @default.
• W2044679375 cites W2045439330 @default.
• W2044679375 cites W2045750170 @default.
• W2044679375 cites W2047420354 @default.
• W2044679375 cites W2050638929 @default.
• W2044679375 cites W2054668289 @default.
• W2044679375 cites W2059428912 @default.
• W2044679375 cites W2063042519 @default.
• W2044679375 cites W2063919679 @default.
• W2044679375 cites W2078588517 @default.
• W2044679375 cites W2081081246 @default.
• W2044679375 cites W2096678905 @default.
• W2044679375 cites W2113922695 @default.
• W2044679375 cites W2115912999 @default.
• W2044679375 cites W2121533147 @default.
• W2044679375 cites W2130079621 @default.
• W2044679375 cites W2133449590 @default.
• W2044679375 cites W2156467838 @default.
• W2044679375 cites W2156602181 @default.
• W2044679375 cites W2172683288 @default.
• W2044679375 cites W2341325028 @default.
• W2044679375 cites W79276580 @default.
• W2044679375 doi "https://doi.org/10.1074/jbc.270.35.20417" @default.
• W2044679375 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/7657617" @default.
• W2044679375 hasPublicationYear "1995" @default.
• W2044679375 type Work @default.
• W2044679375 sameAs 2044679375 @default.
• W2044679375 citedByCount "111" @default.
• W2044679375 countsByYear W20446793752012 @default.
• W2044679375 countsByYear W20446793752013 @default.
• W2044679375 countsByYear W20446793752014 @default.
• W2044679375 countsByYear W20446793752015 @default.
• W2044679375 countsByYear W20446793752016 @default.
• W2044679375 countsByYear W20446793752017 @default.
• W2044679375 countsByYear W20446793752018 @default.
• W2044679375 countsByYear W20446793752019 @default.
• W2044679375 countsByYear W20446793752020 @default.
• W2044679375 countsByYear W20446793752021 @default.
• W2044679375 countsByYear W20446793752022 @default.
• W2044679375 countsByYear W20446793752023 @default.
• W2044679375 crossrefType "journal-article" @default.
• W2044679375 hasAuthorship W2044679375A5072170846 @default.
• W2044679375 hasAuthorship W2044679375A5080668619 @default.
• W2044679375 hasBestOaLocation W20446793751 @default.
• W2044679375 hasConcept C126322002 @default.
• W2044679375 hasConcept C134018914 @default.
• W2044679375 hasConcept C185592680 @default.
• W2044679375 hasConcept C2779306644 @default.
• W2044679375 hasConcept C2780212709 @default.
• W2044679375 hasConcept C55493867 @default.
• W2044679375 hasConcept C71924100 @default.
• W2044679375 hasConcept C79879829 @default.
• W2044679375 hasConcept C86803240 @default.
• W2044679375 hasConcept C90061646 @default.
• W2044679375 hasConcept C95444343 @default.
• W2044679375 hasConceptScore W2044679375C126322002 @default.
• W2044679375 hasConceptScore W2044679375C134018914 @default.
• W2044679375 hasConceptScore W2044679375C185592680 @default.
• W2044679375 hasConceptScore W2044679375C2779306644 @default.
• W2044679375 hasConceptScore W2044679375C2780212709 @default.
• W2044679375 hasConceptScore W2044679375C55493867 @default.

• next