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Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A - PubMed

Distinct molecular events during secretory granule biogenesis revealed by sensitivities to brefeldin A

C J Fernandez et al. Mol Biol Cell. 1997 Nov.

Free PMC article

Abstract

The biogenesis of peptide hormone secretory granules involves a series of sorting, modification, and trafficking steps that initiate in the trans-Golgi and trans-Golgi network (TGN). To investigate their temporal order and interrelationships, we have developed a pulse-chase protocol that follows the synthesis and packaging of a sulfated hormone, pro-opiomelanocortin (POMC). In AtT-20 cells, sulfate is incorporated into POMC predominantly on N-linked endoglycosidase H-resistant oligosaccharides. Subcellular fractionation and pharmacological studies confirm that this sulfation occurs at the trans-Golgi/TGN. Subsequent to sulfation, POMC undergoes a number of molecular events before final storage in dense-core granules. The first step involves the transfer of POMC from the sulfation compartment to a processing compartment (immature secretory granules, ISGs): Inhibiting export of pulse-labeled POMC by brefeldin A (BFA) or a 20 degrees C block prevents its proteolytic conversion to mature adrenocorticotropic hormone. Proteolytic cleavage products were found in vesicular fractions corresponding to ISGs, suggesting that the processing machinery is not appreciably activated until POMC exits the sulfation compartment. A large portion of the labeled hormone is secreted from ISGs as incompletely processed intermediates. This unregulated secretory process occurs only during a limited time window: Granules that have matured for 2 to 3 h exhibit very little unregulated release, as evidenced by the efficient storage of the 15-kDa N-terminal fragment that is generated by a relatively late cleavage event within the maturing granule. The second step of granule biogenesis thus involves two maturation events: proteolytic activation of POMC in ISGs and a transition of the organelle from a state of high unregulated release to one that favors intracellular storage. By using BFA, we show that the two processes occurring in ISGs may be uncoupled: although the unregulated secretion from ISGs is impaired by BFA, proteolytic processing of POMC within this organelle proceeds unaffected. The finding that BFA impairs constitutive secretion from both the TGN and ISGs also suggests that these secretory processes may be related in mechanism. Finally, our data indicate that the unusually high levels of unregulated secretion often associated with endocrine tumors may result, at least in part, from inefficient storage of secretory products at the level of ISGs.

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Figures

Figure 1
Figure 1

Subset of POMC-derived peptides secreted from AtT-20 cells can be labeled with [35S]sulfate. (A) Domain structure of POMC relevant to this study, showing N-linked glycosylation (CHO) sites and the temporal order of proteolytic cleavages at pairs of basic residues. Dark-shaded portion indicates the mature ACTH peptide against which antibodies used in this study were raised. Only POMC peptides that contain the major sulfation sites are shown. SP, signal peptide; ±CHO, the glycosylation site is not always used. ACTH-containing peptides secreted from [35S]methionine-labeled cells (B) and from [35S]sulfate labeled cells (C). Identical cultures of AtT-20 cells were labeled with [35S]methionine for 15 min or [35S]sulfate for 5 min. The cells were then chased in unlabeled DMEM for three consecutive 1-h periods. During the last hour of chase, 8-Br-cAMP was added to one well to stimulate release from mature regulated secretory granules. A parallel culture was chased in medium lacking 8-Br-cAMP as control. Chase medium was collected, ACTH-related peptides were immunoprecipitated with affinity-purified anti-ACTH, and radiolabeled bands in the immunoprecipitates were separated by SDS-PAGE on 18% gels and analyzed by PhosphorImager.

Figure 2
Figure 2

POMC is sulfated primarily on N-linked carbohydrates after the acquisition of endo H resistance. AtT-20 cells were pretreated with 5 μg/ml tunicamycin for 3.5 h and then labeled with [35S]sulfate for 5 min or [35S]methionine for 15 min. The cells were immediately extracted and labeled POMC was immunoprecipitated. One-half of each sample was then subjected to digestion with endo H. Lanes 1–4 are samples from methionine-labeled cells, and lanes 5–8 are from sulfate-labeled cells. For lanes 9 and 10, cells were labeled with [35S]sulfate as in lanes 5 and 6, except that digestion was performed with endo F instead of endo H. CHOS and CHOR, endo H-sensitive and endo H-resistant forms, respectively.

Figure 3
Figure 3

Sulfated POMC products migrate through intracellular compartments with increasing densities. (A) Sulfated POMC was recovered in light-density membranes immediately after pulse labeling. To localize the compartment for sulfation of POMC, AtT-20 cells were labeled with [35S]sulfate for 5 min and homogenized. Membranes from a PNS were fractionated on a D2O-Ficoll density gradient and labeled POMC products in each fraction were analyzed by SDS-PAGE and PhosphorImager. Under this condition, only unprocessed POMC was recovered. □, Relative amount of sulfate-labeled POMC (in arbitrary scanning units). To localize a constitutive secretory marker on this gradient, AtT-20 cells were treated with xyloside for 30 min and then pulse-labeled with [35S]sulfate for 5 min to label GAG chains. Cells were homogenized and membranes were fractionated as above. The amount of labeled GAG chains in each fraction was assayed by a CPC precipitation/filtration assay. •, Amount of GAG-associated radioactivity (in cpm) in each fraction. To localize rab 11-containing membranes on the same gradient, AtT-20 cells stably transfected with an influenza hemagglutinin epitope-tagged rab 11 were used, and rab11 immunoreactivity in each fraction was determined by immunoblotting using anti-hemagglutinin antibody. ○, Relative intensity obtained from scanning the Western blot. (B) Cells were continuously labeled with [35S]sulfate for 3 h. Xyloside was added to the cells during the last 30 min to label newly synthesized [35S]GAG chains. Cells were then homogenized and the membranes were fractionated as above. One half of each fraction was assayed for GAG, and the other half was subjected to immunoprecipitation with anti-ACTH. •, Amount of GAG-associated radioactivity (in cpm) in each fraction; □, distribution of labeled mature ACTH in arbitrary scanning units. Because no chase was performed, a small peak corresponding to newly labeled POMC was also present on the top of this gradient (not shown). POMC, GAG, and rab11 peak at a density of 1.12 g/ml, and mature ACTH peaks at a density of 1.20 g/ml.

Figure 4
Figure 4

Sulfate incorporation precedes endoproteolytic processing. (A) Continuous labeling. AtT-20 cells were labeled with [35S]sulfate for 5, 20, or 60 min. Cells were extracted with detergent, and the samples were immunoprecipitated with anti-ACTH antibodies. The labeled proteins in the immune precipitates were analyzed by SDS-PAGE and PhosphorImager. (B) Pulse–chase analysis. Cells were pulse-labeled with [35S]sulfate for 5 min and then chased for the times indicated. At the end of each chase, labeled POMC products in the cells were extracted, immunoprecipitated, and analyzed as in A. (C) Effect of a 20°C incubation on proteolytic processing; duplicate samples are shown. Lanes 1 and 2, cells pulse-labeled as in B (lane 1) were shifted to 20°C for 2 h before extraction. Under this condition, little processing occurred during the 20°C incubation period. Lanes 3 and 4, pulse-labeled cells were first chased at 37°C for 15 min as in B (lane 2) before being shifted to 20°C for 2 h. The 15-min chase allowed labeled POMC to exit the sulfation compartment, and subsequent incubation at 20°C resulted in the production of mature ACTH. Thus, processing requires POMC exit from the TGN, and the prohormone convertase is not inactivated at 20°C.

Figure 5
Figure 5

Cleavage products of POMC are differentially secreted and stored by AtT-20 cells. (A) The 15-kDa POMC N-terminal fragment is generated from the 18-kDa N-terminal fragment by a cleavage event that is blocked at 15°C. Cells were pulse labeled with [35S]sulfate for 5 min and either extracted immediately (lane 1) or chased for 15 min at 37°C to allow POMC to proceed to the processing compartment. The cells were then incubated either at 15°C for 3 h (lane 2) or at 37°C for 3 h (lane 3). Sulfate-labeled proteins from the cell extracts were acetone precipitated and analyzed by SDS-PAGE and PhosphorImager. Incubation at 15°C blocks conversion of the 18-kDa to the 15-kDa N-terminal fragment. (B) Kinetics of unregulated secretion of all sulfated products recovered by TCA precipitation. Identical wells of cells were pulse-labeled with [35S]sulfate for 5 min and then chased for the times indicated in DMEM containing bovine serum albumin. Medium (M) and cell extracts (C) were collected and processed by TCA precipitation. Material equal to approximately one-third of a 12-well was loaded onto the gel. Note the differences in the time course of production and secretion of the individual POMC-derived products. The total recovery of radioactivity was within 5% during 1- to 3-h chase. (C) Comparison of constitutive secretion of GAG chains and constitutive-like secretion of POMC products from AtT-20 cells. The secretion of POMC and the 18-kDa and the 15-kDa N-termini in B was quantitated by using the ImageQuant program (Molecular Dynamics) and normalized to the total amount of each form recovered at 180 min of chase. To determine kinetics of GAG chain secretion, cells pretreated with xyloside were pulse-labeled with [35S]sulfate for 5 min and the percentage of labeled GAG chains secreted into the medium at times indicated was determined using the CPC filtration assay.

Figure 6
Figure 6

Velocity gradient centrifugation demonstrates that POMC is sulfated in trans-Golgi/TGN but is processed in vesicular fractions corresponding to ISGs. A 15-cm dish of AtT-20 cells were pulse-labeled with [35S]sulfate for 5 min, and the cells were harvested either without chase (A) or after a 15-min chase at 37°C (B). A PNS was prepared and loaded on a 0.3–1.2 M sucrose gradient. Radiolabeled products were recovered from each gradient by acetone precipitation and analyzed by SDS-PAGE (on a 15% gel) and PhosphorImager.

Figure 7
Figure 7

BFA inhibits constitutive GAG chain secretion from AtT-20 cells. Cells were treated with xyloside for 30 min and then pulse-chased with [35S]sulfate for 5 min. BFA was added immediately after the pulse labeling. The cells were allowed to chase for up to 1 h. The amount of labeled GAG secreted into the medium was determined for each time point, and plotted as the percentage of total label recovered in the cell plus the medium for that time point. □, Secretion from control untreated cells; ○, secretion from BFA-treated cells.

Figure 8
Figure 8

BFA completely blocks the exit of POMC from the sulfation compartment and partially inhibits constitutive-like secretion from the processing compartment. The basic design of this experiment is shown at the top. AtT-20 cells were pulse-labeled for 5 min with [35S]sulfate. The cells were then immediately treated with 5 μg/ml BFA (condition b) or chased first for 15 min to allow the majority of labeled POMC to exit the sulfation compartment before the addition of BFA (condition c). Control cells were not treated (condition a). The cells were then chased for up to 135 min, and labeled POMC products during each chase and within the cells at the end of the chases were analyzed by immunoprecipitation, SDS-PAGE, and PhosphorImager analysis. Cell extracts were prepared at the indicated times. Lanes 1 and 2, untreated control cells; lanes 3 and 4, cells treated with BFA immediately after the pulse labeling at time 0; lanes 5–7, cells in which BFA was added after 15 min of chase. Medium samples were collected during the indicated chase intervals. Lanes 1, 0–15 min; lanes 1′, 15–45 min; lanes 2, 45–90 min; lanes 3, 90–135 min. Lanes 8–11 show secretion from untreated cells. Lanes 12–14 show that BFA completely inhibits export of POMC from the sulfation compartment. Lanes 15–17 show that BFA also reduced constitutive-like secretion from the processing compartment.

Figure 9
Figure 9

Condensation of secretory granules continues in the presence of BFA. AtT-20 cells were pulse-labeled with [35S]sulfate for 5 min and then chased for 15 min in unlabeled medium to allow the majority of labeled POMC to exit the sulfation compartment. To study the effect of BFA on subsequent maturation of secretory granules, BFA was then added to 5 μg/ml, final concentration, and the cells were chased for an additional 4 h (with fresh BFA added after 2 h of incubation). Control cells were allowed to chase in the absence of BFA. At the end of the chases, cells were lifted from the dish and a PNS was prepared and fractionated on a D2O-sucrose gradient. Samples from each fraction were immunoprecipitated with anti-ACTH and analyzed by SDS-PAGE and PhosphorImager. □, Distribution of unprocessed POMC after 15 min of chase before BFA addition; ○, distribution of mature ACTH after 4 h of chase in the absence of BFA; ⋄, distribution of mature ACTH after 4 h of chase in the presence of BFA. The amount of labeled POMC or mature ACTH in each fraction was calculated as the percentage of the sum of total label in that form throughout the entire gradient.

Figure 10
Figure 10

Working model for ACTH-granule biogenesis. POMC is transported to the trans-Golgi or TGN where it becomes sulfated. Budding of both constitutive secretory vesicles and nascent regulated granules is inhibited by BFA. Upon exiting the TGN, the pH of the nascent granules drops to about 5.5 to initiate proteolytic processing of POMC to yield intermediate forms, the 18-kDa N-terminal fragment, and mature ACTH. Proteolytic activation in ISGs is a BFA-insensitive process. Some unprocessed POMC, intermediate forms, and the 18-kDa N-terminal fragment exit the cells from ISGs in an unregulated manner, perhaps by incorporation into constitutive-like vesicles (question mark). This process is impaired by BFA. Maturation of ISGs is complete after 2–3 h of chase, at which time unregulated release stops and the mature granules exhibit tightly regulated state of exocytosis. This time coincides with the final cleavage of the 18-kDa N-terminal fragment, generating the 15-kDa N-terminal fragment. The latter is efficiently stored within mature secretory granules.

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