ELAPOR1 is a secretory granule maturation-promoting factor that is lost during paligenosis

A single transcription factor, MIST1 (BHLHA15), maximizes secretory function in diverse secretory cells (like pancreatic acinar cells) by transcriptionally upregulating genes that elaborate secretory architecture. Here, we show that the scantly-studied MIST1 target, ELAPOR1, is an evolutionarily conserved, novel Mannose-6-phosphate receptor (M6PR) domain-containing protein. ELAPOR1 expression was specific to zymogenic cells (ZCs, the MIST1-expressing population in the stomach). ELAPOR1 expression was lost as tissue injury caused ZCs to undergo paligenosis (ie, to become metaplastic and reenter the cell cycle). In cultured cells, ELAPOR1 trafficked with cis-Golgi resident proteins and with the trans-Golgi and late endosome protein: cation-independent M6PR. Secretory vesicle trafficking was disrupted by expression of ELAPOR1 truncation mutants. Mass spectrometric analysis of co-immunoprecipitated proteins showed ELAPOR1 and CI-M6PR shared many binding partners. However, CI-M6PR and ELAPOR1 must function differently, as CI-M6PR co-immunoprecipitated more lysosomal proteins and was not decreased during paligenosis in vivo. We generated Elapor1−/− mice to determine ELAPOR1 function in vivo. Consistent with in vitro findings, secretory granule maturation was defective in Elapor1−/− ZCs. Our results identify a role for ELAPOR1 in secretory granule maturation and help clarify how a single transcription factor maintains mature exocrine cell architecture in homeostasis and helps dismantles it during paligenosis.

Sequential in vivo labeling of insulin secretory granule pools in INS-SNAP transgenic pigs

β cells produce, store, and secrete insulin upon elevated blood glucose levels. Insulin secretion is a highly regulated process. The probability for insulin secretory granules to undergo fusion with the plasma membrane or being degraded is correlated with their age. However, the molecular features and stimuli connected to this behavior have not yet been fully understood. Furthermore, our understanding of β cell function is mostly derived from studies of ex vivo isolated islets in rodent models. To overcome this translational gap and study insulin secretory granule turnover in vivo, we have generated a transgenic pig model with the SNAP-tag fused to insulin. We demonstrate the correct targeting and processing of the tagged insulin and normal glycemic control of the pig model. Furthermore, we show specific single- and dual-color granular labeling of in vivo–labeled pig pancreas. This model may provide unprecedented insights into the in vivo insulin secretory granule behavior in an animal close to humans.

Small disulfide loops in peptide hormones mediate self-aggregation and secretory granule sorting

ABSTRACTUnlike constitutively secreted proteins, peptide hormones are stored in densely packed secretory granules, before regulated release upon stimulation. Secretory granules are formed at the trans-Golgi network (TGN) by self-aggregation of prohormones as functional amyloids. The nonapeptide hormone vasopressin, which forms a small disulfide loop, was shown to be responsible for granule formation of its precursor in the TGN as well as for toxic fibrillar aggregation of unfolded mutants in the endoplasmic reticulum (ER). Several other hormone precursors also contain similar small disulfide loops suggesting their function as a general device to mediate aggregation for granule biogenesis. To test this hypothesis, we studied the capacity of small disulfide loops of different hormone precursors to mediate aggregation in the ER and the TGN. They indeed induced ER aggregation although to different extents in Neuro-2a and COS-1 cells. Fused to a constitutively secreted reporter protein, they also promoted sorting into secretory granules, enhanced stimulated secretion, and increased Lubrol insolubility in AtT20 cells. These results support the hypothesis that small disulfide loops act as novel signals for secretory granule biogenesis and sorting by self-aggregation.

Orchestrated Restructuring Events During Secretory Granule Maturation Mediate Intragranular Cargo Segregation

Regulated secretion is an essential process where proteins are packaged into membranous secretory vesicles. However, the details of cargo packaging and secretory granule maturation are largely unknown. Here, we demonstrate that multiple distinct proteins undergo orchestrated intragranular restructuring during secretory granule maturation in vivo, to allow spatial segregation of distinct components within the same granule. Furthermore, through a combination of genetics and multimodality imaging, we demonstrate the molecular identity of each distinct intragranular structure. We further identify genes that are essential for the temporally-ordered restructuring events, including those controlling pH (vha16.1), Cl- ions (Clic and ClC-c) and Ca2+ ions (fwe). Finally, we show that altered cargo glycosylation influences dimensions of these structures, thereby affecting secretory granule morphology. This study elucidates key steps and factors involved in intragranular, rather than intergranular, segregation of cargo through regulated restructuring events during secretory granule maturation. Understanding how multiple distinct proteins are efficiently packaged into and secreted from the same secretory granule may provide insight into diseases resulting from defects in secretion.

A novel function for Rab1 and Rab11 during secretory granule maturation

Regulated exocytosis is an essential process whereby specific cargo proteins are secreted in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo modifications, including an increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the Drosophila larval salivary glands as a model to characterize a novel role for Rab GTPases during granule maturation. We find that secretory granules increase in size ∼300-fold between biogenesis and release, and loss of Rab1 or Rab11 reduces granule size. Surprisingly, we find that Rab1 and Rab11 localize to secretory granule membranes. Rab11 associates with granule membranes throughout maturation, and Rab11 recruits Rab1. In turn, Rab1 associates specifically with immature granules and drives granule growth. In addition to roles in granule growth, both Rab1 and Rab11 appear to have additional functions during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new role for Rab GTPases in secretory granule maturation.

Insulin mRNA is stored in RNA granules in resting beta cells

The glucose-stimulated biosynthesis of insulin in pancreatic islet beta cells is post-transcriptionally regulated. Several RNA-binding proteins (RBPs) that regulate Insulin mRNA stability and translation also bind mRNAs coding for other insulin secretory granule (ISG) proteins. However, an overview of these interactions and their glucose-induced remodelling is still missing. Here we identify two distinct sets of RBPs which were preferentially pulled down with the 5'-UTRs of mouse Ins1, Ins2, spliced Ins2, Ica512/Ptprn and Pc2/Pcsk2 mRNAs from extracts of either resting or stimulated mouse insulinoma MIN6 cells compared to those recovered with the 5'-UTR of mouse Tubg1 encoding for γ-tubulin. Among RBPs binding in resting conditions to all tested transcripts for ISG components was hnRNP A2/B1. Hnrnpa2b1 KO MIN6 cells contained lower levels of Ins1 mRNA, proinsulin and insulin compared to control cells. In resting cells, both hnRNP A2/B1 and Insulin mRNAs localized to stress granules, which dissolved upon glucose stimulation. Insulin mRNA-positive RNA granules were also found in human pancreatic beta cells in situ. Our results suggest that resting beta cells store mRNAs for insulin secretory granule proteins in stress granules through specific RNA protein interactions. Glucose stimulation remodels these interactions, releasing the transcripts, and another set of RBPs coordinates their translation.