Tumor protein D52 controls trafficking of an apical endolysosomal secretory pathway in pancreatic acinar cells

Abstract

ISG maturation progresses by removal of lysosomal membrane and select content proteins, which enter endosomal intermediates prior to their apical exocytosis. Constitutive and stimulated secretion through this mechanism is termed the constitutive-like and minor-regulated pathways, respectively. However, the molecular components that control membrane trafficking within these endosomal compartments are largely unknown. We show that tumor protein D52 is highly expressed in endosomal compartments following pancreatic acinar cell stimulation and regulates apical exocytosis of an apically directed endolysosomal compartment. Secretion from the endolysosomal compartment was detected by cell-surface antigen labeling of lysosomeassociated membrane protein LAMP1, which is absent from ZGs, and had incomplete overlap with surface labeling of synaptotagmin 1, a marker of ZG exocytosis. Although culturing (16 -18 h) of isolated acinar cells is accompanied by a loss of secretory responsiveness, the levels of SNARE proteins necessary for ZG exocytosis were preserved. However, levels of endolysosomal proteins D52, EEA1, Rab5, and LAMP1 markedly decreased with culture. When D52 levels were restored by adenoviral delivery, the levels of these regulatory proteins and secretion of both LAMP1 (endolysosomal) and amylase was strongly enhanced. These secretory effects were absent in alanine and aspartate substitutions of serine 136, the major D52 phosphorylation site, and were inhibited by brefeldin A, which does not directly affect the ZG compartment. Our results indicate that D52 directly regulates apical endolysosomal secretion and are consistent with previous studies, suggesting that this pathway indirectly regulates ZG secretion of digestive enzymes.TPD52; constitutive-like pathway; minor-regulated pathway; endolysosomal secretion PANCREATIC ACINAR CELLS ARE a traditional model for studying membrane and protein trafficking within the secretory pathway of mammalian cells (53) because they mediate the constitutive and regulated secretion of digestive enzymes necessary for nutrient digestion in the intestine. Pancreatic acini are arranged in clusters of 8 -12 cells with their apical plasma membranes forming the terminal end of pancreatic ductules. Secretagogue stimulation at the basolateral membrane occurs through G protein-coupled receptors to activate PLC and/or adenylyl cyclase pathways that induce the exocytosis of large (ϳ1 m diameter) zymogen granules (ZGs). Acutely isolated acini are typically used to study secretion because they are highly sensitive to secretagogues and remain polarized. However, these features are lost in culture through unknown mechanisms, making use of mRNA silencing techniques to reduce protein expression difficult.In addition to the classic ZG secretory pathway, two unique parallel secretory pathways have been identified in acinar cells from the pancreas and parotid glands known as the constitutive-like (CLP) and minor regulated (MRP) pathways (1,10,11,33,35). Newly synthesized secretory proteins are selec...

Vesicle Associated Membrane Protein 8 (VAMP8)-mediated Zymogen Granule Exocytosis Is Dependent on Endosomal Trafficking via the Constitutive-Like Secretory Pathway

“…EGFP-Rab11a S25N adenovirus was a kind gift from Rejji Kuruvilla (Johns Hopkins University). HA-D52 and GFP adenovirus were previously described (22). TeTx adenovirus was previously described (35).…”

“…Acinar Secretory Assays-Pancreatic acini were stimulated as previously described, and amylase released to the medium was determined by Phadebas amylase assay as previously described (22,36). Secretion was expressed as a percent of total cellular amylase and/or as amylase absorbance units normalized to total cellular DNA.…”

“…Furthermore, expressed D52 was found to strongly colocalize with Rab11a, a primary component of the apical recycling endosomal (RE) compartment. Use of the Golgi-and endosomal-disrupting agent brefeldin A together with cell surface labeling of the endolysosomal protein, lysosome-associated membrane protein 1 (LAMP1), implicated Rab5/D52/EEA1-mediated endosomal trafficking in regulating the secretory activity of the CLP/MRP (22). Results that a complete loss of the CLP/MRP compromised only half of the secretory response raised an important question regarding whether 1) the CLP/MRP are only indirectly involved in ZG exocytosis or 2) acinar cells have two pathways mediating ZG exocytosis, one of which is directly regulated by the CLP/MRP.…”

“…After a series of sucrose dehydrations in 1ϫ PBS at 4°C, tissue was embedded in Tissue Tek and flash-frozen in liquid nitrogen-cooled isopentane. Antibody labeling of cryostat sections (8 m) was from fresh or adenovirally infected lobules as previously described in detail (22).…”

Dropping in on the lipid droplet- tumor protein D52 (TPD52) as a new regulator and resident protein

“…TPD52 overexpression in different cancer types has been broadly reported, as have regulatory functions within signaling pathways. 9,10 However, these types of results can be demonstrated for many cellular proteins, so apart from the analysis of TPD52 functions in secretory processes, 11,12 progress in understanding TPD52 0 s molecular functions has been slow. 10 A lack of structural information has also made it difficult to predict how TPD52 contributes to macromolecular complexes, and what additional functions it might have.…”