Adaptor protein-3: A key player in RBL-2H3 mast cell mediator release

Abstract

Mast cell (MC) secretory granules are Lysosome-Related Organelles (LROs) whose biogenesis is associated with the post-Golgi secretory and endocytic pathways in which the sorting of proteins destined for a specific organelle relies on the recognition of sorting signals by adaptor proteins that direct their incorporation into transport vesicles. The adaptor protein 3 (AP-3) complex mediates protein trafficking between the trans-Golgi network (TGN) and late endosomes, lysosomes, and LROs. AP-3 has a recognized role in LROs biogenesis and regulated secretion in several cell types, including many immune cells such as neutrophils, natural killer cells, and cytotoxic T lymphocytes. However, the relevance of AP-3 for these processes in MCs has not been previously investigated. AP-3 was found to be expressed and distributed in a punctate fashion in rat peritoneal mast cells ex vivo. The rat MC line RBL-2H3 was used as a model system to investigate the role of AP-3 in mast cell secretory granule biogenesis and mediator release. By immunofluorescence and immunoelectron microscopy, AP-3 was localized both to the TGN and early endosomes indicating that AP-3 dependent sorting of proteins to MC secretory granules originates in these organelles. ShRNA mediated depletion of the AP-3 δ subunit was shown to destabilize the AP-3 complex in RBL-2H3 MCs. AP-3 knockdown significantly affected MC regulated secretion of β-hexosaminidase without affecting total cellular enzyme levels. Morphometric evaluation of MC secretory granules by electron microscopy revealed that the area of MC secretory granules in AP-3 knockdown MCs was significantly increased, indicating that AP-3 is involved in MC secretory granule biogenesis. Furthermore, AP-3 knockdown had a selective impact on the secretion of newly formed and newly synthesized mediators. These results show for the first time that AP-3 plays a critical role in secretory granule biogenesis and mediator release in MCs.

Host Retromer Protein Sorting Nexin 2 Interacts with Human Respiratory Syncytial Virus Structural Proteins and is Required for Efficient Viral Production

“…This observation is in agreement with previous demonstration of the recruitment of the adaptor protein complex-3 (AP-3), known to be involved in protein traffic between endosomes and the Golgi (33)(34)(35), to HRSV inclusion bodies and its interaction with the HRSV M protein (36). Furthermore, SNX2 was shown to colocalize with the AP-3 complex, which in turn, colocalizes with Vps26 (34,37). Moreover, a recent study has pointed out the colocalization of HRSV glycosylated G and nonglycosylated proteins in the same vesicles as the glycoprotein G recycles back from the plasma membrane (38).…”

“…The findings of the present study indicate that there is an association of HRSV nonglycosylated proteins with elements of the secretory pathway. This observation is in agreement with previous demonstration of the recruitment of the adaptor protein complex-3 (AP-3), known to be involved in protein traffic between endosomes and the Golgi ( 33 – 35 ), to HRSV inclusion bodies and its interaction with the HRSV M protein ( 36 ). Furthermore, SNX2 was shown to colocalize with the AP-3 complex, which in turn, colocalizes with Vps26 ( 34 , 37 ).…”

ESCRT machinery components are required for Orthobunyavirus particle production in Golgi compartments

“…Cells were then fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (pH 7.4) for 1 h and routinely processed for Electron Microscopy analysis as described further below. For pre-embedding immunoelectron microscopy, cells were processed as previously described [ 58 ]. Briefly, cells were fixed by microwave irradiation in 0.05% glutaraldehyde plus 4% formaldehyde in 0.1 M cacodylate buffer (pH 7.4) and subsequently immunolabeled with anti-OROV antibody and with goat anti-mouse IgG conjugated to nanogold (Nanoprobes).…”

Overlapping Machinery in Lysosome-Related Organelle Trafficking: A Lesson from Rare Multisystem Disorders

“…AP3 plays an essential role also in the biogenesis and release of secretory granule in mast cells [ 128 ]. Disruption of cargo proteins delivery to dense granules could explain platelet deficiency and excessive bleeding in HPS2 and HPS10, but these proteins have yet to be identified.…”