Mannose 6–Phosphate Receptors Are Sorted from Immature Secretory Granules via Adaptor Protein AP-1, Clathrin, and Syntaxin 6–positive Vesicles

Abstract

The occurrence of clathrin-coated buds on immature granules (IGs) of the regulated secretory pathway suggests that specific transmembrane proteins are sorted into these buds through interaction with cytosolic adaptor proteins. By quantitative immunoelectron microscopy of rat endocrine pancreatic β cells and exocrine parotid and pancreatic cells, we show for the first time that the mannose 6–phosphate receptors (MPRs) for lysosomal enzyme sorting colocalize with the AP-1 adaptor in clathrin-coated buds on IGs. Furthermore, the concentrations of both MPR and AP-1 decline by ∼90% as the granules mature. Concomitantly, in exocrine secretory cells lysosomal proenzymes enter and then are sorted out of IGs, just as was previously observed in β cells (Kuliawat, R., J. Klumperman, T. Ludwig, and P. Arvan. 1997. J. Cell Biol. 137:595–608). The exit of MPRs in AP-1/clathrin-coated buds is selective, indicated by the fact that the membrane protein phogrin is not removed from maturing granules. We have also made the first observation of a soluble N-ethylmaleimide–sensitive factor attachment protein receptor, syntaxin 6, which has been implicated in clathrin-coated vesicle trafficking from the TGN to endosomes (Bock, J.B., J. Klumperman, S. Davanger, and R.H. Scheller. 1997. Mol. Biol. Cell. 8:1261–1271) that enters and then exits the regulated secretory pathway during granule maturation. Thus, we hypothesize that during secretory granule maturation, MPR–ligand complexes and syntaxin 6 are removed from IGs by AP-1/clathrin-coated vesicles, and then delivered to endosomes.

Islet Cell Autoantigen of 69 kDa Is an Arfaptin-related Protein Associated with the Golgi Complex of Insulinoma INS-1 Cells

“…7F). Syntaxin 6 participates in the TGN-endosomal recycling pathway and is also found on ISGs (11,57). These observations implied that the perinuclear concentration of ICA69 is microtubule-dependent and emphasized the relationship of ICA69 with the Golgi complex.…”

“…Occasionally, ICA69 was detected on what appeared to be small vesicles pinching off the membrane of SGs. Whereas the presence of a coat was not readily apparent, such profiles could conceivably represent budding clathrin-coated vesicles that remove proteins not destined to MSGs (57,67,68). Protein removal from ISGs is part of the maturation process that leads to the formation of MSGs (69,70).…”

“…which is also associated with ISGs but not with MSG of ␤-cells (11,57). Additional studies will be necessary to determine whether ICA69 plays an active role in the maturation process of ISGs.…”

Clathrin Terminal Domain-Ligand Interactions Regulate Sorting of Mannose 6-Phosphate Receptors Mediated by AP-1 and GGA Adaptors

“…Acute Interference with Clathrin TD Function Impairs MPR Retrieval to the TGN-MPR sorting of newly synthesized lysosomal enzymes to the endolysosomal system is mediated by carriers coated with clathrin, AP-1, and/or GGAs (15)(16)(17). A hallmark of AP-1 dysfunction is the peripheral dispersion of MPRs caused by defective retrograde sorting to the TGN (10).…”

Visualization of TGN to Endosome Trafficking through Fluorescently Labeled MPR and AP-1 in Living Cells

“…MPRs, their bound ligands as well as syntaxin 6, a SNARE protein involved in TGN-endosome trafficking, can be detected in vesicular profiles coated with clathrin and AP-1 assembly proteins (Geuze et al, 1985;Bock et al, 1997;Klumperman et al, 1998) located in close vicinity of the TGN. In polarized cells, AP-1B, the epithelial specific adaptor complex that differs from the ubiquitously expressed AP-1A by exchange of its 1A subunit by the closely related 1B, functions by interacting with its cargo molecules and clathrin in the TGN, where it acts to sort basolateral proteins from proteins destined for the apical surface and from those selected by AP-1A for transport to endosomes and lysosomes (Folsch et al, 2001).…”