Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles - PubMed (original) (raw)

Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles

J Klumperman et al. J Cell Biol. 1998.

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 beta 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 approximately 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 beta 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.

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Figures

Figure 1

Ultrathin cryosections of endocrine pancreatic β cells that were double-immunogold labeled for proinsulin (5-nm gold) and CD-MPR (10-nm gold). Granules that were positively stained for proinsulin were marked as IGs (i), whereas proinsulin-negative granules were considered to be matured (M). Note also that mature β granules have the classically described appearance of an electron lucent halo between their content and limiting membrane. (A and B) CD-MPR label is present in the limiting membrane of IGs, but absent from mature granules. (C) An example of a proinsulin-positive condensing vacuole. The arrow points to a clathrin-coated (the coat is indicated by small arrows) membrane bud containing CD-MPR label. Additional CD-MPR staining is found over clathrin-coated TGN vesicles (large arrowheads) and small transport vesicles (small arrowheads). G, Golgi. Bars, 200 nm.

Figure 2

Ultrathin cryosections of endocrine pancreatic β cells. (A and B) Double-immunogold labeling for proinsulin (5-nm gold) and AP-1 (10-nm gold). AP-1 is present in clathrin-coated (arrows) membrane buds of proinsulin-positive IGs (i) and in clathrin-coated membrane buds evolving from TGN membranes (A, arrowheads). (C) Double-immunogold labeling for proinsulin (5-nm gold) and phogrin (10-nm gold). Gold particles indicating the presence of phogrin (arrowheads) are found in both IGs as well as mature (M) granules. G, Golgi. Bars, 200 nm.

Figure 3

Immunogold localization of CI-MPR (A) and AP-1 (B) in the Golgi (G) region of rat parotid acinar cells. (A) CI-MPR is present in IGs (i) with less electron-dense contents. At some sites, MPR-positive granule membranes display the electron-dense coating characteristic for the presence of clathrin (arrows). (B) The presence of AP-1 in clathrin-coated (arrows) membrane areas of IGs with less electron-dense contents. AP-1 label is also associated with vesicles budding from TGN tubules (arrowheads). Bars, 200 nm.

Figure 4

Ultrathin cryosections of rat pancreatic acinar cells illustrating immunogold labeling in the TGN region for CD-MPR (A), AP-1 (B), and AP-1 + CI-MPR (C). Note the irregular shape of IGs (i). (A) CD-MPR is clearly present in clathrin-coated (arrows) membranes budding from an IG. (B) AP-1 associated with clathrin-coated (arrows) membranes budding from an IG. (C) Double-immunogold labeling showing colocalization of CI-MPR (10-nm gold) and AP-1 (15-nm gold) in clathrin-coated (_arrow_s) membrane areas of an IG. G, Golgi. Bars, 200 nm.

Figure 5

Ultrathin cryosections of rat parotid acinar cells showing immunogold labeling for cathepsin B (catB) in secretory granules. (A) Two secretory granules that markedly differ in the electron densities of their protein cores. Note that the labeling density for cathepsin B tends to be significantly higher in the IG (i) with the least dense contents. (B) Occasional cathepsin B labeling in membrane buds emerging from a forming secretory granule. Note on the lower bud the presence of a clathrin coat (arrows). G, Golgi. Bars, 200 nm.

Figure 6

Releasable and intracellular forms of cathepsin B in the rat exocrine tissues. (A–E) Parotid; (F–H) pancreas. (A–D and F–G) Lobules were pulse labeled with 35S-amino acids for 30 min; chase media were collected from lobules bathed either in the absence (−) or presence (+) of secretagogue, and the media (C, D, and G) and cell lysates (A and G) were immunoprecipitated with an antibody that recognizes both ProB and mature cathepsin B (refer to Materials and Methods). Equal aliquots of chase media were analyzed by SDS-PAGE/fluorography. (B and F): Equal aliquots of chase media were analyzed by SDS-PAGE without previous immunoprecipitation, and the secretion of newly synthesized amylase was identified on fluorographs based on band mobility relative to Coomassie-stainable amylase and molecular weight standards (left). (C and G) Additional aliquots of chase media analyzed in B and F were immunoprecipitated for cathepsin B and then analyzed by SDS-PAGE/fluorography. Note the stimulus-dependent secretion of the lysosomal prohydrolase. (C) The small amount of a ∼35-kD band directly beneath ProB does not represent mature B, was not a consistent finding, and most likely represents partial proteolysis in the medium samples. (G) The small stimulated release of a ∼30-kD band directly beneath ProB most likely represents proteolysis of ProB in the medium sample rather than mature cathepsin B, because mature cathepsin B still had not appeared intracellularly at the 3-h chase time. (D) Immunoprecipitation of cathepsin B yielded only a modest stimulus-dependent secretion of labeled ProB during the 3–5 h chase period. (E and H) Immunoblot of parotid and pancreatic tissue, respectively, with anti-cathepsin B. The mature, lysosomal form predominates at steady state in both tissues.

Figure 7

Golgi (G) region of endocrine pancreatic β cells immunogold labeled for the SNARE protein syntaxin 6. (A) Syntaxin 6-labeling is concentrated in clathrin-coated (arrows) membrane buds of an IG and absent from granules with a mature morphology. (B) Colocalization of CD-MPR (10-nm gold) and syntaxin 6 (15-nm gold) on IGs (arrows) and TGN vesicles (arrowheads). Bars, 200 nm.

Figure 8

Localization of syntaxin 6 in the Golgi (G) region of a rat parotid acinar cell. Label is present on the limiting membrane of an IG (i) with less dense contents, but absent from those with highly condensed contents. Additional label is seen in a CCV (large arrowhead) as well as in noncoated TGN vesicles and tubules (small arrowheads). Inset, Colocalization of AP-1 (10-nm gold) and syntaxin 6 (15-nm gold) in a clathrin-coated (arrows) region of an IG. Bars, 200 nm.

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Mannose 6-phosphate receptors are sorted from immature secretory granules via adaptor protein AP-1, clathrin, and syntaxin 6-positive vesicles - PubMed (original) (raw)