Differential Sorting of Lysosomal Enzymes Out of the Regulated Secretory Pathway in Pancreatic β-Cells

Abstract

In cells specialized for secretory granule exocytosis, lysosomal hydrolases may enter the regulated secretory pathway. Using mouse pancreatic islets and the INS-1 β-cell line as models, we have compared the itineraries of procathepsins L and B, two closely related members of the papain superfamily known to exhibit low and high affinity for mannose-6-phosphate receptors (MPRs), respectively. Interestingly, shortly after pulse labeling INS cells, a substantial fraction of both proenzymes exhibit regulated exocytosis. After several hours, much procathepsin L remains as precursor in a compartment that persists in its ability to undergo regulated exocytosis in parallel with insulin, while procathepsin B is efficiently converted to the mature form and can no longer be secreted. However, in islets from transgenic mice devoid of cation-dependent MPRs, the modest fraction of procathepsin B normally remaining within mature secretory granules is increased approximately fourfold. In normal mouse islets, immunoelectron microscopy established that both cathepsins are present in immature β-granules, while immunolabeling for cathepsin L, but not B, persists in mature β-granules. By contrast, in islets from normal male SpragueDawley rats, much of the proenzyme sorting appears to occur earlier, significantly diminishing the stimulusdependent release of procathepsin B. Evidently, in the context of different systems, MPR-mediated sorting of lysosomal proenzymes occurs to a variable extent within the trans-Golgi network and is continued, as needed, within immature secretory granules. Lysosomal proenzymes that fail to be sorted at both sites remain as residents of mature secretory granules.

Cathepsin L in secretory vesicles functions as a prohormone-processing enzyme for production of the enkephalin peptide neurotransmitter

“…In chromaffin cells, the high degree of colocalization of cathepsin L immunofluorescence with ME, confirmed by dual immunoelectron microscopy, demonstrates trafficking of cathepsin L to the regulated secretory pathway. In fact, earlier studies have indicated that cathepsin L is routed to immature secretory granules of insulinproducing cells, and a significant portion of cathepsin L remains as a resident protein of the mature secretory vesicle (26). In addition, cathepsin L is present in secretory granules of pituitary GH4Cl cells and undergoes regulated secretion induced by the secretagogues forskolin and phorbol 12-myristate 13-acetate (27).…”

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

“…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).…”

Toxoplasma gondii ADP-ribosylation Factor 1 Mediates Enhanced Release of Constitutively Secreted Dense Granule Proteins

“…Because this result was not expected, we considered at the time the alternative that a portion of dense granule proteins was released via a budding process from forming dense granules, analogous to the situation in pancreatic ␤-cells (27). Failure to block dense granule protein release using Brefeldin A was interpreted at the time to provide evidence that all release was from preformed dense granules.…”