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MSGs is ~5.0 [89]. PC1/3 could be active in the TGN as well as in ISGs [93], whereas PC2 is

As ISGs mature, ISGs produce vesicles to remove proteins that are not required for MSGs, whereas insulin is retained in MSGs. For example, ISGs are known to produce constitutive-like vesicles that contain excess C-peptide than insulin to be secreted [95, 96]. Also, in contrast to constitutive secretory proteins, lysosomal enzymes are thought to be segregated from ISGs in pancreatic β-cells [97, 98]. Generally, lysosomal enzymes are synthesized in the ER as well as secretory proteins and transported to the Golgi apparatus (Figure 1). In the TGN and endosomes, lysosomal enzymes are recognized by mannose 6-phosphate receptors (MPRs) and then packaged into clathrin-coated vesicle (CCVs). Clathrin is a coat protein that forms a cage-like structure to produce CCVs in the post-Golgi compartment [99]. AP-1 is a clathrin adaptor that binds to MPRs and clathrin and mediates to form clathrin/AP-1-coated vesicles [100, 101]. Proinsulin ISGs have clathrin and AP-1 on their surfaces as well as MPRs [98]. Also, MSGs lose the signal of cathepsin B, a lysosomal protease, whereas ISGs still have a strong cathepsin B signal. These results suggest that lysosomal enzymes recognized by MPRs are removed from ISGs [98].

Syntaxin 6, a SNARE protein that is reported to be important for the homotypic fusion of ISGs in neuroendocrine cells [102], is also removed from insulin ISGs [98]. In neuroendocrine cells, it is thought that homotypic fusion plays an important role in SG maturation, and the fusion machineries required for homotypic fusion are different than that required for MSGs fusion to the PM [67, 102]. It is proposed that membrane fusion machinery is remodeled in the end of ISG maturation. The role of Syntaxin 6 and homotypic fusion in insulin granule maturation is not clear [103]. However, recent study showed that homotypic fusion could also be important in insulin granule maturation [104]. In islets from HID-1 KO mice, Vamp-4, another SNARE protein that is proposed in ISG-derived vesicle fusion to the PM in neuroendocrine cells [67], is mislocalized. Proinsulin processing and acidification are delayed, and by 3D electron microscopy, there are less homotypic fusion events [104]. Although the role of HID-1 and Vamp-4 in homotypic fusion in pancreatic β-cells should be addressed in the future, it is possible that

The MSGs are fused with the PM, and insulin and C-peptide are secreted upon stimulation. For details about the exocytosis of insulin granules, see the review articles [59, 105, 106].

Because of the importance of insulin in diabetes mellitus, insulin secretory pathway has been extensively studied. Recent advance in the understanding of biosynthetic pathway reveals the importance of ER stress in β-cell dysfunction and novel machineries of secretory granule biogenesis. However, still many questions remain. What are the mechanisms by which ER-stress

homotypic fusion might also be important for insulin granule formation.

thought to be active in ISGs and MSGs [91, 94].

3.2.2. Sorting by retention

48 Ultimate Guide to Insulin

3.2.3. Homotypic fusion

4. Conclusion

Michiko Saito<sup>1</sup> and Yoko Shiba<sup>2</sup> \*

\*Address all correspondence to: shibay@iwate-u.ac.jp

