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Loh et al. Extracell Vesicles Circ Nucleic Acids 2023;4:568-87 https://dx.doi.org/10.20517/evcna.2023.34 Page 572

Figure 2. TGN lumenal sorting mechanisms. Motifs that are required for the sorting of RSP proteins to DCVs. These include (A) the
interaction between vasopressin and neurophysin domains in their precursor form; (B) disulfide bond; (C) charged α-helices; (D) the
sorting signal motif of POMC that is conformation-dependent and comprises of two acidic residues, Asp10 and Glu14, and the two
hydrophobic residues, Leu11 and Leu18. (Figure reproduced from Cawley et al. with permission) [27] ; (E) The sorting mechanism for
POMC, pro-enkephalin, and BDNF use similar sorting motifs comprising of a pair of acidic amino acids binding to a pair of basic amino
acids in a sorting receptor, membrane CPE which associates specifically with cholesterol-sphingolipid-rich lipid raft domains at the TGN
membrane prior to budding off to form a DCV. (F) RSP proteins can also be sorted to the RSP by aggregation at pH 5-6 and 1-10 mM
2+
Ca inside the TGN lumen. TGN: trans-Golgi network; POMC: pro-opiomelanocortin; BDNF: brain-derived neurotrophic factor; CPE:
carboxypeptidase E; DCV: dense core vesicles.

Figure 3. Cytoplasmic contribution to DCV formation (I). (A) HID-1 promotes the acidification of TGN lumen by increasing H-ATPase
activity, subsequently decreasing pH value and facilitating protein aggregation; (B) The µ1A subunit of AP-1A complex is critical for the
sorting of PAM-1 to immature DCVs from TGN. Proteins (yellow), Protein Aggregates (orange), PAM1 (red). HID-1: high-temperature-
induced dauer formation protein 1; AP-1A: adaptor protein 1A; PAM-1: peptidylglycine α-amidating monooxygenase-1; TGN: trans-Golgi
network; DCV: dense core vesicles.

The knockout of HID-1 was reported to inhibit the regulated secretion of insulin in pancreatic β cells upon
[46]
its knockout in mice and reduce the levels of DCV cargo proteins in C. elegans [47,48] . One recent study
showed that HID-1 knockout in PC12 cells blocked the acidification of TGN lumen by the mis-localization
[49]
of the Golgi-targeted H-ATPase subunit 2 . Thus, it appears that HID-1 drives DCV formation by
facilitating the acidification of TGN lumen for the pH-dependent aggregation of RSP-targeted hormones
[Figure 3A].