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Page 573 Loh et al. Extracell Vesicles Circ Nucleic Acids 2023;4:568-87 https://dx.doi.org/10.20517/evcna.2023.34

One player involved in DCV formation at the interface is Adaptor protein 1A (AP-1A) complex. The µ1A
subunit of the complex is crucial for the sorting of two prohormone processing enzymes, carboxypeptidase
D (CPD) and peptidylglycine α-amidating monooxygenase-1 (PAM-1), to immature DCVs from the
[50]
TGN . The reduction of the µ1A subunit decreased the number of immature DCVs and caused the
appearance of RSP proteins in non-DCV vesicles. The AP-1A-mediated formation of DCVs appears to be
mediated by the interaction of the cytoplasmic tail of PAM-1 with AP-1A [Figure 3B].

Mechanisms for DCV formation at the cytoplasmic side of the TGN
A group of cytoplasmic proteins play a role in mediating the formation of DCVs at the cytoplasmic side of
the TGN [Figure 4]. In neurons, Vesicle transport through interaction with t-SNARE 1A/1B (Vti1a/1b)
[51]
mediates the formation of DCV . Upon the loss of Vti1a/1b, the transport of DCV cargo into the axon was
decreased, empty vesicles were accumulated, and the Golgi cisternae were distended. This suggests that
Vti1a/1b is required for the formation of DCVs in neurons. Vti1a also appears to play a role in Ca channel
2+
[52]
trafficking to the plasma membrane in chromaffin cells for DCV formation . In the chromaffin cells, Vti1a
was found at the TGN but not in mature DCVs. Upon the knockout of Vti1a, there were fewer secretory
2+
vesicles of reduced size and fewer Ca channels at the plasma membrane without any effect on the release
kinetics of Ca channels.
2+
2+
Ca -dependent activator protein for secretion 1 (CAPS1) that regulates the exocytosis of DCVs at the
secretion sites of the nerve terminals [53,54] was found to be involved in DCV formation at the TGN via the
interaction of its pleckstrin homology (PH) domain with ADP-ribosylation factor 4 (Arf4)/Arf5 in a GDP-
dependent manner in neuroendocrine cells . The knockdown of CAPS1 caused the accumulation of
[55]
chromogranin in the Golgi complex and reduced the secretion of DCVs. Similarly, the overexpression of
Arf5 mutants deficient in CAPS1-binding did the same, indicating that Arf5 is involved in CAPS1-mediated
DCV formation. In addition, CAPS1 appears to play a role in DCV formation in SgII- and BDNF-
containing DCVs in the brain . In forebrain-specific CAPS1 knockout mice, the protein levels of SgII and
[56]
syntaxin 6 (Stx6) were decreased, resulting in reduced presynaptic DCVs and dilated TGN cisternae. In
cerebellum-specific CAPS1 knockout, the protein levels of SgII and BDNF were reduced and the number of
DCVs at the fiber-Purkinje synapses was decreased significantly. These findings point to the significant role
of CAPS1 in DCV formation in the neurons.

Sorting nexin 19 (Snx19) appears to regulate insulin secretion and DCV formation via its interaction with
insulinoma-associated protein 2 (IA-2) in mouse pancreatic β-cells . In pancreatic MIN6 cells, the
[57]
knockdown of Snx19 not only reduced insulin content and secretion but also significantly decreased the
number and size of DCVs. The reintroduction of Snx19 into Snx19 knockdown cells reversed the abnormal
insulin secretion and DCV formation. This suggests that Snx19 is required for the formation of insulin-
containing DCVs in pancreatic β-cells. Rab2 also plays a major role in DCV formation at the cytoplasmic
side of the TGN. The collaboration of Rab2 with islet cell autoantigen of 69 kD (ICA69) and its GTPase
activating protein, Tre-2/Bub2/Cdc16 protein 8 (TBC-8), was found to be required for early DCV formation
at TGN [Figure 4] [58-62] .

Interestingly, microtubules also appear to be involved in DCV formation at the TGN, namely Golgi-derived
microtubules (GDMTs). In pancreatic β cells, the prevention of microtubule nucleation around the Golgi
complex drastically inhibited the exit of proinsulin from the TGN, resulting in the accumulation of
proinsulin in the Golgi cisternae . This report proposes that the biogenesis of DCVs at the TGN requires
[63]
microtubule nucleation for the generation of a GDMT network around DCV-forming sites.