Interaction of Calcium-dependent Activator Protein for Secretion 1 (CAPS1) with the Class II ADP-ribosylation Factor Small GTPases Is Required for Dense-core Vesicle Trafficking in the trans-Golgi Network*

Ca2+-dependent activator protein for secretion (CAPS) regulates exocytosis of catecholamine- or neuropeptide-containing dense-core vesicles (DCVs) at secretion sites, such as nerve terminals. However, large amounts of CAPS protein are localized in the cell soma, and the role of somal CAPS protein remains unclear. The present study shows that somal CAPS1 plays an important role in DCV trafficking in the trans-Golgi network. The anti-CAPS1 antibody appeared to pull down membrane fractions, including many Golgi-associated proteins, such as ADP-ribosylation factor (ARF) small GTPases. Biochemical analyses of the protein-protein interaction showed that CAPS1 interacted specifically with the class II ARF4/ARF5, but not with other classes of ARFs, via the pleckstrin homology domain in a GDP-bound ARF form-specific manner. The pleckstrin homology domain of CAPS1 showed high affinity for the Golgi membrane, thereby recruiting ARF4/ARF5 to the Golgi complex. Knockdown of either CAPS1 or ARF4/ARF5 expression caused accumulation of chromogranin, a DCV marker protein, in the Golgi, thereby reducing its DCV secretion. In addition, the overexpression of CAPS1 binding-deficient ARF5 mutants induced aberrant chromogranin accumulation in the Golgi and consequently reduced its DCV secretion. These findings implicate a functional role for CAPS1 protein in the soma, a major subcellular localization site of CAPS1 in many cell types, in regulating DCV trafficking in the trans-Golgi network; this activity occurs via protein-protein interaction with ARF4/ARF5 in a GDP-dependent manner.

The Ca 2ϩ -dependent activator protein for secretion (CAPS) 2 family is involved in dense-core vesicle (DCV) exocytosis (1)(2)(3) and, in mammals, consists of two family members, CAPS1 (2) and CAPS2 (4,5). Many previous studies have suggested that CAPS1 plays a role in the secretion of catecholamines (e.g. norepinephrine (2,3)), neuropeptides (e.g. neuropeptide Y (6)), and peptide hormones (e.g. insulin (7)) by binding to phosphatidylinositol 4,5-bisphosphate at the priming step of DCV exocytosis (8,9). On the other hand, recent studies using knock-out (KO) or knockdown (KD) approaches have led to fresh debates concerning the involvement of CAPS1 in the priming of synaptic vesicle exocytosis (10) as well as in the vesicular loading of catecholamine or serotonin (11,12). Although these previous studies focused on the role of CAPS protein in exocytosis at secretion sites, such as nerve terminals or the cell periphery, a large fraction of CAPS protein in many neuronal cell types is actually localized in the soma rather than at these secretion sites (2,13,14). Therefore, the role of somal CAPS proteins, which constitute the largest amount of CAPS proteins in the cell, also needs to be elucidated.
In this study, we have investigated the role of somal CAPS1 proteins in the DCV secretory pathway, including biogenesistrafficking-secretion events. We showed an interaction between CAPS1 and the class II ADP-ribosylation factor (ARF) small GTPase on the Golgi membrane, depending on the ARF-GDP mode. The ARF family consists of three classes and a total of six members: class I, consisting of ARF1 to -3; class II, consisting of ARF4 and -5; and class III, which comprises ARF6 (15). The class I (16 -18) and class III ARFs (19,20) have been implicated as important regulators for membrane trafficking, but little is known about the role of the class II ARFs in membrane trafficking. Knockdown of CAPS1 or of the class II ARFs caused accumulation of a DCV marker protein, chromogranin, in the Golgi, resulting in reduced chromogranin secretion. Overexpression of ARF5 mutants that fail to bind CAPS1 also induced accumulation of chromogranin in the Golgi, resulting in a reduction of chromogranin secretion. These results suggest that CAPS1 has a regulatory role, in concert with GDP/GTP binding state-dependent class II ARFs, in DCV trafficking and/or biogenesis in the trans-Golgi network.
Secretion Assay-Mouse chromogranin A and secretogranin II cDNA was subcloned into pEF-BOS with the C-terminal triple HA tag to create pEF-BOS-ChgA-HA and pEF-BOS-SgII-HA, respectively. Transfections were carried out in 12-well plates using Lipofectamine 2000 reagent (Invitrogen) as described previously (5). In these experiments, we also transfected PC12 cells with Lipofectamine complexed with 0.2 g of pEF-BOS-granin-HA and 20 pmol of CAPS1 stealth siRNA (catalog no. RSS304417, Invitrogen). After incubation with Lipofectamine for 24 h, cells were washed and grown in DMEM containing 100 ng/ml NGF. 48 h after transfection, two different DMEMs were used as a stimulating medium: standard DMEM (catalog no. 11965, Invitrogen) and high KCl DMEM (based on 11965 but with 50 mM KCl, 65 mM NaCl (specially ordered); Invitrogen). Both DMEMs were fully equilibrated in a 5% CO 2 atmosphere at 37°C before use. Culture media were collected after control and high KCl stimulation assays, and cell lysates were also collected.
Immunocytochemistry-A yellow-shifted GFP derivative, YPet (23), was generated from Venus (24). A (GGGGS) 3 linker separated CAPS1 from the C-terminal triple HAtagged YPet in the pEF-BOS-CAPS1-YPet-HA plasmid. Dissociated PC12 cells were plated onto a glass coverslip (12 mm in diameter; Matsunami Glass, Japan) coated with poly-L-lysine (Sigma) and then cultured in DMEM supplemented with 1% horse serum and 5% fetal bovine serum at 37°C in a humidified 5% CO 2 atmosphere. Transfection was carried out using Lipofectamine 2000 reagent (Invitrogen). Forty-eight hours after transfection, cells were fixed with Zamboni's fixative (2% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, containing 0.2% picric acid) at room temperature for 15 min. After washing three times with PBS, cells were permeabilized in PBS containing 0.02% Triton X-100 at room temperature for 5 min. After blocking with 5% normal donkey serum in PBS at room temperature for 60 min, cells were incubated with specific primary antibodies at 4°C overnight, rinsed in PBS, and then incubated with Alexa-conjugated secondary antibodies (1:2,000 dilution; Invitrogen) at room temperature for 1 h and again rinsed in PBS. Immunoreacted cells were mounted with Vectashield (Vector) mounting medium. Images were acquired with a microscope (Olympus BX51) equipped with a CCD camera (VB-7000, Keyence). Digital images were processed using Adobe Photoshop 6.0 software.
Golgi Localization Study-The type II membrane-anchored protein galactosyltransferase, B4galt1, has been used as a marker of the Golgi complex (25). The N-terminal 81 amino acids of mouse B4galt1 were fused to tdTomato (catalog no. 632534, Clontech) to make the Golgi indicator, B4galt1-tdTomato. PC12 cells were transfected with B4galt1-tdTomato together with either control or CAPS1 siRNA. Endogenous ARF5 immunoreactivity merged with immunoreactivities for B4galt1-tdTomato (a marker of the Golgi complex) and whole-cell soma was quantified using Metamorph software (Universal Imaging Corp.).
Immunoaffinity Purification of Microsomal Fractions-To purify the CAPS1-associated subfraction from the mouse cer-ebellar microsomal fractions using immunoaffinity, anti-CAPS1 antibody-coupled magnetic beads were made as follows. 40 l of superparamagnetic, polystyrene beads covalently coated with sheep anti-rabbit IgG (Dynabeads M-280; catalog no. DB11203, Invitrogen) were incubated for 1 h at 4°C in phosphate buffer (PBS, pH 7.4, 1% BSA) containing 2 g of primary rabbit polyclonal anti-CAPS1 antibody (5). Similarly, rabbit polyclonal anti-VAMP2 antibody (13) and control rabbit IgG (Jackson ImmunoResearch, West Grove, PA) were coupled to magnetic beads. P21 mouse cerebella were dissected and homogenized in homogenization buffer (50 mM HEPES, pH 7.4, 5 mM EDTA, 0.32 M sucrose, and protease inhibitor mixture). Homogenates were centrifuged at 2,000 ϫ g for 10 min at 4°C, and microsomal fractions were sedimented from the supernatant at 20,000 ϫ g for 15 min as described elsewhere (26). After incubation with 40 l of the magnetic beads (with no primary antibody) in homogenization buffer for 2 h, the preabsorbed microsomal supernatant was diluted with an equal volume of PBS containing 10% skimmed milk and incubated with the primary antibodybound beads for 2 h at 4°C. The microsome-bound beads were collected and washed three times with PBS containing 5% skimmed milk and 2 mM EDTA and then twice with PBS containing 2 mM EDTA.
Immunoprecipitation-Mouse CAPS1 cDNA was subcloned in frame in front of the triple HA epitope tag sequence in pEF-BOS to create the C-terminally HA-tagged CAPS1 construct, pEF-BOS-CAPS1-HA. Similarly, mouse ARF family cDNAs were subcloned in frame in front of the triple FLAG epitope tag sequence to create the constructs, pEF-BOS-ARF-FLAG. Lipofectamine 2000 reagent was used to transiently transfect 5 ϫ 10 5 cells in 6-well plates with 4 g of pEF-BOS-CAPS1-HA and 1 g of pEF-BOS-ARF-FLAG plasmids. 48 h after transfection, transfected COS-7 cells were harvested and lysed in 1.3 ml of lysis buffer (50 mM HEPES, pH 7.4, 10% glycerol, 100 mM NaCl, 1 mM CaCl 2 , 0.5 mM MgCl 2 , and 0.3% Triton X-100) containing a mixture of protease inhibitors. When required for the CAPS1-ARF binding assay, 10 M GDP or 10 M GTP␥S was included in the lysis buffer (see Fig. 3D). After preabsorption with protein A-Sepharose, the supernatants were divided equally into two tubes and incubated with 0.5 g of anti-HA or anti-FLAG antibody, and the immunocomplexes were then associated with protein A-Sepharose resins. The resins were washed five times with lysis buffer, and the bound proteins were separated on an SDS-polyacrylamide gel and transferred to a nitrocellulose membrane for analysis with anti-HA or anti-FLAG antibodies.
GST Pull-down Assay-The purified GST-fused CAPS1 derivatives and MBP-fused ARF5 protein were incubated with glutathione-Sepharose 4B beads (GE Healthcare) in binding buffer (50 mM HEPES-KOH, pH 7.4, 100 mM NaCl, 0.5 mM MgCl 2 , 2 mM EGTA, 10% glycerol, 0.3% Triton X-100, and 10 M GDP) for 1 h at room temperature. After washing five times with 1 ml of binding buffer, proteins trapped within the beads were analyzed by Western blotting.

CAPS1 Is Associated with the Golgi Membrane via the PH
Domain-CAPS1 consists of four major protein domains: a C2 domain, a PH domain, a Munc13-1 homology domain, and a DCV binding domain (2). An exogenously expressed PH domain (a fusion construct with fluorescent protein YPet) ( Fig. 1A) was colocalized with Stx6 (syntaxin 6) (Fig. 1B) in PC12 cells (Fig. 1C) (14). This suggested involvement of the PH domain in the association of CAPS1 with the trans-Golgi.
To elucidate subcellular function of somal CAPS1, we analyzed the relationship between these protein domains and subcellular localization by expressing a series of domain-specific truncation mutants fused with YPet and HA tag in the PC12 neuroendocrine cell line. Full-length CAPS1 exogenously expressed in PC12 cells was distributed diffusely throughout the cytoplasm, but its large fraction was concentrated around the Golgi apparatus (Fig. 1D). The PH domain alone was localized predominantly in Golgi-like structures (Fig. 1E). The N-terminally truncated protein spanning from the PH domain to the C terminus (⌬N-Ter) showed a Golgilike accumulation pattern (Fig. 1F), similar to that of the PH domain alone (Fig. 1E). However, both the C-terminally truncated protein spanning from the N terminus to the PH domain (⌬C-Ter) (Fig. 1G) and the N/C-terminal truncated protein containing the C2 and PH domains (⌬N/C-Ter) (Fig. 1H) were distributed diffusely throughout the cytoplasm. By contrast, the C2 domain-deleted protein (⌬C2) exhibited a Golgilike accumulation pattern (Fig. 1I). There were slight differences in the expression levels of these constructs in PC12 cells (supplemental Fig. S1). These results suggest that CAPS1 associates with the Golgi complex via the PH domain and that the CAPS1-Golgi association is controlled by the C2 domain.
To investigate whether endogenous CAPS1 associates with the Golgi membrane, we tried purifying the CAPS1-associated membrane fraction from the microsomal fraction of mouse cerebella (postnatal day 21) using CAPS1 antibodycoated magnetic beads. Electron microscopic examination of the affinity-purified fraction revealed the presence of membranous structures bound to CAPS1 affinity beads ( Fig. 2A) but not to normal rabbit IgG-coated magnetic beads (data not shown). It was noteworthy that the membranous structures bound to the CAPS1 affinity beads tended to be larger than the vesicles bound to VAMP2 (a synaptic vesicle marker) affinity beads (Fig. 2B). Western blot analysis showed that various Golgi marker proteins (GM130, GS28, p115, Stx5, Stx6, Stx16, Vti1a, and VAMP4) were included in the membranous fractions purified with CAPS1 affinity beads (Fig. 2C). Moreover, the vesicle trafficking-related proteins SNAP25 and ARFs were detected in the CAPS1 affinity fractions. The mitochondrial protein Bcl-2 was hardly detected in the CAPS1 affinity fraction, and only a trace amount of BiP/GRP78, a marker of the endoplasmic reticulum, was present in this crude fraction (Fig. 2C), indicating that the CAPS1 affinity method concentrated the Golgi membranes. These results indicated that some of the endogenous CAPS1 indeed associates with the Golgi membrane.

CAPS1 Interacts with the GDP-bound Form of Class II
ARFs-Most PH domains of various PH-containing proteins have been shown to localize to the plasma membrane. However, the PH domains of several proteins, including oxysterol-binding protein, Goodpasture antigen-binding protein, and phosphatidylinositol 4-phosphate adaptor protein, have the ability to target the Golgi membrane (27)(28)(29). An interaction between the PH domain of these three proteins and phosphatidylinositol 4-phosphate or the ARF1 small GTPase, a regulator of membrane trafficking, is the common mechanism underlying their targeting to the Golgi membrane (28,29). Membrane fractions that were affinity-purified using anti-CAPS1 antibody contained proteins of the ARF family (Fig. 2C). Therefore, we investigated the possibility that CAPS1 binds ARFs. All members of the ARF family of proteins (class I ARF1 to -3, class II ARF4 and ARF5, and class III ARF6) were expressed ubiquitously in the mouse brain (supplemental Fig. S2). Notably, among all of these ARFs, only the class II ARFs (ARF4 and ARF5) were coimmunoprecipitated with CAPS1 (Fig. 3A).
Similar to other small GTPases, such as Ras, the T31N (Thr 31 3 Asn) and Q71L (Gln 71 3 Leu) substitution mutants   DECEMBER 3, 2010 • VOLUME 285 • NUMBER 49 of ARF4/5 have been shown to give rise to a GDP-bound conformation (30) and a GTP-bound conformation (31), respectively. Interestingly, CAPS1 preferentially coimmunoprecipitated with the GDP-locked form (T31N) of ARF4/5 (Fig. 3B), and the region encompassing the C2 and PH domains alone (C2 ϩ PH) continued to retain this coimmunoprecipitation ability (Fig. 3C). Moreover, the preference of CAPS1 to bind to the GDP-ARF5 form was revealed by coimmunoprecipitation of CAPS1 and ARF5 from cell lysates in GDP-containing lysis buffer but not in GTP␥S-containing lysis buffer (Fig. 3D). To confirm the interaction between CAPS1 and the class II ARFs in vivo, we generated an ARF5-specific antibody (supplemental Fig. S3) and carried out coimmunoprecipitation of endogenous CAPS1 from cell lysates of mouse cerebellum. The results showed that endogenous ARF5 was coimmunoprecipitated with CAPS1 (Fig. 3E). These findings indicate that CAPS1 interacts with class II ARF4/5 via the region containing the C2 and PH domains, depending on the GDP-binding state of ARF4/5.

CAPS1-ARF4/5 Regulates DCV Trafficking
Protein-Protein Interaction between the PH Domain of CAPS1 and the N-terminal Region of ARF5-We also examined the structural and functional properties of the CAPS1-ARF5 interaction. Pull-down experiments using the bacterially expressed fusion proteins GST-CAPS1 and MBP-ARF5 indicated that CAPS1 interacts directly with ARF5 via the PH domain but not the C2 domain (Fig. 4A). We then analyzed the ability of CAPS1 to bind to each of 12 GDP-locked ARF5(T31N) mutants that had single or double amino acid substitutions of 16 residues that were identical only in class II ARF4/5 and not in the other classes of ARFs (supplemental Fig. S4). Among the mutants with Ala residue substitutions of class II-specific residues, those with substitutions of the Nterminal Leu, Thr, and Ser, located at the third, fourth, and sixth positions, respectively (L3A, T4A, and S6A), lacked CAPS1 binding activity in coimmunoprecipitation tests (Fig.  4B). These results suggest that the N-terminal region of ARF5 is critical for CAPS1 binding (supplemental Fig. S4).
CAPS1 KD Induces Chromogranin Accumulation in the Golgi Complex-To investigate the role of somal CAPS1 in the Golgi function, we examined the KD effect of CAPS1 expression by transfecting specific CAPS1 siRNAs. CAPS1 KD in PC12 cells, in which CAPS1 is expressed and CAPS2 is not (5), resulted in the levels of CAPS1 becoming almost undetectable by Western blot analysis (Fig. 5A, bottom). We then compared the protein components of culture media from control and CAPS1 KD cultures using SDS-PAGE analysis, followed by tandem mass spectrometry coupled to liquid chromatography (LC-MS/MS). As a result, three granin family proteins, chromogranin A, chromogranin B, and secretogranin II, all of which are DCV marker proteins released by DCV secretion, were identified to be dramatically decreased in the culture media of the CAPS1 KD cells (Fig. 5A, top). To confirm the reduction of granin protein secretion by CAPS1 KD cells, HA-tagged chromogranin A (ChgA-HA) or HAtagged secretogranin II (SgII-HA) was co-transfected with  CAPS1 siRNA into PC12 cells. High KCl (50 mM) treatment significantly enhanced the amount of ChgA-HA released into the media of control cultures, compared with treatment with a normal KCl (5 mM) concentration (Fig. 5, B and C). However, CAPS1 KD decreased both the spontaneous release and the high KCl-induced release of ChgA-HA (Fig. 5, B and C). SgII-HA release was also reduced in CAPS1 KD cultures (Fig.  5B). To determine whether CAPS1 KD impairs DCV exocytosis, DCV trafficking, or DCV biogenesis from the Golgi complex, we analyzed subcellular localization of ChgA in KD cells. CAPS1 KD altered the subcellular distribution of endogenous ChgA in PC12 cells; ChgA was localized largely in the processes of the control cells, whereas it accumulated in Golgilike structures in the CAPS1 KD cells (Fig. 5D). Abnormal accumulation of exogenously expressed ChgA-HA was also observed in Golgi-like structures in the CAPS1 KD cells, in marked contrast to its predominant distribution in processes in the control cells (Fig. 5E). By contrast, transmembrane proteins, including neurotrophin receptors TrkA (data not shown) and TrkB (Fig. 5F), which are not transported by DCVs, were normally targeted to the cell surface of the CAPS1 KD cells. These results implicate somal CAPS1 in DCV trafficking in the Golgi complex, and thus CAPS1 KD has a consequential effect on DCV secretion of granins into the culture media.
The Interaction between CAPS1 and Class II ARFs Is Indispensable to DCV Trafficking-Class I ARFs are known to regulate membrane trafficking (19,32). To examine whether ARF4/5 plays a role in DCV-Golgi trafficking, we analyzed the KD effects of ARF4/5 expression on the subcellular distribution pattern of ChgA-HA in PC12 cells (Fig. 6, A-D). Exogenously expressed ChgA-HA was densely localized in the tips of processes of control PC12 cells, whereas KD of either ARF4 or ARF5 resulted in stacking of ChgA-HA in Golgi-like structures (Fig. 6, B and C), which was also observed in CAPS1 KD cells (Fig. 5, D and E). Furthermore, when ARF4 and ARF5 were knocked down simultaneously (rather than separately), a greater quantity of ChgA-HA accumulated in Golgi structures (Fig. 6D). These results suggest that ARF4/5 plays a role in DCV-Golgi trafficking.
To investigate whether the association between CAPS1 and class II ARFs has a role in DCV secretion, we compared high  DECEMBER 3, 2010 • VOLUME 285 • NUMBER 49
We then analyzed whether the CAPS1-ARF5 association affects the subcellular localization of exogenous ChgA-HA in PC12 cells. Coexpression of wild-type ARF5 induced no obvious change in the subcellular distribution of ChgA-HA (Fig. 6I). When coexpressed with ARF5(3,4,6A), ChgA-HA accumulated in the Golgi complex (Fig. 6J), as was observed in CAPS1 KD cells (Fig. 5, D and E) and ARF4/5 KD cells (Fig. 6D). Similarly, coexpression of ARF5(3,4,6A) in primary cultured hippocampal cells altered the somal distribution pattern of ChgA-HA from a diffuse distribution throughout the cytoplasm (Fig. 6K) to a Golgi-like accumulation (Fig. 6L). Taken together, these results suggest that the association between CAPS1 and ARF5 plays a role in trafficking chromogranin-containing DCVs in the Golgi network.
The GDP-bound Form of ARF5 Localizes to the Golgi Complex, Depending on the Interaction with the PH Domain of CAPS1, and Is Not Transported to Axons of Hippocampal Neurons-A previous study showed that GDPlocked class II ARFs have a cytoplasmic, diffuse distribution pattern, whereas GTP-locked class II ARFs associate with the Golgi membrane (33). We investigated whether these guanine nucleotide-dependent distribution patterns of GDP-locked ARF5(T31N) were altered by coexpression of CAPS1 constructs in PC12 cells (Fig. 7, A-I).
Coexpression of the CAPS1 PH domain alone (CAPS1(PH)) changed the distribution of ARF5 (T31N) from a diffuse cytosolic pattern to a localized pattern (Fig.  7B), which was merged with the Golgi marker protein VAMP4 (Fig. 7F). On the other hand, coexpression of CAPS1 lacking the PH domain (CAPS1(dPH)) resulted in a diffuse cytoplasmic distribution pattern of ARF5(T31N) (Fig. 7H). We next examined whether CAPS1 KD affected the ARF5 localization in the Golgi membrane (Fig. 7, J-M). Endogenous ARF5 was localized in the Golgi in cells transfected with control siRNA (Fig. 7K). However, endogenous ARF5 showed a diffuse cytosolic pattern in KD cells transfected with CAPS1 siRNA (Fig. 7M). These data suggest that GDP-locked ARF5 is recruited to the Golgi complex by binding to CAPS1 via the PH domain. To quantify Golgi localization, PC12 cells were transfected with the fluorescent Golgi marker construct (Golgi marker B4galt1 fused to tdTomato) together with either control or CAPS1 siRNA. ARF5 immunoreactivity merged with immunoreactivities for the Golgi complex and whole-cell soma images was quantified (Fig. 7N). The results showed a modest effect and suggested that CAPS1 induces accumulation of ARF4/5 in the Golgi complex probably by interaction between CAPS1 and ARF4/5.
Finally, we compared the subcellular localization of GDPlocked and GTP-locked ARF5 exogenously expressed in primary cultured hippocampal neurons. Both ARF5 forms were highly expressed in the neuronal soma, where the Golgi complex is located. Interestingly, the GTP-locked ARF5 was distributed in axons and dendrites, in addition to the soma (Fig.  7O), whereas the GDP-locked ARF5 was transported to dendrites but not to axons (Fig. 7P). Although the biological significance of this differential localization between GDP-locked and GTP-locked ARF5 in hippocampal neurons remains unclear, the function of CAPS1 may be regulated differently for these subcellular compartments of neurons (soma-dendrites versus axons), depending on the localization of GDP-bound class II ARFs.

DISCUSSION
Our findings indicate that CAPS1 interacts with class II ARF4/5 in a GDP/GTP state-dependent manner and that CAPS1 is involved in DCV trafficking in or from the trans-Golgi network. We showed that CAPS1 has the ability to bind GDP-bound, but not GTP-bound, ARF4/5 and the Golgi membrane. ARF4/5 in the GDP-bound state is recruited to the Golgi complex by interaction of its N-terminal domain with the PH domain of CAPS1. This recruitment is indispensable to DCV trafficking for the following reasons: 1) overexpression of CAPS1 binding-deficient ARF5 mutants induced accumulation of ChgA in the Golgi, with a consequent reduction in ChgA release; 2) CAPS1 KD resulted in the accumulation of ChgA in the Golgi network and, thereby, a reduction in ChgA release; and 3) ARF4/5 KD caused abnormal ChgA accumulation in the Golgi network. Collectively, these findings suggest that CAPS1 is required for DCV trafficking in the trans-Golgi network by recruiting class II ARF small GTPases, in a GDP-bound dependent manner, to the Golgi membrane. Because ARF1 reportedly induces vesicle budding in vitro by regulating membrane curvature (34,35), the class II ARFs may also have a role, in concert with CAPS1, in regulating DCV budding in the trans-Golgi network.
The PH domain of CAPS1 is essential for its accumulation in the Golgi. On the other hand, the C2 domain of CAPS1 appears to have a regulatory role in this subcellular localization, because the N/C-terminal truncated protein containing the C2 and PH domains was distributed throughout the cell soma. We also defined the structural determinants of the CAPS1-ARF4/5 interaction; the PH domain of CAPS1 and the N-terminal region of ARF4/5 interact in a GDP-dependent manner. In addition, we confirmed by substitution of the myristoylation consensus site Gly 2 by Ala that the N-terminal myristoylation state of ARF4/5 does not affect CAPS1 interaction (data no shown). These results suggest that the N-terminal region of class II ARFs binds CAPS1 independently of the Golgi membrane anchoring via myristoylation.
The members of the ARF family of proteins are thought to be regulators of membrane trafficking. Various regulators and effectors of the class I and III ARFs have already been identified (16,19,36,37). On the other hand, class II ARF-specific regulators or effectors have not been reported, although previous studies have shown that ARF4 bound Munc18-interacting proteins, which contributed to vesicle trafficking of Alzheimer precursor protein ␤-APP (38,39), and that ARF4 bound the epidermal growth factor receptor, which in turn activated PLD2, leading to transcriptional regulation (38,39). The present study demonstrates that CAPS1 is a novel type of class II ARF target. All ARF guanine nucleotide exchange factors identified to date possess a Sec7 domain, a module of ϳ200 amino acids that is sufficient to catalyze the exchange of GDP for GTP on ARF in vitro (16). However, CAPS1 contains no regions that are homologous with the Sec7 domain, and it has no detectable level of guanine nucleotide exchange factor activity (data not shown). Neither CAPS nor class II ARF orthologs are present in Saccharomyces cerevisiae, but both CAPS and class II ARF orthologs are found in Drosophila melanogaster and Caenorhabditis elegans, as well as in vertebrates (37). Thus, the interaction between CAPS and class II ARFs presumably is required for fine regulation of DCV trafficking in multicellular organisms.
Previous studies have shown that CAPS1 is involved in the priming step of DCV exocytosis (8,9). However, in most cell types (but not hippocampal DG granule cells) (13), the largest amount of endogenous CAPS1 protein is localized in the cytoplasm of the soma rather than in the plasma membrane region near secretion sites. The major role of the cytosolic CAPS proteins present in the cell soma has yet to be determined. The results of this study shed light on another important function of CAPS1 protein in the cell soma, in addition to its regulatory function at DCV secretion sites; CAPS1, together with the class II ARFs, is involved in DCV trafficking in the trans-Golgi network in the cell soma. We therefore suggest that CAPS1 has two functions in the DCV secretion pathway in neurons and neuroendocrine cells: a somal function that regulates DCV-Golgi trafficking and a synaptic (or secretion site-specific) function that regulates DCV exocytosis. In this respect, it seems noteworthy that the GDP-bound ARF5, which is capable of binding to CAPS1, is localized in the soma of cultured hippocampal neurons but is not transported into axons or terminals, where synaptic DCV exocytosis occurs, suggesting that the ARF4/5-CAPS1 interaction is required for the somal function of CAPS1 but not for its synaptic function. Thus, we suggest that the two functions of CAPS1 depend on the presence of its binding partner, GDP-bound class II ARFs, although the basic properties of CAPS1 underlie both functions.