Protein secretion is a fundamental process for communication between eukaryotic cells and therefore, for organismal homeostasis, reproduction, and survival. Two types of secretory processes can be distinguished based on the rate and mode of regulation of secretory vesicle release: constitutive secretion and regulated secretion (Morgan, 1995). In constitutive secretion, secretory vesicles are exocytosed as they are produced. This process takes place in all eukaryotic cells, mainly to maintain homeostasis of the plasma membrane and the extracellular matrix. In contrast, regulated secretion takes place in specialized cell types, such as endocrine and exocrine cells, as well as in cells of the immune system such as platelets and neutrophils (Aggarwal et al., 2023; Ley et al., 2018). These cell types produce specialized secretory vesicles known as secretory granules (SGs), which are released in response to specific stimuli (Kögel and Gerdes, 2010). SGs sprout from the trans-Golgi network (TGN) as immature vesicles, incompetent for fusion with the plasma membrane. Maturation of SGs involves homotypic fusion and acquisition of membrane proteins, which are required for SG delivery and fusion with the plasma membrane. Finally, stimulus-driven fusion of SGs with the plasma membrane results in cargo release to the extracellular milieu (Omar-Hmeadi and Idevall-Hagren, 2021; Sugita, 2008).

The salivary gland of Drosophila melanogaster larvae provides several advantages for the identification of regulators of SG biogenesis, maturation, and exocytosis (Burgess et al., 2012; de la Riva-Carrasco et al., 2021; Neuman and Bashirullah, 2018; Rousso et al., 2016). At late third larval instar, salivary glands synthesize a series of mucins, collectively known as Glue proteins, which become packed in SGs. Approximately 4 hr before the onset of pupariation, concerted exocytosis of mucin-containing SGs, followed by extrusion of the mucins out of the prepupal body are essential for gluing the puparium to the substratum (Borne et al., 2020). Interestingly, these three events of SG development (biogenesis, maturation, and exocytosis) occur sequentially and only once in each cell of the salivary gland, that will later on be degraded during metamorphosis (Duan et al., 2020; Tracy et al., 2016). The tagging of one of these mucins with fluorophores (Sgs3-GFP or Sgs3-dsRed), (Biyasheva et al., 2001) combined with the large size of salivary gland cells and their SGs, have allowed high-resolution imaging and real-time traceability of SG biogenesis, maturation, and secretion, leading to the identification and characterization of dozens of factors required along the exocytic pathway (Burgess et al., 2011; Neuman et al., 2022; Reynolds et al., 2019; Torres et al., 2014; Tran et al., 2015).

The exocyst is a hetero-octameric protein complex identified and initially characterized in the budding yeast, and later found to be conserved across all eukaryotic organisms (Hsu et al., 1996; Novick et al., 1980; Novick et al., 1995; TerBush et al., 1996). Yeast cells bearing mutations in exocyst subunits display intracellular accumulation of secretory vesicles and defects in exocytosis (Govindan et al., 1995; TerBush et al., 1996). Molecularly, the exocyst complex participates in vesicle tethering to the plasma membrane prior to SNARE-mediated fusion (An et al., 2021). Recently, it was shown that it stimulates multiple steps of exocytic SNARE complex assembly and vesicle fusion (Lee et al., 2024). Exocyst complex malfunction has been therefore associated with tumor growth and invasion, as well as with development of ciliopathies, among other pathological conditions (Luo et al., 2013; Mavor et al., 2016; Thapa et al., 2012; Whyte and Munro, 2002; Wu and Guo, 2015).

In this work, we have utilized the Drosophila salivary gland to carry out a methodic analysis of the requirement of each of the eight subunits of the exocyst along the regulated exocytic pathway. By inducing temperature-dependent gradual downregulation of the expression of each of the subunits, we discovered novel functions of the exocyst complex in regulated exocytosis, namely in SG biogenesis, SG maturation and homotypic fusion, as well as in SG fusion with the plasma membrane. We propose that the exocyst complex participates in all these processes as a general tethering factor.

The exocyst was initially identified as crucial for secretion in yeast (Novick et al., 1980; TerBush et al., 1996), and later on, for basolateral trafficking in animal epithelial cells (Grindstaff et al., 1998; Lipschutz et al., 2000; Langevin et al., 2005), with implications in various cellular processes, including cell migration, cytokinesis, ciliogenesis, and autophagy (Bodemann et al., 2011; Park et al., 2010; Rogers et al., 2004; Thapa et al., 2012). Recently, an in-depth study of the mammalian exocyst was performed, showing that each of the eight subunits of the complex is essential for constitutive secretion of soluble proteins (Pereira et al., 2023). However, fewer is known about the function of the exocyst in regulated exocytosis. A recent work indicates that, in cultured pancreatic beta cells, the exocyst mediates tethering of insulin-containing granules to the plasma membrane and to the cortical F-actin network, being required for exocytosis of a subset of these granules (Zhao et al., 2023). Previous studies had implicated specific subunits of the complex in insulin-stimulated exocytosis of the glucose transporter Glut4 in cultured adipocytes (Inoue et al., 2003), as well as in skeletal muscle cells (Fujimoto et al., 2019). In Drosophila, some subunits of the complex (Sec3, Sec5, Sec6, Sec8, and Sec15) were recently suggested to be required for SG development (Ma et al., 2020), although earlier, a role for the Sec10 in salivary gland exocytosis had been disregarded (Andrews et al., 2002). Therefore, a role of the exocyst in regulated exocytosis is less clear. In this study, not only we demonstrate that the eight subunits of the complex are required for regulated exocytosis of SGs of Drosophila larval salivary gland, but also, that the holocomplex participates in multiple steps along the secretory pathway (Figure 10).

Figure 10

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Most studies of the secretory pathway rely on a bimodal readout: Intracellular retention of SGs versus exocytosis of SG content. These approaches set a limit to our understanding of the specific functions that regulators and effectors exert on the exocytic pathway. By using fluorescently labeled versions of the Sgs3 cargo protein in Drosophila salivary glands we have shown that knock-down of any of the exocyst subunits can bring about three distinct phenotypic outcomes: (1) Impairment of SG biogenesis, (2) Impairment of SG homotypic fusion and maturation, or (3) Impairment of SG fusion with the apical plasma membrane. Noteworthy, the frequency at which each of these defects occurs depends on the extent of exocyst downregulation, implying that each of the three functions depends on different levels of the complex: SG–plasma membrane fusion is highly sensitive to even slight reduction of exocyst subunits levels; SG maturation requires intermediate levels of the complex, and SG biogenesis seems to be the most robust of the three processes, and strong reduction of exocyst levels is required to provoke this defect.

Although exocyst subcomplexes or individual subunits have been suggested to play specific roles in cell biology (Mehta et al., 2005), it is generally believed that the functions of the exocyst are carried out by the holocomplex (Ahmed et al., 2018). Studies analyzing the requirement of all eight subunits in a single biological process are not abundant. In Drosophila, a study was performed in which the eight subunits were found to be required for developmentally regulated autophagy during salivary cell death, but not for starvation-induced autophagy in the fat body (Tracy et al., 2016), and also for proper synaptic development at neuromuscular junctions (Kang et al., 2024). However, in other biological settings some subunits of the exocyst (Sec3, Sec5, Sec6, Sec8, and Sec10) but not others (Sec15, Exo70, and Exo84) were shown to be specifically required for general as well as for specific autophagy in yeast (Singh et al., 2019). In mammalian cells, Exo84- and Sec5-induced exocyst complex activation have opposing roles on activation of the autophagy machinery (Bodemann et al., 2011). Our systematic analysis of the requirement of each of the exocyst subunits in biogenesis, maturation, and exocytosis of SGs led us to conclude that all three biological processes are carried out by the holocomplex, and not by individual subunits or subcomplexes.

Consistent with the three biological functions that the exocyst exerts in the secretory pathway, we found that its subcellular localization during salivary gland development is dynamic (Figure 10). Immediately after the onset of Sgs3 synthesis, the exocyst associated with the Golgi complex; later, when SG biogenesis has begun, the exocyst was mostly present at the fusion point between immature SGs, and finally it localized on mature SGs in close proximity to the apical plasma membrane. These dynamic subcellular localization is coherent with the three phenotypes that we have observed after precise modulation of exocyst levels. Exocyst localization at the TGN and at the plasma membrane has been reported before in mammalian cells (Yeaman et al., 2001), and although it was suggested in that study that the exocyst might be required at several steps of the secretory pathway from the TGN to the plasma membrane, the idea was not further explored.

By analyzing SG–plasma membrane fusion markers, we have shown that, paralleling the requirement of the exocyst in constitutive exocytosis, the complex participates in SG fusion with the plasma membrane in regulated exocytosis as well. Moreover, we have found that the Sec15 subunit localizes exactly at contact sites between SGs and the plasma membrane, further supporting the notion that the exocyst plays a role in this fusion process. It is accepted that the function exerted by the exocyst in the fusion between SGs and the plasma membrane depends on Ral GTPases (Brymora et al., 2001; Wang et al., 2004). RalA is the only Ral GTPase described so far in Drosophila, and we have recently reported that RalA is involved in SG–plasma membrane fusion in the salivary gland (de la Riva-Carrasco et al., 2021). Therefore, it seems likely that, paralleling constitutive exocytosis, the interaction between RalA and the exocyst is critical for tethering SGs to the plasma membrane during regulated exocytosis as well. The molecular components that regulate tethering and fusion of SGs with the plasma membrane remain largely unknown. Perhaps, this is because the proteins involved in these processes participate in earlier steps of the secretory pathway as well. Although other potential roles of the exocyst in salivary gland biology that could tangentially impact SG biogenesis cannot be completely ruled out, our temperature-dependent manipulation of the expression of exocyst subunits, in combination with differential requirements of the complex in terms of quantity/activity in each of the processes was instrumental for uncovering three different discrete steps in which the exocyst plays a role along the regulated secretory pathway.

Proteins that will be secreted are co-translationally translocated to the ER, and then transported into the cis-region of the GC in COPII-coated vesicles that are 60–90 nm in diameter and therefore, sufficient to accommodate most membrane and secreted molecules (Raote and Malhotra, 2021). Larger cargos, such as collagen and mucins might use alternative ER-cis-GC communication mechanism that are independent of vesicular carriers. Specifically, direct connections between the ER and cis-GC are formed in a Tango1- and COPII-dependent manner (Reynolds et al., 2019; Yang et al., 2024). Disruption of these connections not only affects SG formation, but also has profound impact on GC structure (Bard and Malhotra, 2006; Ríos-Barrera et al., 2017). We found that strong silencing of any of the exocyst subunits results in retention of Sgs3 at the ER, abrogation of SG biogenesis, and alteration of normal morphology of the GC. In Drosophila, as well as in mammalian cells, the Golgi complex is polarized in cis and trans-cisternae that are held together by tethering complexes of the CATCHR family. Disruption of these connections results in altered Golgi complex morphology, reflected in fragmentation and swelling of cisternae, and impairment of the secretory pathway (D’Souza et al., 2020; Khakurel and Lupashin, 2023; Liu et al., 2019). The fact that exocyst silencing not only affected SG formation, but also had profound impact on GC structure, together with the observation that Sec15 specifically associated with the Golgi complex but not with the ER before the onset of SG biogenesis, argues in favor of a role of the exocyst as a tethering complex between cisternae. We propose that the exocyst might be redundant with other CATCHR complexes in this function, since only severe downregulation of exocyst subunits expression can manifest this phenotype.

Maturation of SGs is a multidimensional process that involves homotypic fusion, acidification, cargo condensation, and acquisition of membrane proteins that will steer SGs to the apical plasma membrane, and contribute to recognition, tethering, and fusion (Boda et al., 2023; Ji et al., 2018; Nagy et al., 2022; Syed et al., 2022). Previously, the exocyst was shown to be required for maturation of Weibel–Palade bodies (Sharda et al., 2020). In the current work, we have shown that the exocyst participates in several aspects of SG maturation. One of such processes is homotypic fusion of immature SGs. We found that an adequate extent of downregulation of the expression of any of the exocyst subunits leads to accumulation of immature SGs. Furthermore, consistent with a function of the exocyst in SG homofusion, we found that Sec15 localizes preferentially at the fusion point between adjacent immature SGs. In support to this notion, we found that overexpression of Sec15 results in unusually large granules, likely derived from uncontrolled homotypic fusion. Therefore, our data suggest that the exocyst complex is the tethering factor responsible to bring immature SGs in close proximity to enable homofusion.

Besides SG growth by homotypic fusion, several factors are recruited to maturing SGs, including the transmembrane proteins CD63 and Syt-1. Our temperature-dependent genetic manipulations revealed that the addition of Syt-1 to SGs occurs immediately after they have emerged from the TGN, and addition of this protein continues after they have attained a mature size. Syt-1 localizes at the basolateral plasma membrane of salivary gland cells before SG biogenesis, and it is not known how Syt-1 reaches maturing SGs. Given that the exocyst is required for loading SGs with Syt-1, one possibility is that a vesicular carrier transports Syt-1 from the basolateral membrane to nascent and maturing SGs, and that the contact between SGs and these vesicles is mediated by the exocyst. CD63, which is also required for SG maturation, localizes instead at the apical plasma membrane before SG biogenesis, and reaches the SGs from the endosomal retrograde pathway through endosomal tubes (Ma et al., 2020). We found that cells in which the exocyst has been knocked-down display reduced levels of CD63 around SGs, suggesting that the exocyst might contribute to the contact between endosomal tubes and SGs as well. Based on our analysis, this maturation event should take place mostly before, and not after, homotypic fusion. Thus, our results support the notion that the exocyst complex might contribute to tethering maturing SGs to different types of membrane-bound carriers that transport maturation factors like Syt-1 and CD63. These observations expand the notion that a crosstalk between the secretory and endosomal pathways occurs (Ma and Brill, 2021a; Ma et al., 2020; Papandreou and Tavernarakis, 2020), and that the exocyst is a critical factor linking the two pathways.

Vesicle maturation, either at the endocytic or the exocytic pathways, usually involves changes in vesicle-bound Rab proteins (Ailion et al., 2014; Ma and Brill, 2021a; Thomas et al., 2021). The mechanisms that mediate these Rab-switches are not completely understood. Specifically, in Drosophila larval salivary glands, Rab1 and Rab11 association to SGs appear to be crucial for SG maturation and timely exocytosis (Neuman et al., 2021). We found that the levels of Rab1 and Rab11 on SGs are, respectively, positively and negatively regulated by the exocyst, and that additionally, Rab11 is itself required for recruitment of Sec15 to immature SGs. These intrincated relationships between Rab11, Rab1 and the exocyst can be explained through a single-negative feedback loop that would guarantee adequate levels of Rab11 and the exocyst on maturing SGs. This possibility is supported by previous evidence from Drosophila and other systems that indicate that Rab11 and Sec15 interact physically and genetically (Escrevente et al., 2021; Guo et al., 1999; Novick, 2016). In fact, the exocyst is an effector of Rab11 (Sec4p in yeasts), as GTP-bound Rab11 recruits Sec15 to secretory vesicles, which in turn triggers formation of the holocomplex on the vesicle membrane as previously shown in yeast, Drosophila and mammalian cells (Wu et al., 2005; Takahashi et al., 2012; Zhang et al., 2004).

An increasing number of publications reveal the complexity and variety of vesicle trafficking routes that feed into the biogenesis, maturation and exocytosis of SGs. Our study has revealed that the exocyst holocomplex acts at several steps of the pathway of regulated exocytosis, maintaining Golgi complex morphology to allow protein exit from the ER, and transit through the early secretory pathway; promoting homotypic fusion of SGs and acquisition of maturation factors; and finally, allowing SG–plasma membrane fusion (Figure 10). We propose that all these events depend on the activity of the exocyst complex as a tethering factor.

All data generated or analyzed during this study are included in the manuscript and supporting files.

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Author details

1. Sofía Suárez Freire

   1. Fundación Instituto Leloir, Buenos Aires, Argentina
   2. Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Buenos Aries, Argentina

   Contribution

   Conceptualization, Investigation, Visualization, Methodology, Writing - original draft, Writing - review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0009-0003-2908-3110

2. Sebastián Perez-Pandolfo

   1. Fundación Instituto Leloir, Buenos Aires, Argentina
   2. Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Buenos Aries, Argentina

   Contribution

   Methodology

   Competing interests

   No competing interests declared

3. Sabrina Micaela Fresco

   Fundación Instituto Leloir, Buenos Aires, Argentina

   Contribution

   Methodology

   Competing interests

   No competing interests declared

4. Julián Valinoti

   Fundación Instituto Leloir, Buenos Aires, Argentina

   Contribution

   Methodology

   Competing interests

   No competing interests declared

5. Eleonora Sorianello

   1. Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Buenos Aries, Argentina
   2. Laboratorio de Regulación Hipofisaria, Instituto de Biología y Medicina Experimental (IBYME-CONICET), Buenos Aires, Argentina

   Contribution

   Methodology

   Competing interests

   No competing interests declared

6. Pablo Wappner

   1. Fundación Instituto Leloir, Buenos Aires, Argentina
   2. Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Buenos Aries, Argentina
   3. Departamento De Fisiología, Biología Molecular Y Celular, Facultad De Ciencias Exactas Y Naturales, Universidad De Buenos Aires, Buenos Aires, Argentina

   Contribution

   Resources, Supervision, Funding acquisition, Project administration, Writing - review and editing

   For correspondence

   pwappner@leloir.org.ar

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0003-1517-0742

7. Mariana Melani

   1. Fundación Instituto Leloir, Buenos Aires, Argentina
   2. Consejo Nacional De Investigaciones Científicas Y Técnicas (CONICET), Buenos Aries, Argentina
   3. Departamento De Fisiología, Biología Molecular Y Celular, Facultad De Ciencias Exactas Y Naturales, Universidad De Buenos Aires, Buenos Aires, Argentina

   Contribution

   Conceptualization, Supervision, Funding acquisition, Investigation, Methodology, Writing - original draft, Writing - review and editing

   For correspondence

   melanimari@gmail.com

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0002-9491-932X

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