A novel function for Rab proteins during maturation of secretory granules

Regulated exocytosis is an essential process whereby professional secretory cells synthesize and secrete specific cargo proteins in a stimulus-dependent manner. Cargo-containing secretory granules are synthesized in the trans-Golgi Network (TGN); after budding from the TGN, granules undergo many modifications, including a dramatic increase in size. These changes occur during a poorly understood process called secretory granule maturation. Here we leverage the professional secretory cells of the Drosophila larval salivary glands as a model system to characterize a novel and unexpected role for Rab GTPases during secretory granule maturation. We find that secretory granules in the larval salivary glands increase in size ~300-fold between biogenesis and release, and loss of Rab1 or Rab11 dramatically reduces granule size. Surprisingly, we find that Rab1 and Rab11 protein localize to secretory granule membranes. Rab11 associates with granule membranes throughout the maturation process, and Rab11 is required for recruitment of Rab1. In turn, Rab1 associates specifically with immature secretory granules and drives granule growth. In addition to their roles in granule growth, both Rab1 and Rab11 appear to have additional roles during exocytosis; Rab11 function is necessary for exocytosis, while the presence of Rab1 on immature granules may prevent precocious exocytosis. Overall, these results highlight a new and unexpected role for Rab GTPases in secretory granule maturation.

Rab proteins are small GTPases that play a role in regulating nearly every aspect of intracellular trafficking, including regulated exocytosis. These proteins are highly conserved throughout eukaryotes, although the number of Rabs increases with the complexity of the organism; the yeast genome contains 11 Rab proteins, while the human genome contains at least 60 (Pfeffer, 2017;Li and Marlin, 2015;Homma et al., 2021). Rab proteins are prenylated at their C-terminus, allowing for membrane localization (Kinsella and Maltese, 1991;Leung et al., 2006;Rossi et al., 1991;Khosravi-Far et al., 1991). Inactive Rab proteins are bound to GDP and GDP-dissociation inhibitor (GDI) and localized to the cytoplasm; upon approaching their target membrane, GDI is displaced by GDI displacement factor (GDF), allowing membrane insertion of the Rab protein (Li and Marlin, 2015;Sasaki et al., 1990;Pfeffer et al., 1995;Müller and Goody, 2018;Sivars et al., 2003). The Rab protein then interacts with a cognate guanine nucleotide exchange factor (GEF), which facilitates the replacement of GDP for GTP, activating the Rab (Blümer et al., 2013;Müller and Goody, 2018;Ullrich et al., 1994;Soldati et al., 1994;Barr and Lambright, 2010). GTP-bound Rabs physically interact with their effector proteins to regulate membrane trafficking, including vesicle budding, movement, and fusion, among other functions (Homma et al., 2021;Li and Marlin, 2015). Rab proteins are inactivated via interaction with GTPase activating proteins (GAPs) which aid in catalyzing the hydrolysis of GTP to GDP, allowing the Rab to be removed from the membrane via interaction with GDI (Ullrich et al., 1993;Soldati et al., 1993;Garrett et al., 1993;Müller and Goody, 2018; Barr and Lambright, 2010). Each Rab protein is localized to specific membrane compartments, allowing these small GTPases to direct and orchestrate many of the complex trafficking events required for regulated exocytosis. Rab3D and Rab27A have previously been reported to localize to secretory granule membranes in mammalian neuroendocrine and endothelial cells, respectively, where they may play a functional role in regulating secretory granule membrane and cargo remodeling during maturation (Kögel and Gerdes, 2010b;Kögel et al., 2013;Hannah et al., 2003). However, a role for other Rab proteins in secretory granule maturation in different cell types has not been described.
The Drosophila larval salivary glands are composed of professional secretory cells that synthesize and secrete massive quantities of mucin-like "glue" proteins via regulated exocytosis (Korge, 1977;Beckendorf and Kafatos, 1976). Mucin biogenesis and secretion are developmentally controlled. Biogenesis begins at the mid-third instar larval transition, and exocytosis begins about 4 h before the onset of metamorphosis, 24 h later (Biyasheva et al., 2001;Kang et al., 2017;Beckendorf and Kafatos, 1976). Once exocytosis is complete, the mucins are expelled out of the lumen of the salivary glands and onto the surface of the animal, where they act like glue, allowing the puparium to adhere to a solid surface during metamorphosis (Biyasheva et al., 2001;Kang et al., 2017). Like secretory granules in many types of mammalian cells, these mucin-containing granules undergo a maturation process, increasing in size over time (Niemeyer and Schwarz, 2000;Reynolds et al., 2019;Neuman and Bashirullah, 2018).
Because mucin production is a developmentally controlled process consisting of a single round of biogenesis, maturation, and exocytosis, we are able to view the events that occur during maturation with a high degree of temporal resolution. Moreover, fluorescent tagging of mucins (Sgs3-GFP or Sgs3-DsRED) (Biyasheva et al., 2001;Costantino et al., 2008) allows us to view these events in living tissue in real-time.
Here, we have leveraged mucin secretion in the larval salivary glands as a model system to examine the role of Rab proteins in secretory granule maturation. We screened Drosophila Rab proteins using stage-and tissue-specific RNAi knockdown for defects in secretory granule size.
This screen demonstrated that RNAi knockdown of Rab1 and Rab11 resulted in a striking small granule phenotype. Surprisingly, we found that Rab1 and Rab11 protein localized to secretory granule membranes; Rab11 was present on granule membranes from biogenesis through secretion, while Rab1 was only present on granule membranes during the process of maturation. We also demonstrate that Rab1 and Rab11 localization to granule membranes requires GTP binding, and that Rab11 is required for recruitment of Rab1. Rab11 function is required for both maturation and exocytosis, as loss of Rab11 results in secretion defects and impairs the ability of granules to dock and fuse with the apical membrane. Finally, surprisingly, we found that small granules lacking Rab1 are able to be secreted, suggesting that Rab1 may have a role in preventing precocious secretion of immature granules. Overall, these results highlight a previously unknown role for Rab proteins on secretory granule membranes during the process of secretory granule maturation and provide new insights into the role of maturation during regulated exocytosis.

Secretory granules dramatically increase in size from biogenesis to secretion
The biogenesis of mucin-containing secretory granules in the Drosophila larval salivary glands begins when animals reach the mid-third instar larval transition and ends with secretion at the onset of metamorphosis (puparium formation) 24 h later (Beckendorf and Kafatos, 1976;Biyasheva et al., 2001). Previous work has shown that mucin-containing granules undergo a maturation process, increasing in size by fusing with one another (Neuman and Bashirullah, 2018;Niemeyer and Schwarz, 2000;Reynolds et al., 2019). To further characterize and quantify changes in secretory granule size that occur during this 24 h period from biogenesis to secretion, we synchronized animals at the onset of mucin biogenesis and used confocal microscopy to analyze mucin granule size in 4 h windows spanning this time period. We defined granules in the 0-4 h timepoint as 'nascent granules,' granules in the 4-20 h period as 'immature granules,' and granules in the 20-24 h period (when exocytosis has begun) as 'mature granules' (Fig. 1A).
We observed that granules dramatically increased in size, with nearly a 300-fold increase in overall volume when comparing nascent vs. mature granules (Fig. 1A, C). Interestingly, the single largest increase in volume occurred between the 0-4 h and 4-8 h timepoints, with ~18-fold Neuman   Live-cell imaging of mucins (red) from staged salivary glands spanning from the onset of biogenesis (0-4 h) to the onset of exocytosis (20-24 h). Timepoints are shown in h after Sgs3. Images shown are a maximum intensity projection of ten optical slices from z-stacks comprising 24-32 total slices at a step size of 0.28 µm. (B) Live-cell imaging of mucin granules (red) at the 20-24 h after Sgs3 timepoint in salivary glands expressing Rab1-or Rab11-RNAi. Full genotypes: Sgs3-GFP/UAS-Rab1-RNAi; Sgs3>/+ and UAS-Rab11-RNAi/+; Sgs3-GFP/+; Sgs3>/+. Scale bars: 5 µm. (C) Box and whisker plot showing the volume of secretory granules from staged salivary glands. Z-stack imaging data was used to identify the medial cross-section of each granule for quantification. x-axis reflects the developmental stages and genotypes analyzed; y-axis shows log2transformed volumes in µm 3 . Boxes outlines the 25th to 75th percentiles; middle line indicates the median. Whiskers extend to the minimum and maximum values. n=50 granules per timepoint/genotype. change in average volume, indicating that the granules doubled in size every 1 h (Fig. 1C).
Thereafter, the secretory granules doubled in size about every 4 h, with a total of ~15-fold change in average volume between the 4-8 h and 20-24 h timepoints (Fig. 1C). This dramatic difference in growth rate between nascent granules and immature granules suggests that there may be independent regulatory mechanisms mediating each of these stages of granule growth.

An RNAi screen for Rab proteins that regulate secretory granule size
The Drosophila genome contains 33 annotated Rab proteins (Zhang et al., 2007). To test if any of these Rabs play a role in regulating secretory granule size, we conducted a salivary glandspecific RNAi screen and looked for a reduction in mature granule size. We used the stage-and tissue-specific Sgs3-GAL4 driver for this screen (Cherbas et al., 2003); since GAL4 expression is driven by the same promoter as mucins, we were able to examine specific effects on mucin granules without affecting development or earlier functions of the larval salivary glands. We tested each of the 30 annotated Drosophila Rab proteins that had one or more RNAi lines publicly available. This screen identified Rab1 and Rab11 as high-confidence hits with a dramatic reduction in secretory granule size validated by multiple independent RNAi lines (Table S1, Fig.   1B, C). We also identified Rab3, Rab5, Rab10, and Rab27 as lower-confidence hits with a moderate reduction in granule size that was only observed in a single RNAi line (Table S1). qPCR analysis in dissected salivary glands confirmed that Rab1-RNAi and Rab11-RNAi significantly reduced Rab1 and Rab11 expression levels, respectively (Fig. S1). The terminal size of mucin granules in salivary glands expressing Rab1-or Rab11-RNAi was similar to that of controls at the 4-8 h timepoint (Fig. 1C), indicating that disruption of Rab1 or Rab11 function severely inhibits mucin granule growth. These screen results highlight a novel role for Rab proteins in the regulation of secretory granule size.

Rab1 and Rab11 localize to secretory granule membranes
Many Rab proteins localize to specific organelle membranes, and this specificity in localization allows them to carry out their roles in intracellular trafficking (Li and Marlin, 2015;Homma et al., 2021;Zhen and Stenmark, 2015). To begin to gain an understanding of how Rab proteins regulate mucin granule size, we used a collection of endogenously regulated, EYFP-tagged Rab proteins (Dunst et al., 2015) to assess the subcellular localization patterns of our screen hits in salivary glands during the 24 h time period spanning mucin biogenesis through secretion.
Surprisingly, we observed that both Rab1 and Rab11 were enriched on secretory granule membranes. Rab11 appeared to be enriched on secretory granule membranes throughout the entire 24 h period from biogenesis through secretion ( Fig. 2A, C). Rab11 has typically been reported to localize to the Golgi and to recycling endosomes (Zhen and Stenmark, 2015;Ullrich et al., 1996;Calhoun and Goldenring, 1996;Welz et al., 2014;Urbé et al., 1993), and we did observe co-localization of Rab11 with a pan-Golgi marker, as well as small Rab11-positive puncta that are likely recycling endosomes (Fig. S2B), indicating that Rab11 localizes both to the expected organelles and to secretory granule membranes. Rab1, in contrast, exhibited stagespecific localization to granule membranes; Rab1 was absent from both nascent (0-4 h) and mature (20-24 h) granules but present on nearly all granules at the 4-8 h and 8-12 h timepoints and present on some granules at the 12-16 h and 16-20 h timepoints ( Fig. 2A  Images shown are a single slice from a z-stack comprising three slices at a 0.30 µm step size. (C) Live-cell imaging analysis of Rab11-EYFP (cyan) localization with mucins (red). Rab11 is present on granule membranes at 0-4, 8-12, and 20-24 h after Sgs3. Images shown are a single slice from a z-stack comprising ten slices at a 0.31 µm step size. Scale bars: 5 µm.
promoting anterograde ER-to-Golgi trafficking (Zhen and Stenmark, 2015;Plutner et al., 1991;Segev et al., 1988;Schmitt et al., 1988). We observed co-localization of Rab1 with Sec31 ( Fig.   S2A), a component of the COPII coat protein complex and marker for ERES (Förster et al., 2010), indicating that Rab1 also localizes to ERES in salivary glands. In contrast, our lower-confidence screen hits did not appear to localize to secretory granule membranes (Fig. S3), suggesting that Rab1 and Rab11 may play a granule-autonomous role in regulating secretory granule size.

GTP binding is required for Rab1 and Rab11 localization and function in maturation
Rab protein function is dependent on binding to GTP; only GTP-bound Rab proteins can recruit their effector proteins to carry out their biological functions (Li and Marlin, 2015;Homma et al., 2021). To examine the role of GTP binding in Rab1 and Rab11 function during mucin granule maturation, we overexpressed YFP-tagged dominant negative Rab1 and Rab11 specifically in the salivary glands using the Sgs3-GAL4 driver. These dominant negative constructs contain a point mutation that changes a serine in the GTP binding domain to an asparagine, effectively preventing association with GTP (Zhang et al., 2007). Localization analysis showed that Rab1 DN -YFP was absent from secretory granule membranes at the 8-12 h timepoint (Fig. 3B), when wildtype Rab1-EFYP is highly enriched on secretory granule membranes (Fig. 3A). Similarly, Rab11 DN -YFP did not appear enriched on secretory granule membranes at the 8-12 h timepoint (compare Fig. 3C vs. D). These results suggest that GTP binding is required for localization of Rab1 and Rab11 to secretory granule membranes. We did find that expression of the Rab11 DN -YFP construct was delayed compared to Rab1 DN -EYFP; we observed robust expression of Rab1 DN -YFP by ~4 h after Sgs3 but did not see significant expression of Rab11 DN -YFP until ~8 h after Sgs3. When examining the effect of Rab1 DN and Rab11 DN overexpression on secretory granule size, we found that salivary gland-specific overexpression of both of these proteins resulted in a substantial decrease in secretory granule size at the 20-24 h timepoint (Fig. 3E).
However, the phenotype appeared slightly weaker and more variable in Rab11 DN -expressing glands, likely due to delayed expression of this transgenic construct. Overall, this data validates our RNAi screen results and indicates that GTP binding is required for Rab1 and Rab11 function during mucin granule maturation.

Rab11 is required for Rab1 localization to secretory granule membranes
Many Rabs function sequentially to regulate membrane trafficking; for example, Rab5 and its effectors are required for Rab7 recruitment to endosomal membranes, regulating the transition from an early endosome to a late endosome (Wandinger-Ness and Zerial, 2014  Rab11 (Fig. 4A, B). qPCR analysis of dissected salivary glands showed that Rab11-RNAi did not significantly alter Rab1 mRNA expression levels ( Fig. S1), suggesting that loss of Rab11 specifically affects Rab1 localization, not expression. In contrast, RNAi knockdown of Rab1 did not alter Rab11 expression levels or localization patterns (Fig. S1, S4A, B). Taken together, these results suggest that Rab11 is required for Rab1 recruitment to the membrane of immature secretory granules.

Rab11 function is required for exocytosis
Interestingly, in control cells, we observed that Rab11 protein continued to associate with the membrane of secretory granules during the process of exocytosis, and Rab11 was deposited on the apical plasma membrane after completion of granule fusion (Movie 1, Fig. 5A), suggesting that Rab11 may play a functional role in exocytosis. Some animals homozygous for the hypomorphic Rab11 93Bi mutation (Giansanti et al., 2007;Eisenberg et al., 1990;Jankovics et al., 2001) survive to enter metamorphosis, making it possible to test for exocytosis defects in this mutant background. All mucin proteins had been secreted by the onset of metamorphosis in the salivary glands of control animals; however, Rab11 93Bi mutant salivary glands still contained a significant amount of mucin granules (Fig. 5B) upon loss of Rab11 function. Notably, the un-secreted granules were quite small, further confirming a functional role for Rab11 in regulating secretory granule size. Similar phenotypes were observed in salivary glands expressing Rab11-RNAi. To further confirm these results, we examined the localization of mucins in whole animals at the onset of metamorphosis. Mucins are expelled out of the salivary gland lumen and onto the surface of control animals at puparium formation (Biyasheva et al., 2001;Kang et al., 2017), forming a visible fluorescent 'halo' around the prepupa (Fig. 5D). In contrast, mucins were present only within the salivary glands and not on the surface of animals upon salivary gland-specific overexpression of Rab11 DN (Fig. 5D), confirming that exocytosis fails upon loss of Rab11 function.
Neuman et al. page 12 We next wanted to understand why loss of Rab11 function inhibits secretory granule maturation and exocytosis. Previous work by us and others demonstrates that defects in secretory granule membrane fusion protein trafficking results in both maturation and exocytosis defects (Neuman and Bashirullah, 2018;Ma et al., 2020;Burgess et al., 2012), prompting us to test if these membrane fusion proteins were present on secretory granule membranes upon loss of Rab11.
The SNARE protein SNAP-24 was present on the membranes of secretory granules in control glands (Fig. S5A), consistent with previously reported results (Niemeyer and Schwarz, 2000;Neuman and Bashirullah, 2018). SNAP-24 was also present on the membranes of small secretory granules in salivary glands with RNAi knockdown of Rab11 (Fig. S5C), indicating that a defect in membrane fusion protein trafficking is not responsible for the maturation or exocytosis defects observed upon loss of Rab11.
As a next step to understanding why exocytosis fails upon loss of Rab11, we examined the machinery required for physical fusion of secretory granule membranes with the apical plasma membrane. Previous work has demonstrated that the actomyosin cytoskeleton is required for exocytosis of these large mucin-containing secretory granules, and assembly of filamentous actin (F-actin) is the first observable event during this process (Rousso et al., 2015;Tran et al., 2015). Consistently, we observed filamentous actin (F-actin), assessed by phalloidin staining, wrapped around granules undergoing exocytosis in control salivary glands, and the presence of mucins in the lumen indicated that other secretory granules had already undergone exocytosis ( Fig. 5C). In contrast, no mucin was visible in the lumen of salivary glands overexpressing Rab11 DN at the 20-24 h timepoint, and no secretory granules near the apical membrane were wrapped in F-actin (Fig. 5C). Note that the variability in secretory granule size in Rab11 DNexpressing glands is likely due to the delayed onset of expression of Rab11 DN -YFP, as described earlier. Taken together, these results suggest that Rab11 may play a functional role in promoting F-actin-dependent fusion of mucin-containing secretory granules during exocytosis.

Small granules lacking Rab1 are secreted
Our next goal was to determine if loss of Rab1 affected exocytosis. Rab1 knockdown did not appear to perturb trafficking of SNAP-24, since this SNARE protein was still present on the membranes of small secretory granules (Fig. S5A, B). We also did not observe any significant secretion defects at the onset of metamorphosis in salivary glands expressing Rab1-RNAi.
Surprisingly, unlike Rab11 DN , salivary glands expressing Rab1 DN had visible mucins in the lumen and tiny secretory granules near the apical membrane wrapped in F-actin at the 20-24 h timepoint (Fig. 6C). To confirm these results, we performed live-cell time-lapse imaging of mucin granules and F-actin dynamics using the fluorescently tagged F-actin binding peptide Lifeact-ruby (Riedl et al., 2008). Consistent with previous reports (Rousso et al., 2015;Tran et al., 2015), we observed F-actin assemble around mucin granules as they were undergoing exocytosis in control cells (Movie 2, Fig. 6A). Similarly, F-actin assembled around small granules in salivary glands expressing Rab1-RNAi, and these small granules were secreted (Movie 3, Fig.   6B). This data shows that immature granules lacking Rab1 can be secreted and suggests that an increase in mucin granule size is not required for exocytosis. Sgs3>/+) glands at 20-24 h after Sgs3. Control image shows a single slice from a z-stack comprising 19 optical slices at a 0.36 µm step size; Rab1 DN image shows a single slice from a z-stack comprising 15 optical slices at a 0.20 µm step size. The salivary gland lumen is on the right side of the images. Scale bars: 5 µm. (D) Summary model. Mucincontaining secretory granules undergo two distinct 'phases' of growth; the first involves a rapid, Rab1-independent fusion of nascent granules to generate an ~18-fold increase in volume in just 4 h. The second phase of growth begins at 4 h after biogenesis and involves a more gradual Rab1-dependent ~15-fold increase in granule volume over the following 16 h. Rab11 is present on nascent granule membranes and recruits Rab1 to immature granule membranes. Rab1 is removed from mature granules, while Rab11 remains on granule membranes and regulates secretory granule "docking" to the plasma membrane and/or F-actin recruitment during exocytosis.

DISCUSSION
Secretory granule maturation is a poorly understood process during regulated exocytosis. Here, we have identified a new role for Rab proteins in the regulation of secretory granule size during maturation. Rab11 is present on nascent secretory granule membranes and is required for recruitment of Rab1; Rab1, in turn, drives growth of immature secretory granules, and Rab1 removal signals the completion of maturation. Rab11 appears to have a second functional role in initiating secretory granule fusion with the plasma membrane during regulated exocytosis (Fig.   6D). Overall, these findings provide unexpected new insights into the function of Rab proteins during secretory granule maturation and regulated exocytosis.
Our results demonstrate that Rab1 plays a critical functional role in promoting growth of immature granules during maturation. However, how Rab1 facilitates fusion of immature granules remains an unanswered question. Secretory granule membrane protein trafficking appears unaffected upon loss of Rab1; therefore, other as-yet-unidentified factors must function downstream of Rab1 to regulate granule-granule fusion. Rab proteins function through direct interaction with effector proteins; therefore, one or more Rab1 effectors likely play a role in secretory granule growth. However, most of the known Rab1 effectors localize to the ER/ERES/ Golgi to regulate ER exit (Homma et al., 2021), raising the possibility that Rab1 may work with a distinct subset of effectors that are specific to secretory granules. What regulates removal of Rab1 from mature granules also remains an open question. It is possible that removal of Rab1 may be triggered by a granule-autonomous process that 'senses' when the granule has reached its final mature size; alternatively, removal of Rab1 could be triggered by an independent developmental signal. Rab GAPs are frequently involved in removal of Rabs from their target membrane (Müller and Goody, 2018); therefore, a change in the localization or developmental expression levels of Rab1 GAPs may facilitate Rab1 removal.
Rab11 appears to have two roles during mucin exocytosis: first in recruiting Rab1 to immature granule membranes, and second in promoting mucin granule fusion with the plasma membrane during secretion. However, we cannot eliminate a possible earlier function for Rab11 in regulating fusion of nascent granules due to technical limitations. The Sgs3-GAL4 driver does not drive robust expression of dsRNA hairpins early enough to interfere with a function during the first growth phase of nascent granules, and the Rab11 93Bi allele is quite weak, with a significant fraction of the animals surviving to adulthood (Jankovics et al., 2001). In contrast, the requirement for Rab11 function during exocytosis, like the role of Rab11 in recruiting Rab1 to immature granules, is much more conclusive. However, we do not yet know how Rab11 promotes exocytosis. During mucin exocytosis, the granules fuse with the apical membrane, forming a 'neck-like' fusion pore; this is followed by the recruitment of F-actin and non-muscle myosin which generate a contractile force to drive cargo release (Rousso et al., 2015;Tran et al., 2015). Rab11 could be required for either of these steps or for an earlier membrane attachment/ tethering step. Sec15, a component of the exocyst complex that plays a role in tethering of vesicles to the plasma membrane, has been reported to be an effector of Rab11 (Wu et al., 2005;Zhang et al., 2004), suggesting that Rab11 function may be required for plasma membrane tethering of mucin-containing vesicles in the larval salivary glands. Given the high penetrance and severity of the secretion defects observed upon loss of Rab11 function, we posit that Rab11 function is likely required for an early membrane attachment or fusion pore formation step during mucin exocytosis. Future studies will be required to test this model.
Secretory granule maturation, including the process of granule fusion, is thought to be essential for regulated exocytosis, as immature granules are not responsive to stimulation with secretagogues (Bonnemaison et al., 2013). However, our data indicates that small granules lacking Rab1 can still undergo regulated exocytosis, suggesting that at least some aspects of secretory granule maturation may be dispensable. Alternatively, this finding raises the possibility that Rab1 may serve as a 'marker' for immature granules, and the presence of Rab1 on secretory granule membranes may prevent those granules from undergoing exocytosis. Our unpublished observations suggest that the mucins secreted from these small granules are abnormal. A number of other processes are known to occur during secretory granule maturation, including refinement of secretory granule membrane and cargo content (Kögel and Gerdes, 2010a;Bonnemaison et al., 2013), suggesting that Rab1 function may be required for other processes during secretory granule maturation beyond granule growth and that the presence of Rab1 on granules may inhibit the precocious release of immature granules.
One of the unique features of mucin granules in the larval salivary glands is their large size; mature granules reach an average volume of 30 µm 3 . This large size presents significant mechanical challenges for membrane fusion events, suggesting that these granules may require additional factors to generate sufficient contractile forces to drive granule-granule fusion during maturation and granule-plasma membrane fusion during exocytosis. However, large secretory granules are not unique to the Drosophila larval salivary glands; several mammalian cell types also produce and secrete large vesicles, including digestive enzymes in the exocrine pancreas (Geron et al., 2013), surfactants in the lungs (Miklavc et al., 2015), and von Willebrand factor in endothelial cells (Nightingale et al., 2011). Future analysis of the localization and function of Rab1 and Rab11 in these systems, as well as in other professional secretory cells, may reveal a shared requirement for these proteins in secretory vesicle trafficking.

Fly stocks and husbandry
The following fly stocks were obtained from the Bloomington Drosophila Stock Center: all UAS-Rab-RNAi lines (stock identifiers listed in Table S1)

Developmental staging
Animals were synchronized at the onset of mucin/glue biogenesis as previously described (Neuman et al., 2021). Given that there is some degree of variability in developmental rates between animals, a second layer of tissue-autonomous synchronization was used to further confirm developmental stages. Since glue protein synthesis begins in the cells at the distal tip of the glands and progresses forward (Biyasheva et al., 2001), the number of cells that synthesized glue was used to confirm developmental timing according to the following criteria: 0-4 h after

Confocal microscopy and immunofluorescence
All images except those in Fig. 5C Vectashield (Vector Laboratories) and imaged as described above. Quantification of granule volume was done in ImageJ, and z-stack data was used to confirm that the diameter was measured at the medial cross-section of each granule. Box and whisker plot was generated using Prism. Quantification of the percentage of granules with Rab1-EFYP was done using FV31S-SW software. Images of whole puparia were obtained using an Olympus SXZ16 stereomicroscope coupled to an Olympus DP72 digital camera with DP2-BSW software. Table S1. Summary of Rab-RNAi screen results. All RNAi lines were crossed to flies containing the Sgs3-GFP and Sgs3-GAL4 transgenes. ++ indicates a strong reduction in granule size; + indicates a moderate reduction in granule size;indicates no size defect. Highlighted stocks were validated for the efficiency of RNAi knockdown via qPCR and used for all detailed experimental analysis (see Fig. S1). Figure S1. Validation of RNAi knockdown efficiency. qPCR analysis of Rab1 and Rab11 expression levels in dissected salivary glands from animals synchronized at the onset of metamorphosis (0 h after puparium formation, PF) shows that Rab1-RNAi knocks down Rab1 expression levels but does not significantly affect Rab11 expression levels. Similarly, Rab11-RNAi reduces Rab11 expression levels but does not significantly alter Rab1 expression levels. y-axis shows relative expression and x-axis shows the genotypes analyzed. Expression was normalized to rp49 and samples were analyzed in biological triplicate. Error bars and statistics were determined using REST analysis. Asterisks indicate p>0.05. Figure S2. Rab1 also localizes to ER exit sites and Rab11 also localizes to Golgi. (A) Live-cell imaging of Rab1-EFYP (cyan) and Sec31-RFP (magenta), a component of the COPII coat complex and marker for ER exit sites (ERES), shows that Rab1 localizes to ERES in addition to localizing to secretory granule membranes at 4-8 h after Sgs3. Image shown is a single slice from a z-stack comprising ten optical slices at a 0.31 µm step size. (B) Live-cell imaging of Rab11-EFYP (cyan) and the pan-Golgi marker Golgi-RFP (magenta), shows that Rab1 localizes to the Golgi in addition to localizing to secretory granule membranes at 4-8 h after Sgs3. Note that small Rab11-positive puncta that do not co-localize with Golgi-RFP are also present; these are likely recycling endosomes. Image shown is a single slice from a z-stack comprising 17 optical slices at a 0.30 µm step size. Scale bars: 5 µm.  Live-cell imaging analysis of Rab5-EYFP (cyan) localization with mucins (red). Rab5 is absent from granule membranes but localizes in small puncta that are likely early endosomes (Zhang et al., 2007;Dunst et al., 2015) at 0-4, 8-12, and 20-24 h after Sgs3. Images shown are a single slice from a z-stack comprising three slices (0-4 h), four slices (8-12 h), or five slices (20-24 h) at a 0.31 µm step size. (C) Live-cell imaging analysis of Rab10-EYFP (cyan) localization with mucins (red). Rab10 is absent from granule membranes but localizes in small puncta that are likely recycling endosomes (Chan et al., 2011;Gaudet et al., 2011) at 0-4, 8-12, and 20-24 h after Sgs3. Images shown are a single slice from a z-stack comprising 16 slices (0-4 h), 13 slices (8-12 h) or six slices (20-24 h) at a 0.31 µm step size. Scale bars: 5 µm. Note that we did not observe substantial expression of Rab3-or Rab27-EFYP in salivary glands; therefore, these proteins were not pursued further.  Figure S4. Rab1 knockdown does not affect Rab11 localization. (A) Live-cell imaging of Rab11-EFYP (cyan) and mucins (red) shows that Rab11 is present on mucin granule membranes at 4-8 h after Sgs3. Image shown is a single slice from a z-stack comprising ten optical slices at a 0.31 µm step size. (B) Live-cell imaging of Rab11-EYFP (cyan) localization with mucins (red) in salivary glands expressing Rab1-RNAi shows that Rab11 is still present around secretory granule membranes upon knockdown of Rab1 expression at 4-8 h after Sgs3. Image shown is a single slice from a z-stack comprising ten optical slices at a 0.31 µm step size. Scale bars: 5 µm.

Figure S5. Knockdown of Rab1 or Rab11 does not affect secretory granule membrane protein localization. (A)
Antibody staining for the SNARE protein SNAP-24 (green) in fixed cells shows that this secretory granule membrane protein is present around mucin granules (red) in control salivary glands at 20-24 h after Sgs3. Image shown is a single slice from a z-stack comprising 13 optical slices at a 0.37 µm step size. (B) SNAP-24 (green) is present around small mucin granules (red) in Rab1-RNAi-expressing cells at 20-24 h after Sgs3. Image shown is a single slice from a z-stack comprising nine optical slices at 0.37 µm step size. (C) SNAP-24 (green) is present around small mucin granules (red) in Rab11-RNAi-expressing cells at 20-24 h after Sgs3. Image shown is a single slice from a z-stack comprising 13 optical slices at 0.37 µm step size. Scale bars: 5 µm.
Movie 1. Rab11 is present on secretory granule membranes during exocytosis. Live-cell time-lapse imaging of Rab11-EYFP (cyan) and mucins (red) shows that Rab11 stays associated with secretory granule membranes and is deposited on the apical plasma membrane during exocytosis. Arrow points to a granule undergoing exocytosis. Movie shows a maximum intensity projection of nine optical slices acquired at a 0.31 um step size and was not deconvolved. A total of 100 frames were captured, comprising ~9 mins total time. Full genotype: Sgs3-DsRED/+; Rab11-EYFP/+.
Movie 2. F-actin dynamics during exocytosis in control cells. Live-cell time-lapse imaging of Lifeact-ruby (gray) to visualize F-actin and mucins (red) during exocytosis in control cells shows that F-actin forms around secreting granules and drives exocytosis. Arrow points to a granule undergoing exocytosis. Movie shows a single slice from a z-stack comprising 16 total optical slices at a 0.75 µm step size. A total of 150 frames were captured, comprising ~14 mins total time. Movie shows 79 frames comprising a total of ~7.3 mins total time. Full genotype: UAS-lifeact-ruby/+; Sgs3>/Sgs3-GFP.
Movie 3. Small granules lacking Rab1 can be secreted. Live-cell time-lapse imaging of Lifeact-ruby (gray) to visualize Factin and mucins (red) during exocytosis in Rab1-RNAi-expressing cells shows that small granules undergo exocytosis. Arrow points to a granule undergoing exocytosis. Movie shows a single slice from a z-stack comprising 13 optical slices at a 1.0 µm step size. A total of 200 frames were captured, comprising ~15 mins total time. Movie shows 69 frames comprising a total of ~5.4 mins total time. Full genotype: UAS-lifeact-ruby/UAS-Rab1-RNAi; Sgs3>/Sgs3-GFP.