We provide compelling evidence of a previously unknown function for clathrin and AP-1 in the formation of mucin-type secretory granules. We show that clathrin and AP-1 localize to the TGN prior to synthesis of secretory cargo, colocalize with newly synthesized secretory cargo, and are required for secretory granule formation. Hence AP-1 and clathrin play a crucial role in early stages of secretory granule formation in salivary gland cells. Consistent with this idea, clathrin becomes delocalized upon AP-1 depletion, indicating that other adaptors cannot recruit clathrin in the absence of AP-1 at this stage of salivary gland development.
Our results suggest that formation of mucin-containing glue granules and Weibel-Palade bodies might be similar. Weibel-Palade bodies have an unusual cigar-shaped appearance and it was proposed that AP-1 and clathrin might participate in their formation at the TGN by allowing lumenal cargo to properly fold and aggregate or by preventing premature scission (Lui-Roberts et al., 2005
; Metcalf et al., 2008
). Indeed, depletion of AP-1 in endothelial cells results in the formation of small, round von Willebrand factor–containing organelles lacking other Weibel-Palade body markers. Our data demonstrate that the requirement for clathrin and AP-1 is not restricted to one specific type of granule. Depletion of clathrin or AP-1 in Drosophila
salivary glands resulted in the accumulation of glue protein both at the TGN and in small organelles of aberrant morphology. This finding extends the role of AP-1 and clathrin to the formation of granules containing mucoprotein cargo and suggests a broader requirement for this coat complex in granule production.
How might AP-1 participate in glue granule formation? One possibility is that AP-1 and clathrin are directly involved in packaging glue granule cargo at the TGN. In mammalian cells, several transmembrane proteins are targeted to regulated secretory granules, including peptidyl-α-amidating monooxygenase, muclin, and phogrin (Bell-Parikh et al., 2001
; Wasmeier et al., 2002
; Boulatnikov and De Lisle, 2004
; Dikeakos and Reudelhuber, 2007
). Indeed, phogrin has been shown to bind to AP-1 and AP-2 through well-conserved tyrosine and dileucine sorting motifs present in its cytosolic tail (Torii et al., 2005
; Wasmeier et al., 2005
). How AP-1, a cytosolic coat protein, might interact with lumenal glue proteins in salivary cells remains to be determined. Because none of the known granule proteins contains a predicted transmembrane domain, a yet-unidentified transmembrane receptor might mediate this interaction.
A distinct possibility is that AP-1 might be required to maintain a steady-state distribution of proteins that shuttle between the TGN and endosomes such that they are available at the TGN during granule formation. For instance, the protein convertase furin recycles between the TGN and endosomes and is required to process numerous secreted proteins such as von Willebrand factor (Creemers et al., 1993
). Importantly, furin is no longer concentrated at the TGN in μ1A-deficient fibroblasts (Fölsch et al., 2001
). Thus failure to recycle transmembrane enzymes that play a crucial role in processing secreted cargo could also contribute to defective granule formation.
Reduced levels of AP-1 resulted in intermediate-sized granules, suggesting AP-1 might have an additional role during glue granule maturation. The development of Drosophila
glue granules is characterized by an overall increase in size and decrease in number, consistent with homotypic fusion of smaller granules over time (Farkas and Suakova, 1999
). Whether small and large granules are equally capable of fusing and whether fusion events are temporally regulated is not known. AP-1 might regulate granule maturation by sorting or retrieving membrane proteins required for homotypic fusion and eventual exocytosis. Additionally, AP-1 might function directly on maturing granules to remove missorted proteins, such as lysosomal hydrolases, similar to what has been reported for other types of secretory granules (Dittie et al., 1996
; Klumperman et al., 1998
). In support of this view, live imaging revealed a dynamic association of AP-1 with immature granules. Further studies are needed to resolve whether AP-1 functions in the addition and/or removal of proteins from maturing glue granules.
On the basis of the small size of mutant cells, AP-1 likely participates in additional trafficking pathways. In mammalian cells, AP-1A is ubiquitously expressed and required for trafficking between TGN and endosomes, whereas AP-1B is present only in polarized epithelial cells and is required for basolateral sorting from recycling endosomes (Fölsch et al., 1999
; Cancino et al., 2007
; Gravotta et al., 2007
; Deborde et al., 2008
). The sole AP-1 complex in Drosophila
might mediate both functions in a single cell type. Interestingly, depletion of AP-1γ in salivary glands after granule formation caused the basolateral protein Discs large to redistribute to the apical surface (Peng et al., 2009
), suggesting that AP-1 is required for basolateral targeting of proteins in this tissue. However, an independent analysis of AP-1μ null cells in the dorsal thorax epithelium failed to reveal a similar polarity defect (Benhra et al., 2011
). This discrepancy might be due to cell type–specific requirements for AP-1 or to differences in RNAi versus mutant clones.
The observation that the abundance of Sgs3-DsRed protein and several Sgs mRNAs is reduced upon AP-1 knockdown suggests the existence of a negative-feedback loop, whereby a block in anterograde secretory trafficking results in down-regulation of secretory genes. A block in secretion at the TGN could potentially induce the unfolded protein response, analogous to what happens upon depletion of the Arf1 GEF GBF1 (Citterio et al., 2008
). However, GBF1 functions early in the secretory pathway, and knockdown of two Arf-GEFs that act on the TGN did not elicit a similar response (Citterio et al., 2008
). Alternatively, a block in anterograde trafficking might repress transcriptional activation of secretory genes by Drosophila
CrebA and Forkhead (Fkh) by some as-yet-unknown mechanism (Abrams and Andrew, 2005
; Abrams et al., 2006
In addition to the AP-1 complex, the Drosophila
genome encodes two other Golgi-localized clathrin adaptor proteins, EpsinR/LqfR and Golgi-localized, γ-ear–containing, ADP-ribosylation factor–binding (GGA) protein (Drosophila
has only one GGA) (Hirst et al., 2009
; Lee et al., 2009
; Kametaka et al., 2010
). LqfR partially colocalizes with AP-1 at the TGN in salivary gland cells and lqfR
mutants exhibit small salivary glands, suggesting defects in granule biogenesis (Lee et al., 2009
). It will be interesting to determine whether LqfR and GGA participate in glue granule biogenesis, especially since these clathrin adaptors might facilitate sorting of other types of cargo. For example, EpsinR has been shown to bind SNARE proteins and could function to provide vesicle identity to nascent glue-containing granules (Miller et al., 2007
; Chidambaram et al., 2008
). SNAP-24 was previously identified as a glue granule–specific SNARE, although whether this SNARE mediates homotypic fusion of granules or functions during exocytosis of granules at the plasma membrane is unclear (Niemeyer and Schwarz, 2000
). Given the apparent similarities between glue granule and Weibel-Palade body biogenesis, as well as the high degree of conservation of TGN sorting machinery in Drosophila
, our findings suggest that Drosophila
salivary glands are of great utility to further elucidate the mechanisms of biogenesis of regulated secretory granules.