The EARP Complex and Its Interactor EIPR-1 Are Required for Cargo Sorting to Dense-Core Vesicles

The dense-core vesicle is a secretory organelle that mediates the regulated release of peptide hormones, growth factors, and biogenic amines. Dense-core vesicles originate from the trans-Golgi of neurons and neuroendocrine cells, but it is unclear how this specialized organelle is formed and acquires its specific cargos. To identify proteins that act in dense-core vesicle biogenesis, we performed a forward genetic screen in Caenorhabditis elegans for mutants defective in dense-core vesicle function. We previously reported the identification of two conserved proteins that interact with the small GTPase RAB-2 to control normal dense-core vesicle cargo-sorting. Here we identify several additional conserved factors important for dense-core vesicle cargo sorting: the WD40 domain protein EIPR-1 and the endosome-associated recycling protein (EARP) complex. By assaying behavior and the trafficking of dense-core vesicle cargos, we show that mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants in the RAB-2 pathway. Genetic epistasis data indicate that RAB-2, EIPR-1 and EARP function in a common pathway. In addition, using a proteomic approach in rat insulinoma cells, we show that EIPR-1 physically interacts with the EARP complex. Our data suggest that EIPR-1 is a new interactor of the EARP complex and that dense-core vesicle cargo sorting depends on the EARP-dependent trafficking of cargo through an endosomal sorting compartment.


Author Summary
Animal cells package and store many important signaling molecules in specialized compartments called dense-core vesicles. Molecules stored in dense-core vesicles include peptide hormones like insulin and small molecule neurotransmitters like dopamine. Defects in the release of these compounds can lead to a wide range of metabolic and mental disorders in humans, including diabetes, depression, and drug addiction. However, it is not well understood how dense-core vesicles are formed in cells and package the appropriate trans-Golgi, suggesting that they act in the neuron cell body to mediate proper cargo-sorting as dense-core vesicles bud off from the trans-Golgi or undergo post-Golgi maturation.
Here we investigate the function of several additional molecules identified in our genetic screen for dense-core vesicle mutants: the conserved WD40 domain protein EIPR-1 and the VPS-52 and VPS-53 subunits shared by the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes. We show that EIPR-1 physically and genetically interacts with the EARP complex. Mutants that lack EIPR-1 or EARP have defects in dense-core vesicle cargo-sorting similar to those of mutants that lack RAB-2, RUND-1 or CCCP-1, and genetic epistasis data indicate that they function in the same pathway. The requirement of EARP for proper sorting of cargos to dense-core vesicles suggests that dense-core vesicle cargo sorting is achieved in part through the trafficking of dense-core vesicle cargos or cargo receptors through an endosomal sorting compartment. While there is ample prior evidence that cargos are removed during dense-core vesicle maturation and trafficked to endosomes, our experiments suggest that there is also trafficking from endosomes to maturing dense-core vesicles.

eipr-1 encodes a conserved WD40 domain protein
To identify proteins required for dense-core vesicle biogenesis, we performed a forward genetic screen in the nematode C. elegans based on suppression of the hyperactive locomotion of an activated Gq mutant [23] (S1A Fig). We previously characterized the function of two novel conserved proteins identified from this screen, RUND-1 and CCCP-1. Both RUND-1 and CCCP-1 interact with the small GTP-binding protein RAB-2 when it is in its activated GTPbound state [23]. Mutants in rab-2, rund-1 and cccp-1 have a stereotypical defect in locomotion behavior characterized by little spontaneous movement on food, but coordinated slow locomotion when stimulated. One additional uncloned mutant from our screen (ox316) had defects in locomotion and egg-laying very similar to rab-2, rund-1, and cccp-1 mutants. We cloned this mutant by single nucleotide polymorphism (SNP) mapping and transgenic rescue experiments (Materials and Methods). We named the gene eipr-1 (EARP-interacting-protein, see below). The eipr-1 mutant phenotype is fully rescued by microinjection of the single gene Y87G2A.11, and the ox316 mutation leads to a premature stop codon in this gene (Fig 1A-1C). We subsequently obtained a deletion allele (tm4790) that causes identical locomotion and egg-laying phenotypes ( Fig 1C and S1B Fig). eipr-1 encodes a novel WD40 domain protein of 362 amino acids ( Fig 1B). Like RUND-1 and CCCP-1, EIPR-1 has a single conserved ortholog in other metazoans. However, unlike RUND-1 and CCCP-1, EIPR-1 is also found outside metazoans in plants and some fungi and protozoa (S2 Fig). Interestingly, EIPR-1 is not present in all protozoa and fungi, suggesting an ancestral origin and loss in specific lineages including the budding yeast Saccahromyces cerevisiae. The mouse Eipr1 cDNA could also rescue the worm eipr-1 mutant (Fig 1C and S1B Fig), demonstrating functional conservation of the mammalian ortholog.
EIPR-1 acts in the RAB-2 pathway to control locomotion and dense-core vesicle cargo trafficking The phenotypic similarities between eipr-1 and rab-2, rund-1, and cccp-1 mutants suggests that these genes act in a common pathway. To test this, we built double mutants between eipr-1 and either rab-2 or rund-1 and assayed locomotion and egg-laying behavior. In both cases, the double mutants do not show enhanced locomotion or egg-laying defects compared to the single mutants ( Fig 1D and S1C Fig). Thus, eipr-1 acts in the rab-2 pathway to control locomotion and egg-laying behaviors.
Mutants in rab-2, rund-1 and cccp-1 have reduced axonal levels of dense-core vesicle cargos [19,22,23]. To test whether eipr-1 also acts in dense-core vesicle cargo trafficking, we examined trafficking of both luminal and membrane cargos of dense-core vesicles. For luminal cargos, we assayed the Venus-tagged peptides NLP-21, FLP-3, and INS-22. NLP-21 and FLP-3 are endogenous neuropeptides that undergo proteolytic processing [25,26], while INS-22 is an EIPR-1 is a 362 amino acid protein with four predicted WD40 repeats. The part of the protein deleted by tm4790 is marked, but because this deletion starts and ends in introns (Fig 1A), its precise effect on the protein is unknown, though it would cause a frameshift if exon 3 splices to exon 5 in this mutant. The protein structure was drawn using DOG 1.0 [24]. WD40 repeat positions were as predicted by SMART (http://smart.embl-heidelberg.de/). (C) eipr-1 acts in neurons to control locomotion. The eipr-1 mutant has slow locomotion that is rescued by panneuronal expression of either the worm gene (yakEx1 transgene) or its mouse ortholog (yakEx38 transgene) (***, P<0.001). Error bars = SEM; n = 10. (D) eipr-1 acts in the same genetic pathway as rab-2 and rund-1 to control locomotion. Double mutants of eipr-1(tm4790) with rab-2(nu415) or rund-1(tm3622) do not have stronger locomotion defects than the single mutants (***, P<0.001 compared to wild type; ns, not significant, P>0.05). Error bars = SEM; n = 10.
insulin-family peptide that does not undergo proteolytic processing [27]. For a membrane cargo, we assayed GFP tagged IDA-1, the worm ortholog of the dense-core vesicle transmembrane protein phogrin [28]. Like the rab-2 pathway mutants, we found that eipr-1 mutants also had reduced axonal levels of all luminal cargos and the transmembrane cargo IDA-1::GFP (Fig  2A-2D and S3A-S3C Fig). Double mutants of eipr-1 with either rab-2 or rund-1 did not show an enhanced defect compared to the single mutants (Fig 2A), again suggesting that eipr-1 acts in the rab-2 pathway. To determine whether the eipr-1 defect is specific to dense-core vesicle cargos, we examined trafficking of the synaptic vesicle cargo UNC-17, the vesicular acetylcholine transporter. eipr-1 mutants showed no reduction in axonal levels of UNC-17 ( Fig 2E). eipr-1 mutants also showed no reduction in axonal levels of the synaptic vesicle SNARE protein synaptobrevin (SNB-1) (S3D Fig). We conclude that EIPR-1 acts in the RAB-2 pathway to specifically mediate trafficking of dense-core vesicle cargos.
A mutation in the egl-3 proprotein convertase blocks neuropeptide processing and leads to an increase in axonal levels of luminal dense-core vesicle cargos [19,22]. As with rab-2 and rund-1, there is increased axonal fluorescence of Venus-tagged NLP-21 in eipr-1; egl-3 double mutants as compared to the eipr-1 single mutant ( Fig 2F). However, the egl-3 mutant on its own has increased axonal fluorescence compared to wild-type, and the eipr-1 mutant decreases axonal fluorescence about 50% in either wild-type or egl-3 backgrounds (Fig 2F), indicating that eipr-1, like rab-2 and rund-1, acts in parallel to egl-3. Thus, eipr-1 mutants have reduced axonal levels of both processed and unprocessed cargos.

EIPR-1 acts in neurons to control locomotion and dense-core vesicle biogenesis
The behavioral and dense-core vesicle phenotypes of eipr-1 mutants suggest that eipr-1 acts in neurons. Given that eipr-1 is the fourth gene in an operon, it has been difficult to determine the endogenous expression pattern of eipr-1 (see Materials and Methods for details and S4A Fig). Thus, we instead expressed the eipr-1 cDNA under the control of neuronal-specific promoters to determine whether the gene functions in neurons. Expression of eipr-1(+) under the neuronal specific rab-3 promoter fully rescued eipr-1 mutant locomotion, dense-core vesicle trafficking, and egg-laying (Figs 3A, 3B and S1B). Expression of eipr-1(+) under the cholinergic unc-17 promoter also rescued the locomotion defect. Expression in cholinergic motor neurons (using the acr-2 promoter) did not rescue the locomotion phenotype, but expression driven by a head-specific derivative of the unc-17 promoter (unc-17Hp) rescued the locomotion phenotype, indicating that eipr-1 acts mainly in head cholinergic neurons to regulate locomotion behavior ( Fig 3A). The dense-core vesicle trafficking defect of eipr-1 mutants is cell autonomous, as expression of eipr-1(+) driven by the unc-129 promoter fully rescued the axonal trafficking defect of NLP-21::Venus expressed by the same unc-129 promoter ( Fig 3B). Thus, EIPR-1 acts in neurons to control dense-core vesicle trafficking at the cellular level and locomotion behavior at the organismal level.

EIPR-1 interacts with members of the GARP and EARP trafficking complexes
WD40 domain proteins typically act as scaffolds that mediate multiple protein-protein interactions [30]. Given the genetic interactions seen between eipr-1 and members of the rab-2 pathway, we asked whether the EIPR-1 protein shows physical interactions with any of the RAB-2 pathway proteins. Using yeast two-hybrid assays, we did not detect interactions between EIPR-1 and RAB-2, RUND-1 or CCCP-1. RAB-2 and RUND-1 were also localized normally in eipr-1 mutants. Thus, neither cellular localization nor physical interactions connect EIPR-1 directly to RAB-2 or its interactors, suggesting that EIPR-1 may act at a distinct step in this trafficking pathway.
To identify interactors of EIPR-1, we expressed GFP-tagged rat EIPR1 in the rat insulinoma cell line 832/13 and performed mass spectrometry on GFP pulldowns. 832/13 cells package insulin into dense-core secretory granules that closely resemble neuronal dense-core vesicles and share similar mechanisms of biogenesis, maturation, and release [31,32]. Mammalian orthologs of RAB-2, RUND-1, CCCP-1 and EIPR-1 are all expressed endogenously in 832/13 cells (Fig 4A), so these cells are a reasonable place to look for interactors of these proteins.
We performed two independent pulldowns of GFP-tagged rat EIPR1. The top four hits identify the proteins VPS51, VPS50 (also known as syndetin or VPS54L), VPS52, and VPS53 ( Fig 4B and S5A Fig). Also high on the list is VPS54. These five proteins define the related GARP and EARP trafficking complexes that share the VPS51, VPS52, and VPS53 proteins, but use either VPS54 or VPS50 as a fourth subunit [33][34][35]. Thus, EIPR1 is likely tied closely to one or both of these complexes, neither of which has been previously linked to dense-core vesicle biogenesis. These proteomic results are especially striking because, as described below, we had independently identified the vps-52 and vps-53 genes in our forward genetic screen for dense-core vesicle mutants.
To confirm the interactions detected by mass spectrometry, we performed coimmunoprecipitation experiments. GFP-tagged EIPR1 expressed in 832/13 cells coimmunoprecipitated with either mCherry-tagged VPS51 or with endogenous VPS50 (Fig 4C), validating the interactions. Since no other proteins showed as strong an interaction by mass spectrometry as the GARP/ EARP subunits, EIPR1 is a specific interactor of the GARP/EARP complexes.
The GARP/EARP subunits VPS-51, VPS-52 and VPS-53 act in the RAB-2 pathway to control locomotion and dense-core vesicle cargo trafficking Further supporting the model that EIPR-1 functions with either the GARP or EARP complex, we isolated mutants in the worm orthologs of vps-52 and vps-53 in our original forward genetic screen. We cloned these mutants by mapping, whole-genome sequencing, and transgenic rescue (see Materials and Methods). Both the vps-52(ox345) and vps-53(ox339) mutants are predicted to be molecular nulls, an early stop and deletion respectively, and both mutants phenocopy other deletion mutants in the same genes. These mutants also resemble a deletion mutant in the vps-51 gene. Neuronal roles of vps-51, vps-52, and vps-53 have not been previously described in C. elegans.
We tested whether vps-51, vps-52 and vps-53 mutants have behavioral and cellular phenotypes similar to those of eipr-1 and rab-2. Like eipr-1 and members of the rab-2 pathway, vps- interacts with VPS51 and VPS50. EGFP-tagged rat EIPR1 or EGFP was coexpressed with mCherry-tagged rat VPS51 in 832/13 cells. Immunoprecipitation of EIPR1::EGFP pulled down VPS51::mCherry and 51, vps-52 and vps-53 mutants have slow but coordinated locomotion ( Fig 5A) and an egg-laying defect. A double mutant between rab-2 and vps-53 did not have a stronger locomotion phenotype than the rab-2 single mutant (Fig 5B), indicating that rab-2 and vps-53 act in the same pathway to control locomotion. These mutants also exhibit defects in trafficking dense-core vesicle cargos. Like rab-2 and eipr-1, vps-51, vps-52 and vps-53 mutants all show reduced axonal levels of Venus-tagged NLP-21 or FLP-3 peptides (Figs 5C, 5D and S6C). A rab-2; vps-53 double mutant did not show an enhanced NLP-21::Venus trafficking defect compared to the single mutants ( Fig 5E), also suggesting that rab-2 and vps-53 act in the same pathway. However, unlike the rab-2 and eipr-1 mutants which show reduced axonal accumulation of the densecore vesicle transmembrane protein IDA-1, mutants in vps-51, vps-52 and vps-53 all show increased axonal accumulation of IDA-1 (Fig 5F), indicating that these genes have at least some function distinct from rab-2 or eipr-1. Since VPS-51, VPS-52, and VPS-53 are common to both GARP and EARP, these data suggest that GARP, EARP, or both are required for normal dense-core vesicle biogenesis and regulation of worm locomotion.
To determine the expression pattern of vps-52 and vps-53, we made transgenic worms expressing the YFP-derivative Citrine under either the vps-52 or vps-53 promoter. Both vps-52 and vps-53 were expressed strongly and widely in neurons and were also expressed in several other tissues (Fig 6A and 6B, and Materials and Methods for details). These data are consistent with the reported expression patterns of vps-51, vps-52, and vps-53 [36]. To determine where VPS-52 and VPS-53 localize in neurons, we tagged them with tagRFP at their C-termini and expressed them under their own promoters as single-copy transgenes to achieve endogenous expression levels. Both VPS-52 and VPS-53 localized mainly to two or three perinuclear puncta in neuronal cell bodies (Fig 6C and 6D), reminiscent of the localization of RAB-2, RUND-1 and CCCP-1 to the trans-Golgi [23]. We examined colocalization of VPS-52::tagRFP or VPS-53::tagRFP with GFP-tagged RAB-2 and found that they are partially overlapping in worm neurons ( Fig 6C and 6D), consistent with the reported localization of GARP to the trans-Golgi in other cell types and other systems [36][37][38][39].
The EARP complex controls cargo sorting to dense-core vesicles To determine whether it is GARP or EARP (or both) that is required for dense-core vesicle biogenesis, we obtained deletion mutations in the GARP-specific vps-54 and EARP-specific vps-50 subunits. Unlike the other GARP mutants, vps-54 mutants did not have defects in locomotion or axonal accumulation of the luminal dense-core vesicle cargos NLP-21 and FLP-3 ( The EARP Complex Controls Dense-Core-Vesicle Biogenesis was used in the genetic screen for dense-core vesicle mutants, similar to the suppression seen by eipr-1, vps-52 and vps-53 (S1A Fig). However, a vps-54 mutation did not suppress this activated Gq mutant. Taken together, these data support a role for EARP, but not GARP, in densecore vesicle biogenesis and regulation of worm locomotion.
reduced levels of FLP-3::Venus (ceIs61 transgene) fluorescence in the dorsal nerve cord. Scale bar: 10 μm. ***, P<0.001; **, P<0.01 compared to wild type. Error bars = SEM; n = 10. (E) vps-53 acts in the same genetic pathway as rab-2 to control FLP-3::Venus (ceIs61 transgene) trafficking. A rab-2(nu415); vps-53 (ox339) double mutant does not have a stronger phenotype than rab-2(nu415) or vps-53(ox339) single mutants. Scale bar: 10 μm. ***, P<0.001 compared to wild type. Error bars = SEM; n = 10. (F) vps-51, vps-52 and vps-53 mutants have increased levels of IDA-1::GFP (ceIs72 transgene) in the dorsal cord. vps-51 (tm4275) was assayed in a separate experiment from vps-52(ox345) and vps-53(ox339), each with independent matched wild type controls. Scale bar: 10 μm. ***, P<0.001; *, P<0.05 compared to wild type. Error bars = SEM; n = 10.   The decreased axonal levels of luminal dense-core vesicle cargos like NLP-21::Venus could be due either to decreased levels of the cargo in dense-core vesicles, or to increased release of the cargo. Exocytosis of dense-core vesicles and release of their cargos is reflected by accumulation of NLP-21::Venus in scavenger cells called coelomocytes that reside in the body cavity of the worm [40,41]. Thus, we measured coelomocyte fluorescence as an indirect measure of dense-core vesicle release. Like rab-2 mutants, eipr-1, vps-50, and vps-52 mutants all showed reductions in the accumulation of Venus fluorescence in coelomocytes that is approximately proportional to the decrease in axonal fluorescence seen in these mutants (S6B Fig). This suggests that these mutants are not defective in dense-core vesicle release per se, but release reduced amounts of dense-core vesicle cargo because the vesicles contain less cargo. Additionally, double mutants between eipr-1 and rab-2 or vps-50 did not have stronger coelomocyte uptake phenotypes than the single mutants, providing further evidence that these genes all act in the same dense-core vesicle cargo sorting pathway.
Two independent vps-50 mutants exhibited either a slight decrease or no decrease in axonal levels of the dense-core vesicle transmembrane cargo IDA-1 (Fig 7D). Thus, vps-50 and eipr-1 have a similar IDA-1 trafficking phenotype that is distinct from the increased axonal accumulation of IDA-1 seen in vps-51, vps-52 and vps-53 mutants. Interestingly, vps-54 mutants showed no change in axonal accumulation of IDA-1 (Fig 7D). These results suggest that vps-51, vps-52, and vps-53 may have a function in IDA-1 trafficking independent of vps-50 and vps-54.
Previously, C. elegans GARP mutants, including vps-54, were shown to have enlarged lysosomes in coelomocytes [36]. We examined eipr-1 and vps-50 mutants for this phenotype using the lysosomal marker LMP-1::GFP expressed in coelomocytes. Unlike GARP mutants, neither eipr-1 nor vps-50 had enlarged lysosomes (S3E Fig), indicating that EARP is not required for normal lysosomal morphology. Thus, GARP is required for normal lysosomal morphology and EARP is required for dense-core vesicle biogenesis.

Discussion
In this study, we identified several new factors important for dense-core vesicle biogenesis: the WD40 protein EIPR-1 and the EARP trafficking complex with which it interacts. EIPR-1 and EARP are required for normal trafficking of dense-core vesicle cargos, and act in the same genetic pathway as the small GTP-binding protein RAB-2 and its interactors RUND-1 and CCCP-1. Loss of any of these proteins results in reduced levels of cargo in mature dense-core vesicles and associated behavioral defects. The identification of EARP, an endosomal recycling complex, suggests that dense-core vesicle cargo trafficking involves not only the trafficking of cargo into nascent dense-core vesicles as they bud off from the trans-Golgi, but also the retrieval or recycling of cargo or sorting factors from endosomal compartments (Fig 8).

The WD40 domain protein EIPR1 is a new interactor of the EARP complex
Through a convergence of worm genetics and proteomics in rat 832/13 cells, we identified the WD40 domain protein EIPR1 as a direct physical and functional interactor of the EARP complex. The EARP complex was recently identified as a new complex structurally related to the GARP complex [34,35]. EARP shares the VPS51, VPS52 and VPS53 subunits with the GARP complex, but uses VPS50 instead of VPS54 as a fourth subunit. VPS50 was also recently shown to interact with GARP subunits in three large-scale mass spectrometry interactome data sets, demonstrating that the EARP complex is present in various cell types in several species [42][43][44]. In two of these data sets, EIPR1 was shown to interact with EARP subunits in human HEK293T or HeLa cells [42,43], providing support for our results and indicating that the EIPR1 interaction with EARP is robust and conserved. In fact, using VPS50 as a bait protein, VPS51, VPS52, VPS53 and EIPR1 were pulled down as a stoichiometric complex [42], indicating that EIPR1 may form part of a stable complex with EARP. No VPS54 was pulled down. This is consistent with our genetic experiments showing that eipr-1 and vps-50 mutants have similar locomotion and dense-core vesicle cargo trafficking phenotypes that are shared by vps-51, vps-52, and vps-53 mutants, but not vps-54. Thus, we propose that EIPR1 is a new interactor of the EARP complex.
The human Eipr1 gene was originally named Tssc1 for "tumor-suppressing subtransferable candidate 1" because it was first identified as one of several genes thought to be candidates for a tumor-suppressing activity on chromosome 11 [45]. However, when the human genome was completed, Tssc1 was located on chromosome 2, and thus could not be the putative tumor suppressor. Because the TSSC1 protein appears to be both physically and functionally connected to the EARP complex, we propose that it be renamed EIPR1 (EARP-Interacting PRotein).
WD40 domain proteins typically act as scaffolds that provide several interaction surfaces for the assembly of larger protein complexes [30]. Though EIPR1 clearly interacts with EARP, it is unclear whether EIPR-1 is required for the localization or stability of the EARP complex. In an eipr-1 mutant, VPS-52 still showed a punctate localization (S4F Fig), though this could also reflect its participation in the GARP complex. In C. elegans (unlike yeast), the remaining members of the GARP complex were shown to be stable and localized in the absence of one of the subunits [36], so the same may hold true for EARP. Surprisingly, GFP-tagged EIPR-1 was Model for dense-core vesicle cargo sorting. The EARP complex localizes to endosomal compartments and together with RAB-2 and its effectors (e.g CCCP-1) is involved in regulating cargo sorting to mature dense core vesicles. Dense-core vesicle cargo (green dots) is packaged into immature dense-core vesicles as they bud off from the trans-Golgi. During the process of dense-core vesicle maturation, EARP/ EIPR-1 and RAB-2 regulate the trafficking of cargo or sorting factors through or from endosomal compartments. In mutants of RAB-2 or EARP/EIPR-1, increased trafficking of dense-core vesicle cargo to endosomes or the failure to retrieve cargo or sorting factors from endosomes may lead to the ultimate loss of cargos to lysosomes, and hence reduced cargo levels in mature DCVs. GARP acts in a distinct endosome to Golgi trafficking pathway. doi:10.1371/journal.pgen.1006074.g008 The EARP Complex Controls Dense-Core-Vesicle Biogenesis found throughout the cytoplasm when overexpressed in C. elegans neurons and not associated with a membrane-bound compartment. However, overexpression of membrane-localized proteins can sometimes lead to a diffuse cytoplasmic appearance [46]. Because we could not detect an endogenously tagged EIPR-1::GFP fusion protein, we presume that eipr-1 is normally expressed at low levels. This conclusion is supported by the few cDNAs or RNAseq reads reported for eipr-1 on Wormbase (http://www.wormbase.org/species/c_elegans/gene/ WBGene00013599#0df9-gec6-2).
One other hit found in both of our EIPR1 pulldowns is SNAP29, a late Golgi/endosomal SNARE protein that has been shown to function in endocytic recycling and autophagosome fusion [47][48][49][50]. SNAP29 was also identified as an interactor of EIPR1 or EARP subunits (but not VPS54) in other recent mass spectrometry studies [35,42,43], and we confirmed that SNAP29 interacts with EIPR1 by a coimmunoprecipitation experiment (S5B Fig). GARP has been shown to interact with several late-Golgi SNARE proteins and has been proposed to both tether vesicles and promote SNARE assembly that leads to fusion [36,[51][52][53][54]. We propose that SNAP29 is part of a SNARE complex that interacts with EARP.
A new role for the GARP/EARP complex in mediating dense-core vesicle cargo sorting EARP was originally shown to be localized to a Rab4 positive endosomal compartment in both fly S2 cells and rat hippocampal neurons [34,35], and depletion of the EARP-specific VPS50 subunit led to a modest defect in the endocytic recycling of the transferrin receptor back to the cell surface [35]. Here we identify a new neuronal role of this complex in the sorting of cargos to dense-core vesicles.
The GARP complex was originally characterized as an endosome to Golgi retrograde trafficking complex that may tether endosome-derived vesicles at the trans-Golgi [37,51,53,55]. Loss or depletion of GARP subunits leads to a variety of phenotypes that are presumed to be secondary to a primary defect in retrograde trafficking, including missorting of lysosomal proteins, abnormal lysosomal morphology, defective autophagy, defective lipid and sterol transport, defective anterograde transport of glycosyl phosphatidylinositol (GPI)-anchored and transmembrane proteins, and defects in sphingolipid homeostasis [33,36,37,52,[55][56][57].
GARP and EARP subunits are expressed especially strongly in the nervous system in both mice and worms [35,36,58,59], but until now, neuronal phenotypes of GARP/EARP mutants have not been carefully characterized. However, several mutations in GARP/EARP subunits have been connected to neurological diseases. Mutations in VPS54 and VPS52 lead to neurological disorders in mice that have been studied as models of the human diseases amyotrophic lateral sclerosis (ALS) and the seizure disorder high-pressure nervous syndrome, respectively [60,61]. Also, mutations in the human VPS53 gene cause progressive cerebello-cerebral atrophy type 2 (PCCA2), a neurodegenerative disease characterized by severe mental retardation and early-onset epilepsy [62]. It will be interesting to determine whether the GARP/EARP mutations cause these diseases due to one of their previously characterized roles such as sorting lysosomal cargos [37,55], or due to their newly identified role in dense-core vesicle biogenesis. It will also be interesting to determine whether neurological diseases associated with VPS52 and VPS53 are caused by dysfunction of GARP or EARP or both. The overt behavioral phenotypes of C. elegans mutants affecting the GARP/EARP common subunits (vps-51, vps-52 and vps-53) are very similar to those of vps-50 and eipr-1, suggesting that EARP is more important than GARP for neuronal function in C. elegans.
Our identification of EIPR-1 and EARP as important for dense-core vesicle cargo sorting supports a recent study that showed that C. elegans vps-50 mutants have locomotion and dense-core vesicle cargo trafficking defects [59]. Like eipr-1, the slow locomotion of vps-50 mutants on food was partially rescued by expression in cholinergic neurons [59], suggesting that eipr-1 and vps-50 function together in the same neurons to control locomotion behavior. VPS-50 was also shown to physically interact with the vacuolar-ATPase (v-ATPase) complex that acidifies dense-core vesicles, and vps-50 mutants had defects in vesicle acidification and assembly of the v-ATPase complex [59]. These defects may be indirectly caused by a primary defect of EARP mutants in missorting dense-core vesicle cargos, possibly including subunits of the v-ATPase. One discrepancy between the two studies is that we found a modest reduction in axonal accumulation of the dense-core vesicle transmembrane cargo IDA-1 in the vps-50 (ok2627) mutant and no change in vps-50(n4022) (Fig 7D). In contrast, Paquin et al. reported a modest increase in axonal accumulation of IDA-1 in vps-50(n4022) [59]. Though we do not know the basis of this discrepancy, one difference between the two studies is that Paquin et al. imaged a more posterior section of the dorsal nerve cord [59].
How does EARP contribute to the sorting of dense-core vesicle cargos? EARP has been reported to localize to endosomal compartments [34,35], suggesting that dense-core vesicle cargo sorting may involve trafficking through endosomes. We propose a speculative model based on the idea that the RAB-2/EARP pathway proteins may contribute to dense-core vesicle cargo sorting by a passive mechanism that does not involve direct interactions with cargo ( Fig  8). We propose that EARP and the RAB-2 pathway may control the trafficking of cargo into or out of the endosomal compartment, perhaps as a way to remove non-aggregated cargo from the dense-core vesicle. In mutants in this pathway, increased trafficking of cargos to endosomes or reduced retrieval of cargos from endosomes leads to their ultimate loss to lysosomes and hence reduced cargo levels in mature dense-core vesicles. In this model, EARP and the RAB-2 pathway do not act instructively to sort cargos in the way a sorting receptor would, but instead have a permissive role. Such a permissive role for EARP and the RAB-2 pathway would be consistent with the fact that none of these mutants has a complete loss of cargo sorted to the mature dense-core vesicles that accumulate in axons.
Several previous studies are consistent with our model. Following dense-core vesicle exocytosis, the dense-core vesicle transmembrane protein peptidylglycine α-amidating monooxygenase (PAM) has been shown to be recycled back to dense-core vesicles via endosomal compartments [63], demonstrating that there must be trafficking of cargos from endosomes to dense-core vesicles. Our model suggests that such endosomal trafficking occurs not only following exocytosis, but also during the biogenesis of dense-core vesicles. Another study demonstrated that soluble dense-core vesicle cargos are segregated from self-aggregating cargos in immature dense-core vesicles near the trans-Golgi [64], consistent with the idea that aggregation of cargos at this point in vesicle maturation can be sufficient to drive sorting into mature dense-core vesicles.

A growing number of proteins important for dense-core vesicle biogenesis
The identification of EIPR-1 and EARP adds to a growing list of proteins that have been recently identified as being involved in mediating aspects of dense-core vesicle biogenesis. In C. elegans, we and others previously identified the small GTPase RAB-2; the putative RAB-2 GAP TBC-8; the RAB-2 effectors RIC-19, RUND-1 and CCCP-1; and the membrane-associated Golgi-localized protein HID-1 [19,20,22,23,65,66]. Mutations in all of these proteins result in similar defects in dense-core vesicle cargo trafficking, and genetic epistasis data support the idea that they function in a common pathway.
In other systems, a number of factors have been recently identified as having roles in densecore vesicle biogenesis. These include factors that belong to protein families implicated in various aspects of membrane trafficking such as BAR domain proteins [18,21,67,68], members of the Arf family of small GTPases [69], the clathrin adaptor complexes AP-1 and AP-3 [70][71][72][73], members of the BLOC-1 and HOPS complexes that are important for the biogenesis of lysosome-related organelles [74][75][76], and members of the sortilin family of lysosomal sorting receptors [77,78]. Among these, there are a few possible connections to the RAB-2/EARP pathway we have described in C. elegans. AP-1 is involved in trafficking between various cellular compartments, most notably between the trans-Golgi and endosomes [79], and has been shown to be associated with immature dense-core vesicles [6,8], so it may be involved in the endosomal recycling pathway proposed by our model. The BLOC-1 subunit dysbindin was identified as an interactor of EIPR1, VPS51 and VPS53 by mass spectrometry [43], and the possible dense-core vesicle coat protein VPS41 (along with other HOPS subunits) was identified as an interactor of Rab2 [34].
One final factor shown to be important for dense-core vesicle biogenesis in mouse chromaffin cells is the SNARE protein Vti1a [80]. Interestingly, Vti1a is one of the components of a trans-Golgi SNARE complex that binds GARP [53], and it also localizes to immature densecore vesicles near the trans-Golgi [80]. Syntaxin 6, another member of this SNARE complex, has also been shown to be associated with immature dense-core vesicles and small vesicles near the trans-Golgi or endosomes [8,11,81]. Blocking antibodies to syntaxin 6 were shown to inhibit the homotypic fusion of immature dense-core vesicles in an in vitro fusion assay [11], but the physiological importance of syntaxin 6 for dense-core vesicle biogenesis in vivo remains unclear. Interestingly, the RAB-2 effector RUND-1 is tightly colocalized with syntaxin 6 in C. elegans neurons [23] and the BAR domain protein PICK1 tightly colocalized with syntaxin 6 in mouse pituitary cells [21]. Also, syntaxin 6 physically interacts with SNAP29 [11], a SNARE protein that we and others identified as an EIPR1 or EARP interactor [35,42,43]. It will be interesting to determine whether SNAP29, Vti1a, and syntaxin 6 are members of a SNARE complex that interacts with EARP, and to identify the compartment where they function.

Strains
Worm strains were cultured and maintained using standard methods [82]. A complete list of strains and mutations used is provided in the Strain List (S1 Table).

Isolation and identification of eipr-1, vps-52, vps-53, and vps-50 mutations
The eipr-1(ox316), vps-52(ox345) and vps-53(ox339) mutants were isolated in an ENU screen for suppressors of the hyperactive locomotion of an activated Gq mutant, egl-30(tg26) [23] (S1A Fig). When outcrossed away from the egl-30(tg26) mutation, all three of these mutants had sluggish unmotivated locomotion and defects in egg-laying. The phenotypes of all three of these mutants are partially maternally rescued; for example, the phenotype of an eipr-1 mutant is weaker if its parent is heterozygous (eipr-1/+) than if its parent is homozygous (eipr-1/eipr-1). The basis of this maternal rescue is not clear, but indicates the likely maternal deposition of these proteins in the egg.
We mapped these mutations using single nucleotide polymorphisms (SNPs) in the Hawaiian strain CB4856 as described [83]. The eipr-1(ox316) mutation was mapped to a 205 kb region on the right arm of chromosome I with 31 predicted protein-coding genes. Because there was no coverage of this region by C. elegans cosmids, we obtained C. briggsae BACs (from CHORI) carrying orthologs of the genes in this region and injected them into eipr-1 (ox316) mutants. The BAC RPCI94_01L06 rescued the eipr-1 locomotion defect, while the BAC RPCI94_13K10 did not. RPCI94_01L06 carries orthologs of six C. elegans genes in the mapped region. We sequenced the best two candidate genes in the ox316 mutant and found a C to T transition mutation in exon 4 of Y87G2A.11, leading to a premature stop codon at Q310. We obtained and sequenced the eipr-1 cDNA from the ORFeome library, confirming the gene structure predicted on Wormbase. The clone carried a single mutation that we fixed by Quick Change mutagenesis. We then cloned the mutation-free cDNA into a Gateway entry vector for use in rescue experiments. We rescued the ox316 mutant with a transgene carrying only Y87G2A.11, confirming the gene identification.
The vps-52(ox345) mutation was mapped to a 720 kb region in the middle of the X chromosome with 122 genes. The vps-53(ox339) mutation was mapped to a 92 kb region in the middle of chromosome III with 23 genes. We constructed a double mutant strain carrying both the ox339 and ox345 mutations and performed Illumina whole-genome sequencing. Within the region where ox339 mapped, we found no SNPs predicted to alter protein function, but did identify a 687 bp deletion within the T05G5.8 (vps-53) ORF. Within the region where ox345 mapped, we found only a single SNP predicted to alter protein function, a C to T transition mutation in the F08C6.3 (vps-52) ORF that created a premature stop codon at S119. The mutations in the vps-53 and vps-52 genes were confirmed by Sanger sequencing. We confirmed the gene identifications by performing complementation tests with other deletion mutations in each gene and by performing single-gene rescue experiments.
The vps-50(ox476) mutation was isolated as a background mutation in a strain carrying the linked ced-6(n2095) mutation. It was shown to be an allele of vps-50 by a complementation test with vps-50(ok2627), and by sequencing vps-50 to identify the molecular lesion [59]. vps-50 (ox476) was separated from the ced-6 mutation before further analysis.

Molecular biology and transgenes
A complete list of constructs is provided in the Plasmid List (S2 Table). Most of the constructs were made using the three slot multisite Gateway system (Invitrogen). Typically, a promoter, a coding sequence (genomic DNA or cDNA), and an N-or C-terminal fluorescent tag (GFP or tagRFP-T) were cloned along with a 3'UTR into the pCFJ150 destination vector used for Mos1-mediated single copy insertion (MosSCI) on chromosome II at ttTi5605 [84,85]. All insertions were made by the direct injection MosSCI method. For most constructs, we isolated multiple independent insertions that behaved similarly. Extrachromosomal arrays were made by standard transformation methods [86].
Expression of eipr-1, vps-52, and vps-53 eipr-1 is the fourth gene in an operon about 30 kb downstream from the start of the first gene in the operon, so it is not clear what promoter regulates eipr-1. We made a construct driving expression of GFP by the promoter region upstream of the first gene of the operon (1348 bp upstream of the W09C5.1 start codon). Extrachromosomal arrays of this construct showed expression in several tissues including the intestine, pharynx and hypodermis (S4A Fig), but not neurons, suggesting that this promoter fusion does not represent the endogenous expression pattern of eipr-1. We also tagged the chromosomal eipr-1 gene with GFP using the CRISPR/Cas9 method [87]. However, neither N-terminal nor C-terminal GFP-tagged eipr-1 was bright enough to detect, even with the use of an anti-GFP antibody to amplify the signal, indicating that eipr-1 is likely to be expressed endogenously at low levels.
We made constructs driving expression of the yellow-fluorescent protein Citrine under the vps-52 and vps-53 promoters and generated worms with extrachromosomal arrays. For vps-52, 822 bp upstream of the start codon was used as a promoter. vps-53 is the second gene in an operon downstream of the gene gcc-2. For vps-53, 4502 bp upstream of the gcc-2 start codon was used as the promoter. vps-52 showed strong expression in neurons in the head, ventral cord, and tail (Fig 6A), as well as expression in the pharynx, intestine, uterus, spermatheca, skin, and several muscle types (body wall, vulval). Other than the skin and muscle expression, this expression pattern matches that of rund-1 [23]. vps-53 was not as strongly expressed, but was clearly visible in many head neurons (though possibly not all), ventral cord neurons, tail neurons, the pharynx, and weakly in the body wall muscles (Fig 6B).

Locomotion and egg-laying assays
To measure locomotion, first-day adults were picked to thin lawns of OP50 bacteria, allowed to rest for 30 seconds, and then body bends were counted for one minute. A body bend was defined as the movement of the worm from maximum to minimum amplitude of the sine wave [88].
To measure egg-laying, L4 larvae were placed on plates with OP50 and allowed to mature at 25°C overnight. The next day, five adult animals were moved to a fresh plate and allowed to lay eggs at 25°C for 2.5 hours. The number of eggs present on the plate was then counted.

Expression and purification of anti-GFP nanobody
The anti-GFP nanobody was expressed and purified as previously described [90] with few modifications. In brief, bacterial expression vector pLaG16 containing the anti-GFP nanobody was transformed in Arctic Express (DE3) cells (Agilent). Cells were induced with 0.1 mM IPTG for 16 h at 8°C and centrifuged at 5,000g for 10-min at 4°C. To isolate the periplasmic fraction by osmotic shock [91], cells were incubated for 1h in TES buffer (0.2 M Tris-HCl pH 8, 0.2 mM EDTA and 0.5 M sucrose). Cells were spun down for 10 min at 6,000g at 4°C and the pellet was suspended in TE buffer (0.2 M Tris-HCl pH 8 and 0.5 mM EDTA) following a 45 min incubation. Cells were pelleted by a 30 minutes spin at 30,000g at 4°C. The supernatant (periplasmic fraction) was bound to PerfectPro Ni-NTA Agarose affinity resin (5 Prime) for 1h at 4°C. The resin was washed once with wash buffer I (20 mM sodium phosphate pH 8.0, 0.9 M NaCl) and twice with wash buffer II (20 mM sodium phosphate pH 8.0, 150 mM NaCl and 10 mM imidazole) and eluted with His elution buffer (20 mM sodium phosphate pH 8.0, 150 mM NaCl, 250 mM imidazole). The eluent was then dialyzed with PBS. Recombinant anti-GFP nanobody was conjugated to epoxy-activated magnetic Dynabeads M270 (Life Technologies) using 10 μg recombinant protein per 1 mg of Dynabeads, with conjugations carried out in 0.1 M sodium phosphate pH 8.0 and 1 M ammonium sulfate, with an 18 h incubation at 30°C. Beads were washed once with 100 mM Glycine pH 2.5, once with 10 mM Tris pH 8.8, 4 times with PBS and twice with PBS plus 0.5% Triton X-100. Beads were stored at -20°C in PBS with 50% glycerol.

Mass spectrometry
For immunoprecipitation, 4x10 6 832/13 cells were plated onto 10 cm petri dishes. Twenty-four hours later, cells were transfected with either EIPR1::GFP or GFP. After 24 hours, cells were washed with PBS and harvested in lysis buffer [50 mM Tris pH 7.5, 150 mM NaCl, 1% NP-40, Protease inhibitor cocktail (Pierce)]. Lysates were passed 10 times through a 20G needle and incubated for 30 min at 4°C. Lysates were pre-cleared by a 20,000 g centrifugation at 4°C for 15 minutes and the supernatant was incubated with 30 μg of anti-GFP nanobody bound to magnetic beads, for two hours at 4°C. The beads were washed three times with 250 μL wash buffer (50 mM Tris pH 7.5, 300 mM NaCl, 1% NP-40). Samples were eluted in two steps with (1) 0.2 M glycine pH 2.5 and (2) 1 M NH4OH. Eluents were neutralized with 0.33 M Tris-HCl pH 7.5 and stored at -80°C.
Samples were processed using modified FASP (filter aided sample preparation) to remove mass spec incompatible substances [92]. Samples were filtered with 10K Amicon Ultra 0.5 ml filters (Millipore). Samples were buffer exchanged with 8 M urea in 50 mM ammonium bicarbonate pH 7.8, reduced with TCEP (Pierce), alkylated with IAA (Sigma) and digested with trypsin (Pierce). Samples were concentrated by speed vacuum.
A total of 0.15 μg of peptides was chromatographically separated onto a Thermo LTQ-Velos Pro mass spectrometer coupled with a Waters nanoACQUITY liquid chromatography system. A 75 μm fused silica column (Polymicro Technologies) was loaded with 30 cm C12 Jupiter (Phenomenex), 4 μm reverse-phase beads, and a 100 μm fused silica Kasil frit trap (PQ Corporation) loaded with 4 cm of C12 Jupiter. Peptides were separated by a 110 minute gradient consisting of buffer A (95% water, 5% acetonitrile and 0.1% formic acid) and buffer B (95% acetonitrile, 5% water and 0.1% formic acid). Two analytical replicates were run for each sample with each set of replicates randomized. Quality control samples were run every sixth sample and at the beginning of the runs to assess column chromatography stability.
Data were searched with SEQUEST [93] against a Rattus norvegicus FASTA database containing contaminants. False discovery rates were determined via a decoy database using Percolator [94] at a q-value threshold of 0.01 and peptides were assembled into protein identifications using ID Picker [95]. Lists of EIPR1::GFP hits were assembled after first subtracting hits found in a GFP control pulldown (S5A Fig, Left) or subtracting hits found in the pulldowns of two other proteins, CCCP1::GFP and RUNDC1::GFP (S5A Fig, Right), performed in parallel.

Imaging and image analysis
Worms were mounted on 2% agarose pads and anesthetized with 50 mM sodium azide. Images were obtained using a Nikon 80i wide-field compound microscope except for the images shown in Fig 6A and 6B which were obtained on an Olympus confocal microscope, and the images in Fig 6C and 6D which were obtained on a Deltavision deconvolution microscope. To image the dorsal nerve cords, young adult animals were oriented with dorsal side up by exposure to the anesthetic for ten minutes on the slide before placing the cover slip. For quantitative imaging of dorsal cord fluorescence, all strains in a given experiment were imaged on the same days and all microscope settings were kept constant. The same section of the dorsal cord around the vulva was imaged in all worms. Maximum intensity projections were quantified using ImageJ software, measuring the total fluorescence in a region of interest encapsulating the cord and subtracting the background fluorescence of a region of identical size adjacent to the cord.
Immunostaining of 832/13 cells 2x10 5 832/13 cells per well were seeded onto sterilized cover slips placed in 12 well cell culture plates. The next day, cells were rinsed twice with ice-cold PBS and fixed with 4% paraformaldehyde in PBS for 20 minutes at room temperature. The cells were rinsed twice with PBS and permeabilized with 0.5% Triton X-100 in PBS for 5 minutes at room temperature. The cells were again washed twice with PBS and then placed in 5% milk in PBS for 1 hour at room temperature, followed by immunofluorescence staining with rabbit polyclonal anti-CCDC132 (1:50, Sigma #HPA026679) and mouse monoclonal anti-GFP (1:350, Santa Cruz #sc-9996) in 0.5% milk in PBS at room temperature for 1 hour. The cells were then washed with PBS three times for 5 minutes each, and incubated with Rhodamine anti-rabbit secondary antibody (1:1000, Jackson Immunoresearch #111-025-144) and Alexa Fluor 488 anti-mouse secondary antibody (1:1000, Jackson Immunoresearch #115-545-146) at room temperature for 1 hour. The cells were washed with PBS three times for 5 min each and examined by fluorescence microscopy (Nikon 80i wide-field compound microscope). The anti-CCDC132 antibody recognizes VPS50.
Statistics P values were determined using GraphPad Prism 5.0d (GraphPad Software). Data sets with multiple comparisons were analyzed by a one-way ANOVA followed by a Bonferroni posthoc test to examine selected comparisons or by Dunnett's test if all comparisons were to the wild type control. Pairwise data comparisons were analyzed by two-tailed unpaired t tests.
Supporting Information S1 Fig. eipr-1 mutants suppress the locomotion phenotype of an activated Gq mutant and have egg laying defects. (A) The activated Gq mutant egl-30(tg26) has hyperactive locomotion and deep body bends. The egl-30(tg26) locomotion phenotype is suppressed by the eipr-1 (ox316), vps-50(ok2627), vps-52(ox345), and vps-53(ox339) mutations. (B) eipr-1 acts in neurons to control egg laying. The graph shows the number of eggs laid by 5 animals in a 2.5 hour period. eipr-1 mutants show egg-laying defects and this phenotype is rescued by panneuronal expression of either the worm gene or its mouse ortholog ( ÃÃÃ , P<0.001). Error bars = SEM; n = 10 plates of 5 worms each. (C) eipr-1 acts in the same pathway as rab-2 to control egg-laying. Double mutants of eipr-1(tm4790) with rab-2(nu415) or rund-1(tm3622) do not have stronger egg-laying defects than the single mutants. Though rund-1(tm3622) mutants have a visible Egl (egg-laying defective) phenotype indicating that they retain more eggs, they did not have a significantly reduced egg-laying rate as measured by this assay. Error bars = SEM; n = 5 plates of 5 worms each. ns, not significant, P>0.05. (TIF) S2 Fig. Alignment of EIPR-1. Alignment of C. elegans EIPR-1 (worm, accession # NP_493383.1) and its orthologs from Arabidopsis thaliana (Arabidopsis, accession # NP_173478.2), Drosophila melanogaster (fly, CG10646, accession # NP_648581.1), and Homo sapiens (human, TSSC1, accession # NP_003301.1). Identical residues are shaded in black and similar residues are shaded in gray. The WD40 repeats (from SMART, using the worm protein; http://smart.emblheidelberg.de/) are marked with single black bars. Using SMART, worm EIPR-1 has four predicted WD40 repeats, Arabidopsis has six, fly has three, and human has five. WD40 repeats are difficult to identify by primary sequence and are often missed by prediction programs. The position of the ox316 stop mutation is marked with an asterisk. Alignment was made with MUSCLE (http://www.ebi.ac.uk/Tools/msa/muscle/) using default parameters and exhibited with List of top hits from two independent experiments performing mass spectrometry of a pulldown of rEIPR1::GFP in 832/13 cells. On the left side of the table, we show the list of hits found in a mass spec experiment after subtracting hits found in a GFP control pulldown. On the right side of the table, we show the list of hits found in an independent mass spec experiment after subtracting hits found in either the pulldown of CCCP1::GFP or RUNDC1::GFP. In both cases, EIPR1::GFP pulldowns were performed in parallel to controls. # seq = number of unique peptides from each protein. All proteins with two or more unique peptides are shown. (B) EGFPtagged rat EIPR1 or EGFP was expressed in 832/13 cells. Immunoprecipitation of EIPR1:: EGFP pulled down more SNAP29 than untagged EGFP. IN: input; IP: immunoprecipitation. (TIF) S6 Fig. EARP, not GARP, is important for locomotion and trafficking dense-core vesicle cargos. (A) vps-50 acts in the same genetic pathway as eipr-1 to control FLP-3::Venus trafficking. Left: representative images. Right: quantification. vps-50(ok2627) and eipr-1(tm4790) have reduced FLP-3::Venus (ceIs61) fluorescence, but an eipr-1(tm4790); vps-50(ok2627) double mutant does not have a stronger phenotype than either single mutant indicating that eipr-1 and vps-50 act in the same genetic pathway. A vps-54(ok1473) mutant does not have decreased axonal levels of FLP-3::Venus. ÃÃÃ , P<0.001 compared to wild type. Error bars = SEM; n = 9-14. (B) eipr-1 and GARP/EARP mutants have reduced secretion of NLP-21::Venus. NLP-21::Venus (nuIs183) fluorescence levels in the coelomocytes of the indicated strains. Like rab-2(nu415) mutants, eipr-1(tm4790), vps-50(ok2627), and vps-52(ox345) mutants show reductions in the accumulation of Venus fluorescence in coelomocytes that is approximately proportional to the decrease in axonal fluorescence seen in these mutants, suggesting that these mutants are not defective in dense-core vesicle release. Double mutants between eipr-1 (tm4790) and rab-2(nu415) or vps-50(ok2627) do not have stronger phenotypes than the single mutants, suggesting that these genes all act in the same dense-core vesicle cargo sorting pathway. ÃÃ   Davis for the GFP nanobody and advice on mass spectrometry; Jihong Bai and the Fred Hutch Antibody Technology Resource for the anti-mCherry antibody; Aude Peden, Christian Frøkjoer-Jensen, Eric Bend, Gunther Hollopeter, and Susan Dalton for worm promoter plasmids; Suzanne Hoppins for the pmCherry-N1 vector; Colin Thacker and Richard Clark for assistance with Illumina sequencing; Jihong Bai, Gunther Hollopeter, and Alex Merz for comments on the manuscript; and Erik Jorgensen, in whose lab early phases of this project were performed. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440).