EH Domain Research Articles

Selection and stabilization of endocytic sites by Ede1, a yeast functional homologue of human Eps15.

During clathrin-mediated endocytosis (CME), endocytic-site maturation can be divided into two stages corresponding to the arrival of the early and late proteins at the plasma membrane. The early proteins are required to capture cargo and position the late machinery, which includes proteins involved in actin assembly and membrane scission. However, the mechanism by which early-arriving proteins select and stabilize endocytic sites is not known. Ede1, one of the earliest proteins recruited to endocytic sites, facilitates site initiation and stabilization. Deletion of EDE1 results in fewer CME initiations and defects in the timing of vesicle maturation. Here we made truncation mutants of Ede1 to better understand how different domains contribute to its recruitment to CME sites, site selection, and site maturation. We found that the minimal domains required for efficient Ede1 localization at CME sites are the third EH domain, the proline-rich region, and the coiled-coil region. We also found that many strains expressing ede1 truncations could support a normal rate of site initiation but still had defects in site-maturation timing, indicating separation of Ede1 functions. When expressed in yeast, human Eps15 localized to the plasma membrane, where it recruited late-phase CME proteins and supported productive endocytosis, identifying it as an Ede1 functional homologue.

Eps15 membrane-binding and -bending activity acts redundantly with Fcho1 during clathrin-mediated endocytosis.

Clathrin coat assembly on membranes requires cytosolic adaptors and accessory proteins, which bridge triskeleons with the lipid bilayer and stabilize lattice architecture throughout the process of vesicle formation. In Caenorhabditis elegans, the prototypical AP-2 adaptor complex, which is activated by the accessory factor Fcho1 at the plasma membrane, is dispensable during embryogenesis, enabling us to define alternative mechanisms that facilitate clathrin-mediated endocytosis. Here we uncover a synthetic genetic interaction between C. elegans Fcho1 (FCHO-1) and Eps15 (EHS-1), suggesting that they function in a parallel and potentially redundant manner. Consistent with this idea, we find that the FCHO-1 EFC/F-BAR domain and the EHS-1 EH domains exhibit highly similar membrane-binding and -bending characteristics in vitro. Furthermore, we demonstrate a critical role for EHS-1 when FCHO-1 membrane-binding and -bending activity is specifically eliminated in vivo. Taken together, our data highlight Eps15 as an important membrane-remodeling factor, which acts in a partially redundant manner with Fcho proteins during the earliest stages of clathrin-mediated endocytosis.

Novel insights on ENTH domain-containing proteins in apicomplexan parasites.

The phylum Apicomplexa includes a large group of early branching eukaryotes having significant medical and economical importance. The molecular machinery responsible for protein trafficking is poorly understood in these apicomplexans. One of the most important proteins involved in clathrin-mediated protein trafficking is Epsin, which contains ENTH domain, a conserved domain crucial for membrane bending leading to vesicle formation. We undertook homology searching and phylogenetic analyses to produce a rigorously annotated set of Epsin homologs retrieved from diverse apicomplexan genomes. Genomic and phylogenetic comparisons revealed that apicomplexans contain unusual Epsin homologs that are distinct from those observed in mammals and yeast. Although there are four Epsin genes in mammalian system and five in the yeast genome, apicomplexan parasites consist only a single Epsin gene. The apicomplexan Epsin contains the conserved ENTH domain consisting of phosphoinositide (PtdIns)-binding sites which indicate about their functional significance in the formation of vesicles; however, the absence of ubiquitin-interacting motif (UIM) suggests a possible different mechanism for protein trafficking. The existence of dileucine motif in Plasmodium, Cryptosporidum parvum and Eimeria tenella Epsins might solve their functionality while lacking a lot of conserved motifs as this motif is known to interact with different adaptor protein complexes (AP1, AP2 and AP3). Other Epsin homologs are also shown to have different peptide motifs reported for possible interaction with α-ear appendage, γ-ear appendage and EH domain present in different adaptors. Bioinformatic and phylogenetic analyses suggest that the apicomplexan Epsins have unusual functionality from that of the mammalian Epsins. This detailed study may greatly facilitate future molecular cell biological investigation for the role of Epsins in these parasites.

EHBP1L1 coordinates Rab8 and Bin1 to regulate apical-directed transport in polarized epithelial cells.

The highly conserved Rab guanosine triphosphatase (GTPase) Rab8 plays a role in exocytosis toward the polarized plasma membrane in eukaryotic cells. In murine Rab8-deficient small intestine cells, apical proteins are missorted into lysosomes. In this study, we identified a novel Rab8-interacting protein complex containing an EH domain-binding protein 1-like 1 (EHBP1L1), Bin1/amphiphysin II, and dynamin. Biochemical analyses showed that EHBP1L1 directly bound to GTP-loaded Rab8 and Bin1. The spatial dependency of these complexes at the endocytic recycling compartment (ERC) was demonstrated through overexpression and knockdown experiments. EHBP1L1- or Bin1-depleted or dynamin-inhibited small intestine organoids significantly accumulated apical membrane proteins but not basolateral membrane proteins in lysosomes. Furthermore, in EHBP1L1-deficient mice, small intestine cells displayed truncated and sparse microvilli, suggesting that EHBP1L1 maintains the apical plasma membrane by regulating apical transport. In summary, our data demonstrate that EHBP1L1 links Rab8 and the Bin1-dynamin complex, which generates membrane curvature and excises the vesicle at the ERC for apical transport.

Molecular dynamics simulation of the interactions between EHD1 EH domain and multiple peptides.

To provide essential information for peptide inhibitor design, the interactions of Eps15 homology domain of Eps15 homology domain-containing protein 1 (EHD1 EH domain) with three peptides containing NPF (asparagine-proline-phenylalanine), DPF (aspartic acid-proline-phenylalanine), and GPF (glycine-proline-phenylalanine) motifs were deciphered at the atomic level. The binding affinities and the underlying structure basis were investigated. Molecular dynamics (MD) simulations were performed on EHD1 EH domain/peptide complexes for 60 ns using the GROMACS package. The binding free energies were calculated and decomposed by molecular mechanics/generalized Born surface area (MM/GBSA) method using the AMBER package. The alanine scanning was performed to evaluate the binding hot spot residues using FoldX software. The different binding affinities for the three peptides were affected dominantly by van der Waals interactions. Intermolecular hydrogen bonds provide the structural basis of contributions of van der Waals interactions of the flanking residues to the binding. van der Waals interactions should be the main consideration when we design peptide inhibitors of EHD1 EH domain with high affinities. The ability to form intermolecular hydrogen bonds with protein residues can be used as the factor for choosing the flanking residues.

SUMOylation of EHD3 Modulates Tubulation of the Endocytic Recycling Compartment.

Endocytosis defines the entry of molecules or macromolecules through the plasma membrane as well as membrane trafficking in the cell. It depends on a large number of proteins that undergo protein-protein and protein-phospholipid interactions. EH Domain containing (EHDs) proteins formulate a family, whose members participate in different stages of endocytosis. Of the four mammalian EHDs (EHD1-EHD4) EHD1 and EHD3 control traffic to the endocytic recycling compartment (ERC) and from the ERC to the plasma membrane, while EHD2 modulates internalization. Recently, we have shown that EHD2 undergoes SUMOylation, which facilitates its exit from the nucleus, where it serves as a co-repressor. In the present study, we tested whether EHD3 undergoes SUMOylation and what is its role in endocytic recycling. We show, both in-vitro and in cell culture, that EHD3 undergoes SUMOylation. Localization of EHD3 to the tubular structures of the ERC depends on its SUMOylation on lysines 315 and 511. Absence of SUMOylation of EHD3 has no effect on its dimerization, an important factor in membrane localization of EHD3, but has a dominant negative effect on its appearance in tubular ERC structures. Non-SUMOylated EHD3 delays transferrin recycling from the ERC to the cell surface. Our findings indicate that SUMOylation of EHD3 is involved in tubulation of the ERC membranes, which is important for efficient recycling.

Ca2+ does not affect the binding properties of ITSN1 EH domains

Ca2+ does not affect the binding properties of ITSN1 EH domains

UIM domain-dependent recruitment of the endocytic adaptor protein Eps15 to ubiquitin-enriched endosomes.

BackgroundEps15 is an endocytic adaptor protein that stimulates clathrin-mediated endocytosis. Among other interactions, Eps15 binds ubiquitin via UIM domains, recruiting ubiquitinated cargo into clathrin-coated vesicles. In EGF-treated cells, Eps15 also localizes to endosomes. The basis of this localization is not known.ResultsWe show that accumulation of ubiquitinated cargo can recruit Eps15 to endosomes via UIM domain interactions. First, treatment of SK-Br-3 breast cancer cells, which overexpress the EGFR family member ErbB2, with geldanamycin to promote receptor ubiquitination and endosomal transport, recruited FLAG-Eps15 to endosomes. Two in-frame ubiquitin constructs, PM-GFP-Ub (retained in endosomes after endocytosis), and GFP-FYVE-UbΔGG (targeted directly to endosomes) also recruited Eps15 to endosomes, as did slowing endosome maturation with constitutively-active Rab5-Q79L. Endosomal recruitment required the UIM domains, but not the N-terminal EH domains or central coiled-coil domains, of Eps15. Silencing of the endosomal Eps15 binding partner Hrs did not affect recruitment of Eps15 to ubiquitin-enriched endosomes. In fact, Hrs silencing itself modestly recruited Eps15 to endosomes, probably by accumulating endogenous ubiquitinated cargo. Eps15 silencing did not affect lysosomal degradation of ubiquitinated ErbB2; however, GFP-FYVE-UbΔGG overexpression inhibited internalization of EGFR and transferrin receptor.ConclusionsWe show for the first time that ubiquitin is sufficient for Eps15 recruitment to endosomes. We speculate that Eps15 recruitment to ubiquitin-rich endosomes may reduce the level of Eps15 at the plasma membrane, slowing endocytosis to allow time for processing of ubiquitinated cargo in endosomes.

Structured cyclic peptides that bind the EH domain of EHD1.

EHD1 mediates long-loop recycling of many receptors by forming signaling complexes using its EH domain. We report the design and optimization of cyclic peptides as ligands for the EH domain of EHD1. We demonstrate that the improved affinity from cyclization allows fluorescence-based screening applications for EH domain inhibitors. The cyclic peptide is also unusually well-structured in aqueous solution, as demonstrated using nuclear magnetic resonance-based structural models. Because few EH domain inhibitors have been described, these more potent inhibitors will improve our understanding of the roles of EHD1 in the context of cancer invasion and metastasis.

Upregulation of EHD2 after Intracerebral Hemorrhage in Adult Rats

EHD2, a member of the Eps15 homology domain (EH domain) family, is important for protein interactions during vesicular trafficking. Previous studies have proved that EHD2 can regulate trafficking from the plasma membrane in the process of endocytosis. However, its function in central nervous system diseases is still with limited understanding. In this frame, we found that EHD2 expression was upregulated in the perihematomal caudate in adult rats after intracerebral hemorrhage (ICH). Double immunofluorescence staining revealed that EHD2 was colocalized with neurons and activated microglias after ICH. Besides, we detected that neuronal apoptosis markers (TUNEL and caspase-3), and microglial activation marker (CD68), also known as a marker of macrophage, were colocated with EHD2. The vitro study also indicated that EHD2 was linked with neuronal apoptosis and microglial phagocytosis. All our findings suggested that EHD2 might be involved in the pathophysiology of ICH.

The function of EHD2 in endocytosis and defense signaling is affected by SUMO

Post-translational modification of target proteins by the small ubiquitin-like modifier protein (SUMO) regulates many cellular processes. SUMOylation has been shown to regulate cellular localization and function of a variety of proteins, in some cases affecting nuclear import or export. We have previously characterized two EHDs (EH domain containing proteins) in Arabidospis and showed their involvement in plant endocytosis. AtEHD2 has an inhibitory effect on endocytosis of transferrin, FM-4-64, and the leucine rich repeat receptor like protein LeEix2, an effect that requires and intact coiled-coil domain. Inhibition of endocytosis of LeEix2 by EHD2 is effective in inhibiting defense responses mediated by the LeEix2 receptor in response to its ligand EIX. In the present work we demonstrate that SUMOylation of EHD2 appears to be required for EHD2-induced inhibition of LeEix2 endocytosis. Indeed, we found that a mutant form of EHD2, possessing a defective SUMOylation site, has an increased nuclear abundance, can no longer be SUMOylated and is no longer effective in inhibiting LeEix2 endocytosis or defense signaling in response to EIX.

Role of Phosphatidylinositol 4,5-Bisphosphate in Regulating EHD2 Plasma Membrane Localization

The four mammalian C-terminal Eps15 homology domain-containing proteins (EHD1-EHD4) play pivotal roles in endocytic membrane trafficking. While EHD1, EHD3 and EHD4 associate with intracellular tubular/vesicular membranes, EHD2 localizes to the inner leaflet of the plasma membrane. Currently, little is known about the regulation of EHD2. Thus, we sought to define the factors responsible for EHD2’s association with the plasma membrane. The subcellular localization of endogenous EHD2 was examined in HeLa cells using confocal microscopy. Although EHD partner proteins typically mediate EHD membrane recruitment, EHD2 was targeted to the plasma membrane independent of two well-characterized binding proteins, syndapin2 and EHBP1. Additionally, the EH domain of EHD2, which facilitates canonical EHD protein interactions, was not required to direct overexpressed EHD2 to the cell surface. On the other hand, several lines of evidence indicate that the plasma membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) plays a crucial role in regulating EHD2 subcellular localization. Pharmacologic perturbation of PIP2 metabolism altered PIP2 plasma membrane distribution (as assessed by confocal microscopy), and caused EHD2 to redistribute away from the plasma membrane. Furthermore, overexpressed EHD2 localized to PIP2-enriched vacuoles generated by active Arf6. Finally, we show that although cytochalasin D caused actin microfilaments to collapse, EHD2 was nevertheless maintained at the plasma membrane. Intriguingly, cytochalasin D induced relocalization of both PIP2 and EHD2 to actin aggregates, supporting a role of PIP2 in controlling EHD2 subcellular localization. Altogether, these studies emphasize the significance of membrane lipid composition for EHD2 subcellular distribution and offer new insights into the regulation of this important endocytic protein.

Pan1 is an intrinsically disordered protein with homotypic interactions

The yeast scaffold protein Pan1 contains two EH domains at its N-terminus, a predicted coiled-coil central region, and a C-terminal proline-rich domain. Pan1 is also predicted to contain regions of intrinsic disorder, characteristic of proteins that have many binding partners. In vitro biochemical data suggest that Pan1 exists as a dimer, and we have identified amino acids 705 to 848 as critical for this homotypic interaction. Tryptophan fluorescence was used to further characterize Pan1 conformational states. Pan1 contains four endogenous tryptophans, each in a distinct region of the protein: Trp(312) and Trp(642) are each in an EH domain, Trp(957) is in the central region, and Trp(1280) is a critical residue in the Arp2/3 activation domain. To examine the local environment of each of these tryptophans, three of the four tryptophans were mutagenized to phenylalanine to create four proteins, each with only one tryptophan residue. When quenched with acrylamide, these single tryptophan mutants appeared to undergo collisional quenching exclusively and were moderately accessible to the acrylamide molecule. Quenching with iodide or cesium, however, revealed different Stern-Volmer constants due to unique electrostatic environments of the tryptophan residues. Time-resolved fluorescence anisotropy data confirmed structural and disorder predictions of Pan1. Further experimentation to fully develop a model of Pan1 conformational dynamics will assist in a deeper understanding of the mechanisms of endocytosis.

Genetic Susceptibility Loci, Pesticide Exposure and Prostate Cancer Risk

Uncovering SNP (single nucleotide polymorphisms)-environment interactions can generate new hypotheses about the function of poorly characterized genetic variants and environmental factors, like pesticides. We evaluated SNP-environment interactions between 30 confirmed prostate cancer susceptibility loci and 45 pesticides and prostate cancer risk in 776 cases and 1,444 controls in the Agricultural Health Study. We used unconditional logistic regression to estimate odds ratios (ORs) and 95% confidence intervals (CIs). Multiplicative SNP-pesticide interactions were calculated using a likelihood ratio test. After correction for multiple tests using the False Discovery Rate method, two interactions remained noteworthy. Among men carrying two T alleles at rs2710647 in EH domain binding protein 1 (EHBP1) SNP, the risk of prostate cancer in those with high malathion use was 3.43 times those with no use (95% CI: 1.44–8.15) (P-interaction = 0.003). Among men carrying two A alleles at rs7679673 in TET2, the risk of prostate cancer associated with high aldrin use was 3.67 times those with no use (95% CI: 1.43, 9.41) (P-interaction = 0.006). In contrast, associations were null for other genotypes. Although additional studies are needed and the exact mechanisms are unknown, this study suggests known genetic susceptibility loci may modify the risk between pesticide use and prostate cancer.

The Human CD8β M-4 Isoform Dominant in Effector Memory T Cells Has Distinct Cytoplasmic Motifs That Confer Unique Properties

The CD8 co-receptor influences T cell recognition and responses in both anti-tumor and anti-viral immunity. During evolution in the ancestor of humans and chimpanzees, the CD8B gene acquired two additional exons. As a result, in humans, there are four CD8β splice variants (M1 to M4) that differ in their cytoplasmic tails. The M-1 isoform which is the equivalent of murine CD8β, is predominantly expressed in naïve T cells, whereas, the M-4 isoform is predominantly expressed in effector memory T cells. The characteristics of the M-4 isoform conferred by its unique 36 amino acid cytoplasmic tail are not known. In this study, we identified a dihydrophobic leucine-based receptor internalization motif in the cytoplasmic tail of M-4 that regulated its cell surface expression and downregulation after activation. Further the M-4 cytoplasmic tail was able to associate with ubiquitinated targets in 293T cells and mutations in the amino acids NPW, a potential EH domain binding site, either enhanced or inhibited the interaction. In addition, the M-4 tail was itself mono-ubiquitinated on a lysine residue in both 293T cells and a human T cell line. When peripheral blood human T cells expressed CD8αβ M-4, the frequency of MIP-1β secreting cells responding to antigen presenting cells was two-fold higher as compared to CD8αβ M-1 expressing T cells. Thus, the cytoplasmic tail of the CD8β M-4 isoform has unique characteristics, which likely contributed to its selective expression and function in human effector memory T cells.

Vertebrate Intersectin1 Is Repurposed to Facilitate Cortical Midline Connectivity and Higher Order Cognition

Invertebrate studies have highlighted a role for EH and SH3 domain Intersectin (Itsn) proteins in synaptic vesicle recycling and morphology. Mammals have two Itsn genes (Itsn1 and Itsn2), both of which can undergo alternative splicing to include DBL/PH and C2 domains not present in invertebrate Itsn proteins. To probe for specific and redundant functions of vertebrate Itsn genes, we generated Itsn1, Itsn2, and double mutant mice. While invertebrate mutants showed severe synaptic abnormalities, basal synaptic transmission and plasticity were unaffected at Schaffer CA1 synapses in mutant mice. Surprisingly, intercortical tracts-corpus callosum, ventral hippocampal, and anterior commissures-failed to cross the midline in mice lacking Itsn1, but not Itsn2. In contrast, tracts extending within hemispheres and those that decussate to more caudal brain segments appeared normal. Itsn1 mutant mice showed severe deficits in Morris water maze and contextual fear memory tasks, whereas mice lacking Itsn2 showed normal learning and memory. Thus, coincident with the acquisition of additional signaling domains, vertebrate Itsn1 has been functionally repurposed to also facilitate interhemispheric connectivity essential for high order cognitive functions.

Identification and Selected Reaction Monitoring (SRM) Quantification of Endocytosis Factors Associated with Numb

Numb is an endocytic adaptor protein that regulates the endocytosis and trafficking of transmembrane receptors including Notch, E-cadherin, and integrins. Vertebrate Numb is alternatively spliced at exons 3 and 9 to give rise to four protein isoforms. Expression of these isoforms varies at different developmental stages, and although the function of Numb isoforms containing exon 3 has been studied, the role of exon 9 inclusion has not been shown. Here we use affinity purification and tandem mass spectrometry to identify Numb associated proteins, including novel interactions with REPS1, BMP2K, and BCR. In vitro binding measurements indicated exon 9-independent Numb interaction with REPS1 and Eps15 EH domains. Selected reaction monitoring mass spectrometry was used to quantitatively compare the proteins associated with the p72 and p66 Numb isoforms, which differ by the exon 9 region. This showed that significantly more EPS15 and three AP-2 subunit proteins bound Numb isoforms containing exon 9. The EPS15 preference for exon 9-containing Numb was confirmed in intact cells by using a proximity ligation assay. Finally, we used multiplexed selected reaction monitoring mass spectrometry to assess the dynamic regulation of Numb association with endocytic proteins. Numb hyper-phosphorylation resulted in disassociation of Numb endocytic complexes, while inhibition of endocytosis did not alter Numb association with the AP-2 complex but altered recruitment of EPS15, REPS1, and BMP2K. Hence, quantitative mass spectrometric analysis of Numb protein-protein interactions has provided new insights into the assembly and regulation of protein complexes important in development and cancer.

EHD1 Functions in Endosomal Recycling and Confers Salt Tolerance

Endocytosis is a crucial process in all eukaryotic organisms including plants. We have previously shown that two Arabidopsis proteins, AtEHD1 and AtEHD2, are involved in endocytosis in plant systems. Knock-down of EHD1 was shown to have a delayed recycling phenotype in mammalians. There are many works in mammalian systems detailing the importance of the various domains in EHDs but, to date, the domains of plant EHD1 that are required for its activity have not been characterized. In this work we demonstrate that knock-down of EHD1 causes a delayed recycling phenotype and reduces Brefeldin A sensitivity in Arabidopsis seedlings. The EH domain of EHD1 was found to be crucial for the localization of EHD1 to endosomal structures. Mutant EHD1 lacking the EH domain did not localize to endosomal structures and showed a phenotype similar to that of EHD1 knock-down seedlings. Mutants lacking the coiled-coil domain, however, showed a phenotype similar to wild-type or EHD1 overexpression seedlings. Salinity stress is a major problem in current agriculture. Microarray data demonstrated that salinity stress enhances the expression of EHD1, and this was confirmed by semi quantitative RT-PCR. We demonstrate herein that transgenic plants over expressing EHD1 possess enhanced tolerance to salt stress, a property which also requires an intact EH domain.

Oligomers of the ATPase EHD2 confine caveolae to the plasma membrane through association with actin

Caveolae are specialized domains present in the plasma membrane (PM) of most mammalian cell types. They function in signalling, membrane regulation, and endocytosis. We found that the Eps-15 homology domain-containing protein 2 (EHD2, an ATPase) associated with the static population of PM caveolae. Recruitment to the PM involved ATP binding, interaction with anionic lipids, and oligomerization into large complexes (60-75S) via interaction of the EH domains with intrinsic NPF/KPF motifs. Hydrolysis of ATP was essential for binding of EHD2 complexes to caveolae. EHD2 was found to undergo dynamic exchange at caveolae, a process that depended on a functional ATPase cycle. Depletion of EHD2 by siRNA or expression of a dominant-negative mutant dramatically increased the fraction of mobile caveolar vesicles coming from the PM. Overexpression of EHD2, in turn, caused confinement of cholera toxin B in caveolae. The confining role of EHD2 relied on its capacity to link caveolae to actin filaments. Thus, EHD2 likely plays a key role in adjusting the balance between PM functions of stationary caveolae and the role of caveolae as vesicular carriers.

Rabankyrin‐5 Interacts with EHD1 and Vps26 to Regulate Endocytic Trafficking and Retromer Function

Rabankyrin-5 (Rank-5) has been implicated as an effector of the small GTPase Rab5 and plays an important role in macropinocytosis. We have now identified Rank-5 as an interaction partner for the recycling regulatory protein, Eps15 homology domain 1 (EHD1). We have demonstrated this interaction by glutathione S-transferase-pulldown, yeast two-hybrid assay, isothermal calorimetry and co-immunoprecipitation, and found that the binding occurs between the EH domain of EHD1 and the NPFED motif of Rank-5. Similar to EHD1, we found that Rank-5 colocalizes and interacts with components of the retromer complex such as vacuolar protein sorting 26 (Vps26), suggesting a role for Rank-5 in retromer-based transport. Indeed, depletion of Rank-5 causes mislocalization of Vps26 and affects both the retrieval of mannose 6-phosphate receptor transport to the Golgi from endosomes and biosynthetic transport. Moreover, Rank-5 is required for normal retromer distribution, as overexpression of a wild-type Rank-5-small interfering RNA-resistant construct rescues retromer mislocalization. Finally, we show that depletion of either Rank-5 or EHD1 impairs secretion of vesicular stomatitis virus glycoprotein. Overall, our data identify a new interaction between Rank-5 and EHD1, and novel endocytic regulatory roles that include retromer-based transport and secretion.