Endocytic recycling proteins EHD1 and EHD2 interact with fer-1-like-5 (Fer1L5) and mediate myoblast fusion

The mammalian ferlins are calcium-sensing, C2 domain-containing proteins involved in vesicle trafficking. Myoferlin and dysferlin regulate myoblast fusion and muscle membrane resealing, respectively. Correspondingly, myoferlin is most highly expressed in singly nucleated myoblasts, whereas dysferlin expression is increased in mature, multinucleated myotubes. Myoferlin also mediates endocytic recycling and participates in trafficking the insulin-like growth factor receptor. We have now characterized a novel member of the ferlin family, Fer1L5, because of its high homology to dysferlin and myoferlin. We found that Fer1L5 protein is expressed in small myotubes that contain only two to four nuclei. We also found that Fer1L5 protein binds directly to the endocytic recycling proteins EHD1 and EHD2 and that the second C2 domain in Fer1L5 mediates this interaction. Reduction of EHD1 and/or EHD2 inhibits myoblast fusion, and EHD2 is required for normal translocation of Fer1L5 to the plasma membrane. The characterization of Fer1L5 and its interaction with EHD1 and EHD2 underscores the complex requirement of ferlin proteins and mediators of endocytic recycling for membrane trafficking events during myotube formation.     

The endocytic recycling protein EHD2 interacts with myoferlin to regulate myoblast fusion

Skeletal muscle is a multinucleated syncytium that develops and is maintained by the fusion of myoblasts to the syncytium. Myoblast fusion involves the regulated coalescence of two apposed membranes. Myoferlin is a membrane-anchored, multiple C2 domain-containing protein that is highly expressed in fusing myoblasts and required for efficient myoblast fusion to myotubes. We found that myoferlin binds directly to the eps15 homology domain protein, EHD2. Members of the EHD family have been previously implicated in endocytosis as well as endocytic recycling, a process where membrane proteins internalized by endocytosis are returned to the plasma membrane. EHD2 binds directly to the second C2 domain of myoferlin, and EHD2 is reduced in myoferlin null myoblasts. In contrast to normal myoblasts, myoferlin null myoblasts accumulate labeled transferrin and have delayed recycling. Introduction of dominant negative EHD2 into myoblasts leads to the sequestration of myoferlin and inhibition of myoblast fusion. The interaction of myoferlin with EHD2 identifies molecular overlap between the endocytic recycling pathway and the machinery that regulates myoblast membrane fusion.     

Recycling to the plasma membrane is delayed in EHD1 knockout mice

EHD1 is a member of the EHD family that contains four mammalian homologs. Among the invertebrate orthologs are a single Drosophila and Caenorhabditis elegans proteins and two plant members. They all contain three modules, a N-terminal domain that contains nucleotide-binding motifs, a central coiled-coil domain involved in oligomerization and a C-terminal region that harbors the EH domain. Studies in C. elegans and EHD1 depletion by RNA interference in human cells have demonstrated that it regulates recycling of membrane proteins. We addressed the physiological role of EHD1 through its inactivation in the mouse. Ehd1 knockout mice were indistinguishable from normal mice, had a normal life span and showed no histological abnormalities. Analysis of transferrin uptake in Ehd1(-/-) embryonic fibroblasts demonstrated delayed recycling to the plasma membrane with accumulation of transferrin in the endocytic recycling compartment. Our results corroborate the established role of EHD1 in the exit of membrane proteins from recycling endosomes in vivo in a mouse model.     

A tubular EHD1-containing compartment involved in the recycling of major histocompatibility complex class I molecules to the plasma membrane

The Eps15 homology (EH) domain-containing protein, EHD1, has recently been ascribed a role in the recycling of receptors internalized by clathrin-mediated endocytosis. A subset of plasma membrane proteins can undergo internalization by a clathrin-independent pathway regulated by the small GTP-binding protein ADP-ribosylation factor 6 (Arf6). Here, we report that endogenous EHD proteins, as well as transgenic tagged EHD1, are associated with long, membrane-bound tubules containing Arf6. EHD1 appears to induce tubule formation, which requires nucleotide cycling on Arf6 and intact microtubules. Mutations in the N-terminal P-loop domain or deletion of the C-terminal EH domain of EHD1 prevent association of EHD1 with tubules or induction of tubule formation. The EHD1 tubules contain internalized major histocompatibility complex class I (MHC-I) molecules that normally traffic through the Arf6 pathway. Recycling assays show that overexpression of EHD1 enhances MHC-I recycling. These observations suggest an additional function of EHD1 as a tubule-inducing factor in the Arf6 pathway for recycling of plasma membrane proteins internalized by clathrin-independent endocytosis.     

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.     

Drosophila exocyst components Sec5, Sec6, and Sec15 regulate DE-Cadherin trafficking from recycling endosomes to the plasma membrane

The E-Cadherin-catenin complex plays a critical role in epithelial cell-cell adhesion, polarization, and morphogenesis. Here, we have analyzed the mechanism of Drosophila E-Cadherin (DE-Cad) localization. Loss of function of the Drosophila exocyst components sec5, sec6, and sec15 in epithelial cells results in DE-Cad accumulation in an enlarged Rab11 recycling endosomal compartment and inhibits DE-Cad delivery to the membrane. Furthermore, Rab11 and Armadillo interact with the exocyst components Sec15 and Sec10, respectively. Our results support a model whereby the exocyst regulates DE-Cadherin trafficking, from recycling endosomes to sites on the epithelial cell membrane where Armadillo is located.     

EHD2 mediates trafficking from the plasma membrane by modulating Rac1 activity

EHDs [EH (Eps15 homology)-domain-containing proteins] participate in different stages of endocytosis. EHD2 is a plasma-membrane-associated EHD which regulates trafficking from the plasma membrane and recycling. EHD2 has a role in nucleotide-dependent membrane remodelling and its ATP-binding domain is involved in dimerization, which creates a membrane-binding region. Nucleotide binding is important for association of EHD2 with the plasma membrane, since a nucleotide-free mutant (EHD2 T72A) failed to associate. To elucidate the possible function of EHD2 during endocytic trafficking, we attempted to unravel proteins that interact with EHD2, using the yeast two-hybrid system. A novel interaction was found between EHD2 and Nek3 [NIMA (never in mitosis in Aspergillus nidulans)-related kinase 3], a serine/threonine kinase. EHD2 was also found in association with Vav1, a Nek3-regulated GEF (guanine-nucleotide-exchange factor) for Rho GTPases. Since Vav1 regulates Rac1 activity and promotes actin polymerization, the impact of overexpression of EHD2 on Rac1 activity was tested. The results indicated that wt (wild-type) EHD2, but not its P-loop mutants, reduced Rac1 activity. The inhibitory effect of EHD2 overexpression was partially rescued by co-expression of Rac1 as measured using a cholera toxin trafficking assay. The results of the present study strongly indicate that EHD2 regulates trafficking from the plasma membrane by controlling Rac1 activity.     

Mgat5 and Pten interact to regulate cell growth and polarity

Phosphatase and tensin homolog (Pten) phosphatase opposes intracellular phosphoinositide 3-kinase (PI3K)/Akt signaling and is a potent tumor suppressor, while Golgi beta1,6 N-acetylglucosaminyltransferase V (Mgat5) is positively associated with cancer progression and metastasis. beta1,6GlcNAc-branched N-glycans on receptor glycoproteins promote their surface residency and sensitizes cells to growth factor signaling. Here we demonstrate that the Pten heterozygosity in mouse embryonic fibroblasts enhances cell adhesion-dependent PI3K/Akt signaling, cell spreading, and proliferation, while Pten/Mgat5 double mutant cells are normalized. However, planar asymmetry typical of fibroblasts and invasive carcinomas is not fully rescued, suggesting that Mgat5 and Pten function together to regulate the membrane dynamics of PI3K/Akt signaling typical of motile cells. Pten heterozygosity was associated with increased surface beta1,6GlcNAc-branched N-glycans, suggesting positive feedback from PI3K signaling to N-glycan branching. In vivo, Mgat5(-/-) Pten(+/-) and Mgat5(+/-)Pten(+/-)mutant mice showed a small but significant increase in longevity compared with Pten(+/-) mice. Taken together, our results reveal that Mgat5 and Pten interact in an opposing manner to regulate cellular sensitivities to extracelluar growth cues.     

The EHD protein Past1 controls postsynaptic membrane elaboration and synaptic function

Membranes form elaborate structures that are highly tailored to their specialized cellular functions, yet the mechanisms by which these structures are shaped remain poorly understood. Here, we show that the conserved membrane-remodeling C-terminal Eps15 Homology Domain (EHD) protein Past1 is required for the normal assembly of the subsynaptic muscle membrane reticulum (SSR) at the Drosophila melanogaster larval neuromuscular junction (NMJ). past1 mutants exhibit altered NMJ morphology, decreased synaptic transmission, reduced glutamate receptor levels, and a deficit in synaptic homeostasis. The membrane-remodeling proteins Amphiphysin and Syndapin colocalize with Past1 in distinct SSR subdomains and collapse into Amphiphysin-dependent membrane nodules in the SSR of past1 mutants. Our results suggest a mechanism by which the coordinated actions of multiple lipid-binding proteins lead to the elaboration of increasing layers of the SSR and uncover new roles for an EHD protein at synapses.     

Endocytosis and the recycling of plasma membrane

For study of the time order of glycosylation, formation of complex oligosaccharides and proteolytic maturation as well as the site of proteolytic maturation of cathepsin D, fibroblasts were subjected to pulse-chase labeling, and cathepsin D was isolated from either total cell extracts or subcellular fractions by immune precipitation and analyzed for its molecular forms and sensitivity to endo-beta-N- acetylglucosaminidase H. After a 10-min pulse, cathepsin D was detected in its glycosylated precursor form, indicating an early, probably a cotranslational, N-glycosylation of cathepsin D. Conversion of the high- mannose oligosaccharide side chains into forms resistant to endo-beta-N- acetylglucosaminidase H started after approximately 40 min, indicating that transport of cathepsin D from the endoplasmic reticulum to the trans-Golgi apparatus requires approximately 40 min. Processing of the 53-kdalton precursor polypeptide of cathepsin D to a 47-kdalton intermediate followed about 20 min after the formation of complex oligosaccharides, and, another 30 min later, 31-kdalton mature forms of cathepsin D were detected. Processing of cathepsin D was first observed in light membranes as a partial conversion of the 53-kdalton precursor into the 47-kdalton intermediate. Both the precursor and the intermediate are transferred into the high density-class lysosomes. After 8 h, the processing to the mature 31-kdalton form of cathepsin D is mostly completed.     

SecY and SecA interact to allow SecA insertion and protein translocation across the Escherichia coli plasma membrane.

SecA, the preprotein-driving ATPase in Escherichia coli, was shown previously to insert deeply into the plasma membrane in the presence of ATP and a preprotein; this movement of SecA was proposed to be mechanistically coupled with preprotein translocation. We now address the role played by SecY, the central subunit of the membrane-embedded heterotrimeric complex, in the SecA insertion reaction. We identified a secY mutation (secY205), affecting the most carboxyterminal cytoplasmic domain, that did not allow ATP and preprotein-dependent productive SecA insertion, while allowing idling insertion without the preprotein. Thus, the secY205 mutation might affect the SecYEG 'channel' structure in accepting the preprotein-SecA complex or its opening by the complex. We isolated secA mutations that allele-specifically suppressed the secY205 translocation defect in vivo. One mutant protein, SecA36, with an amino acid alteration near the high-affinity ATP-binding site, was purified and suppressed the in vitro translocation defect of the inverted membrane vesicles carrying the SecY205 protein. The SecA36 protein could also insert into the mutant membrane vesicles in vitro. These results provide genetic evidence that SecA and SecY specifically interact, and show that SecY plays an essential role in insertion of SecA in response to a preprotein and ATP and suggest that SecA drives protein translocation by inserting into the membrane in vivo.     

The plastid protein THYLAKOID FORMATION1 and the plasma membrane G-protein GPA1 interact in a novel sugar-signaling mechanism in Arabidopsis

Mutations in genes encoding components of the heterotrimeric G-protein complex were previously shown to confer altered sensitivity to increased levels of D-glucose. This suggests that G-protein coupling may be a novel sugar-signaling mechanism in Arabidopsis thaliana. THYLAKOID FORMATION1 (THF1) is here demonstrated in vivo as a Galpha interaction partner that functions downstream of the plasma membrane-delimited heterotrimeric G-protein (GPA1) in a D-glucose signaling pathway. THF1 is a plastid protein localized to both the outer plastid membrane and the stroma. Contact between root plastidic THF1 and GPA1 at the plasma membrane occurs at sites where the plastid membrane abuts the plasma membrane, as demonstrated by F?rster resonance energy transfer (FRET). A probable role for THF1 in sugar signaling is demonstrated by both biochemical and genetic evidence. Root growth in the thf1-1 null mutant is hypersensitive to exogenous D-glucose, and THF1-overexpressing roots are resistant to inhibition of growth rate by high D-glucose. Additionally, THF1 levels are rapidly degraded by D-glucose but not L-glucose. The interaction between THF1 and GPA1 has been confirmed by in vitro and in vivo coimmunoprecipitation, FRET analysis, and genetic epistasis and provides evidence of a sugar-signaling mechanism between plastids and the plasma membrane.     

Shc and CEACAM1 interact to regulate the mitogenic action of insulin.

CEACAM1, a tumor suppressor (previously known as pp120), is a plasma membrane protein that undergoes phosphorylation on Tyr(488) in its cytoplasmic tail by the insulin receptor tyrosine kinase. Co-expression of CEACAM1 with insulin receptors decreased cell growth in response to insulin. Co-immunoprecipitation experiments in intact NIH 3T3 cells and glutathione S-transferase pull-down assays revealed that phosphorylated Tyr(488) in CEACAM1 binds to the SH2 domain of Shc, another substrate of the insulin receptor. Overexpressing Shc SH2 domain relieved endogenous Shc from binding to CEACAM1 and restored MAP kinase activity, growth of cells in response to insulin, and their colonization in soft agar. Thus, by binding to Shc, CEACAM1 sequesters this major coupler of Grb2 to the insulin receptor and down-regulates the Ras/MAP kinase mitogenesis pathway. Additionally, CEACAM1 binding to Shc enhances its ability to compete with IRS-1 for phosphorylation by the insulin receptor. This leads to a decrease in IRS-1 binding to phosphoinositide 3'-kinase and to the down-regulation of the phosphoinositide 3'-kinase/Akt pathway that mediates cell proliferation and survival. Thus, binding to Shc appears to constitute a major mechanism for the down-regulatory effect of CEACAM1 on cell proliferation.     

Ypt31/32 GTPases and their novel F-box effector protein Rcy1 regulate protein recycling

Ypt/Rab GTPases control various aspects of vesicle formation and targeting via their diverse effectors. We report a new role for these GTPases in protein recycling through a novel effector. The F-box protein Rcy1, which mediates plasma membrane recycling, is identified here as a downstream effector of the Ypt31/32 GTPase pair because it binds active GTP-bound Ypt31/32 and colocalizes with these GTPases on late Golgi and endosomes. Furthermore, Ypt31/32 regulates the polarized localization and half-life of Rcy1. This suggests that Ypt/Rabs can regulate the protein level of their effectors, in addition to the established ways by which they control their effectors. We show that like Rcy1, Ypt31/32 regulate the coupled phosphorylation and recycling of the plasma membrane v-SNARE Snc1. Moreover, Ypt31/32 and Rcy1 regulate the recycling of the furin-homolog Kex2 to the Golgi. Therefore, Ypt31/32 and Rcy1 mediate endosome-to-Golgi transport, because this is the only step shared by Snc1 and Kex2. Finally, we show that Rcy1 physically interacts with Snc1. Based on this result and because F-box proteins serve as adaptors between specific substrates and ubiquitin ligases, we propose that Ypt31/32 GTPases regulate the function of Rcy1 in the phosphorylation and/or ubiquitination of proteins that recycle through the Golgi.     

Multiple signaling pathways and a selector protein sequentially regulate Drosophila wing development

Drosophila wing development is a useful model to study organogenesis, which requires the input of selector genes that specify the identity of various morphogenetic fields (Weatherbee, S. D. and Carroll, S. B. (1999) Cell 97, 283-286) and cell signaling molecules. In order to understand how the integration of multiple signaling pathways and selector proteins can be achieved during wing development, we studied the regulatory network that controls the expression of Serrate (Ser), a ligand for the Notch (N) signaling pathway, which is essential for the development of the Drosophila wing, as well as vertebrate limbs. Here, we show that a 794 bp cis-regulatory element located in the 3' region of the Ser gene can recapitulate the dynamic patterns of endogenous Ser expression during wing development. Using this enhancer element, we demonstrate that Apterous (Ap, a selector protein), and the Notch and Wingless (Wg) signaling pathways, can sequentially control wing development through direct regulation of Ser expression in early, mid and late third instar stages, respectively. In addition, we show that later Ser expression in the presumptive vein cells is controlled by the Egfr pathway. Thus, a cis-regulatory element is sequentially regulated by multiple signaling pathways and a selector protein during Drosophila wing development. Such a mechanism is possibly conserved in the appendage outgrowth of other arthropods and vertebrates.     

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.