Transferrin Recycling Research Articles

Rab GTPase–Myo5B complexes control membrane recycling and epithelial polarization

The Rab GTPases are the largest family of proteins regulating membrane traffic. Rab proteins form a nidus for the assembly of multiprotein complexes on distinct vesicle membranes to regulate particular membrane trafficking pathways. Recent investigations have demonstrated that Myosin Vb (Myo5B) is an effector for Rab8a, Rab10, and Rab11a, all of which are implicated in regulating different pathways for recycling of proteins to the plasma membrane. It remains unclear how specific interactions of Myo5B with individual Rab proteins can lead to specificity in the regulation of alternate trafficking pathways. We examined the relative contributions of Rab/Myo5B interactions with specific pathways using Myo5B mutants lacking binding to either Rab11a or Rab8a. Myo5B Q1300L and Y1307C mutations abolished Rab8a association, whereas Myo5B Y1714E and Q1748R mutations uncoupled association with Rab11a. Expression of Myo5B tails containing these mutants demonstrated that Rab11a, but not Rab8a, was required for recycling of transferrin in nonpolarized cells. In contrast, in polarized epithelial cyst cultures, Myo5B was required for apical membrane trafficking and de novo lumen formation, dependent on association with both Rab8a and Rab11a. These data demonstrate that different combinations of Rab GTPase association with Myo5B control distinct membrane trafficking pathways.

Arp2/3- and Cofilin-coordinated Actin Dynamics Is Required for Insulin-mediated GLUT4 Translocation to the Surface of Muscle Cells

GLUT4 vesicles are actively recruited to the muscle cell surface upon insulin stimulation. Key to this process is Rac-dependent reorganization of filamentous actin beneath the plasma membrane, but the underlying molecular mechanisms have yet to be elucidated. Using L6 rat skeletal myoblasts stably expressing myc-tagged GLUT4, we found that Arp2/3, acting downstream of Rac GTPase, is responsible for the cortical actin polymerization evoked by insulin. siRNA-mediated silencing of either Arp3 or p34 subunits of the Arp2/3 complex abrogated actin remodeling and impaired GLUT4 translocation. Insulin also led to dephosphorylation of the actin-severing protein cofilin on Ser-3, mediated by the phosphatase slingshot. Cofilin dephosphorylation was prevented by strategies depolymerizing remodeled actin (latrunculin B or p34 silencing), suggesting that accumulation of polymerized actin drives severing to enact a dynamic actin cycling. Cofilin knockdown via siRNA caused overwhelming actin polymerization that subsequently inhibited GLUT4 translocation. This inhibition was relieved by reexpressing Xenopus wild-type cofilin-GFP but not the S3E-cofilin-GFP mutant that emulates permanent phosphorylation. Transferrin recycling was not affected by depleting Arp2/3 or cofilin. These results suggest that cofilin dephosphorylation is required for GLUT4 translocation. We propose that Arp2/3 and cofilin coordinate a dynamic cycle of actin branching and severing at the cell cortex, essential for insulin-mediated GLUT4 translocation in muscle cells.

The myotubularin phosphatase MTMR4 regulates sorting from early endosomes

Phosphatidylinositol 3-phosphate [PtdIns(3)P] regulates endocytic trafficking and the sorting of receptors through early endosomes, including the rapid recycling of transferrin (Tfn). However, the phosphoinositide phosphatase that selectively opposes this function is unknown. The myotubularins are a family of eight catalytically active and six inactive enzymes that hydrolyse PtdIns(3)P to form PtdIns. However, the role each myotubularin family member plays in regulating endosomal PtdIns(3)P and thereby endocytic trafficking is not well established. Here, we identify the myotubularin family member MTMR4, which localizes to early endosomes and also to Rab11- and Sec15-positive recycling endosomes. In cells with MTMR4 knockdown, or following expression of the catalytically inactive MTMR4, MTMR4(C407A), the number of PtdIns(3)P-decorated endosomes significantly increased. MTMR4 overexpression delayed the exit of Tfn from early endosomes and its recycling to the plasma membrane. By contrast, expression of MTMR4(C407A), which acts as a dominant-negative construct, significantly accelerated Tfn recycling. However, in MTMR4 knockdown cells Tfn recycling was unchanged, suggesting that other MTMs might also contribute to recycling. MTMR4 regulated the subcellular distribution of Rab11 and, in cells with RNAi-mediated knockdown of MTMR4, Rab11 was directed away from the pericentriolar recycling compartment. The subcellular distribution of VAMP3, a v-SNARE protein that resides in recycling endosomes and endosome-derived transport vesicles, was also regulated by MTMR4. Therefore, MTMR4 localizes at the interface of early and recycling endosomes to regulate trafficking through this pathway.

Differential Effects of TBC1D15 and Mammalian Vps39 on Rab7 Activation State, Lysosomal Morphology, and Growth Factor Dependence

The small GTPase Rab7 promotes fusion events between late endosomes and lysosomes. Rab7 activity is regulated by extrinsic signals, most likely via effects on its guanine nucleotide exchange factor (GEF) or GTPase-activating protein (GAP). Based on their homology to the yeast proteins that regulate the Ypt7 GTP binding state, TBC1D15, and mammalian Vps39 (mVps39) have been suggested to function as the Rab7 GAP and GEF, respectively. We developed an effector pull-down assay to test this model. TBC1D15 functioned as a Rab7 GAP in cells, reducing Rab7 binding to its effector protein RILP, fragmenting the lysosome, and conferring resistance to growth factor withdrawal-induced cell death. In a cellular context, TBC1D15 GAP activity was selective for Rab7. TBC1D15 overexpression did not inhibit transferrin internalization or recycling, Rab7-independent processes that require Rab4, Rab5, and Rab11 activation. TBC1D15 was thus renamed Rab7-GAP. Contrary to expectations for a Rab7 GEF, mVps39 induced lysosomal clustering without increasing Rab7 GTP binding. Moreover, a dominant-negative mVps39 mutant fragmented the lysosome and promoted growth factor independence without decreasing Rab7-GTP levels. These findings suggest that a protein other than mVps39 serves as the Rab7 GEF. In summary, although only TBC1D15/Rab7-GAP altered Rab7-GTP levels, both Rab7-GAP and mVps39 regulate lysosomal morphology and play a role in maintaining growth factor dependence.

Loss of SNAP29 Impairs Endocytic Recycling and Cell Motility

Intracellular membrane trafficking depends on the ordered formation and consumption of transport intermediates and requires that membranes fuse with each other in a tightly regulated and highly specific manner. Membrane anchored SNAREs assemble into SNARE complexes that bring membranes together to promote fusion. SNAP29 is a ubiquitous synaptosomal-associated SNARE protein. It interacts with several syntaxins and with the EH domain containing protein EHD1. Loss of functional SNAP29 results in CEDNIK syndrome (Cerebral Dysgenesis, Neuropathy, Ichthyosis and Keratoderma). Using fibroblast cell lines derived from CEDNIK patients, we show that SNAP29 mediates endocytic recycling of transferrin and β1-integrin. Impaired β1-integrin recycling affected cell motility, as reflected by changes in cell spreading and wound healing. No major changes were detected in exocytosis of VSVG protein from the Golgi apparatus, although the Golgi system acquired a dispersed morphology in SNAP29 deficient cells. Our results emphasize the importance of SNAP29 mediated membrane fusion in endocytic recycling and consequently, in cell motility.

MICAL-L1

A key regulator of the slow recycling of receptors and lipids that occurs from the endocytic recycling compartment (ERC) back to the cell surface is EHD1. We have recently identified the Rab8a-interacting protein, MICAL-L1, as a novel binding partner for EHD1 that both recruits and interacts with EHD1 on tubular recycling endosomes. MICAL-L1 belongs to the MICAL-family of proteins that are highly expressed in neurons and involved in plexin-mediated repulsive axon guidance. Interestingly, MICAL-L1 contains a coiled coil region in its C-terminus that is both necessary and sufficient for its localization to the EHD1-containing long tubular membranes of the ERC. Furthermore, MICAL-L1-depletion also impaired recycling of both transferrin and integrin receptors from the ERC back to the plasma membrane. In conclusion, our studies implicate MICAL-L1 as a novel regulator of endocytic recycling, and raises the possibility that additional neuronal-expressed proteins may mediate endocytic events in non-neuronal cells.

The Connecdenn DENN Domain: A GEF for Rab35 Mediating Cargo-Specific Exit from Early Endosomes

The DENN domain is an evolutionarily ancient protein module. Mutations in the DENN domain cause developmental defects in plants and human diseases, yet the function of this common module is unknown. We now demonstrate that the connecdenn/DENND1A DENN domain functions as a guanine nucleotide exchange factor (GEF) for Rab35 to regulate endosomal membrane trafficking. Loss of Rab35 activity causes an enlargement of early endosomes and inhibits MHC class I recycling. Moreover, it prevents early endosomal recruitment of EHD1, a common component of tubules involved in endosomal cargo recycling. Our data reveal an enzymatic activity for a DENN domain and demonstrate that distinct Rab GTPases can recruit a common protein machinery to various sites within the endosomal network to establish cargo-selective recycling pathways.

The Arp2/3 Activator WASH Controls the Fission of Endosomes through a Large Multiprotein Complex

The Arp2/3 complex generates branched actin networks when activated by Nucleation Promoting Factors (NPFs). Recently, the WASH family of NPFs has been identified, but its cellular role is unclear. Here, we show that WASH generates an actin network on a restricted domain of sorting and recycling endosomes. We found that WASH belongs to a multiprotein complex containing seven subunits, including the heterodimer of capping protein (CP). In vitro, the purified WASH complex activates Arp2/3-mediated actin nucleation and binds directly to liposomes. WASH also interacts with dynamin. WASH depletion gives rise to long membrane tubules pulled out from endosomes along microtubules, as does dynamin inhibition. Accordingly, WASH is required for efficient transferrin recycling. Together, these data suggest that the WASH molecular machine, integrating CP with a NPF, controls the fission of endosomes through an interplay between the forces generated by microtubule motors and actin polymerization.

D-AKAP2 Interacts with Rab4 and Rab11 through Its RGS Domains and Regulates Transferrin Receptor Recycling

Dual-specific A-kinase-anchoring protein 2 (D-AKAP2/AKAP10), which interacts at its carboxyl terminus with protein kinase A and PDZ domain proteins, contains two tandem regulator of G-protein signaling (RGS) domains for which the binding partners have remained unknown. We show here that these RGS domains interact with Rab11 and GTP-bound Rab4, the first demonstration of RGS domains binding small GTPases. Rab4 and Rab11 help regulate membrane trafficking through the endocytic recycling pathways by recruiting effector proteins to specific membrane domains. Although D-AKAP2 is primarily cytosolic in HeLa cells, a fraction of the protein localizes to endosomes and can be recruited there to a greater extent by overexpression of Rab4 or Rab11. D-AKAP2 also regulates the morphology of the Rab11-containing compartment, with co-expression causing accumulation of both proteins on enlarged endosomes. Knockdown of D-AKAP2 by RNA interference caused a redistribution of both Rab11 and the constitutively recycling transferrin receptor to the periphery of cells. Knockdown also caused an increase in the rate of transferrin recycling, suggesting that D-AKAP2 promotes accumulation of recycling proteins in the Rab4/Rab11-positive endocytic recycling compartment.

Defects in cellular sorting and retroviral assembly induced by GGA overexpression.

BackgroundWe previously demonstrated that overexpression of Golgi-localized, γ-ear containing, Arf-binding (GGA) proteins inhibits retrovirus assembly and release by disrupting the function of endogenous ADP ribosylation factors (Arfs). GGA overexpression led to the formation of large, swollen vacuolar compartments, which in the case of GGA1 sequestered HIV-1 Gag.ResultsIn the current study, we extend our previous findings to characterize in depth the GGA-induced compartments and the determinants for retroviral Gag sequestration in these structures. We find that GGA-induced structures are derived from the Golgi and contain aggresome markers. GGA overexpression leads to defects in trafficking of transferrin receptor and recycling of cation-dependent mannose 6-phosphate receptor. Additionally, we find that compartments induced by GGA overexpression sequester Tsg101, poly-ubiquitin, and, in the case of GGA3, Hrs. Interestingly, brefeldin A treatment, which leads to the dissociation of endogenous GGAs from membranes, does not dissociate the GGA-induced compartments. GGA mutants that are defective in Arf binding and hence association with membranes also induce the formation of GGA-induced structures. Overexpression of ubiquitin reverses the formation of GGA-induced structures and partially rescues HIV-1 particle production. We found that in addition to HIV-1 Gag, equine infectious anemia virus Gag is also sequestered in GGA1-induced structures. The determinants in Gag responsible for sequestration map to the matrix domain, and recruitment to these structures is dependent on Gag membrane binding.ConclusionThese data provide insights into the composition of structures induced by GGA overexpression and their ability to disrupt endosomal sorting and retroviral particle production.

Early endosomes and endosomal coatomer are required for autophagy

Autophagy, an intracellular degradative pathway, maintains cell homeostasis under normal and stress conditions. Nascent double-membrane autophagosomes sequester and enclose cytosolic components and organelles, and subsequently fuse with the endosomal pathway allowing content degradation. Autophagy requires fusion of autophagosomes with late endosomes, but it is not known if fusion with early endosomes is essential. We show that fusion of AVs with functional early endosomes is required for autophagy. Inhibition of early endosome function by loss of COPI subunits (β′, β, or α) results in accumulation of autophagosomes, but not an increased autophagic flux. COPI is required for ER-Golgi transport and early endosome maturation. Although loss of COPI results in the fragmentation of the Golgi, this does not induce the formation of autophagosomes. Loss of COPI causes defects in early endosome function, as both transferrin recycling and EGF internalization and degradation are impaired, and this loss of function causes an inhibition of autophagy, an accumulation of p62/SQSTM-1, and ubiquitinated proteins in autophagosomes.

D‐AKAP2 interacts with Rab4 and Rab11 through its RGS domains and regulates transferrin recycling

Dual‐specificity A‐Kinase Anchoring Protein 2 (D‐AKAP2/AKAP10) has been linked genetically to heart disease and breast cancer, but its biological function has remained unknown. Besides PKA‐ and PDZ‐binding motifs, D‐AKAP2 has two tandem Regulator of G protein Signaling (RGS) domains with no known binding partners. In HeLa cells, D‐AKAP2 is primarily cytosolic but can localize to membranes of the endocytic recycling compartment where it co‐localizes with endocytosed transferrin. Rab4 and Rab11, two small GTPases known to regulate membrane recycling, co‐localize with D‐AKAP2 and recruit it to endosomes. Co‐expression of D‐AKAP2 and Rab11 causes both proteins to accumulate on enlarged endosomes. Co‐immunoprecipitation and co‐localization studies demonstrated that D‐AKAP2 forms protein complexes through the RGS domains with both Rabs, preferring GTP‐locked Rab4 and wild‐type Rab11. Yeast two‐hybrid analysis confirmed that the tandem RGS domains were sufficient for interactions with both Rabs. Knockdown of D‐AKAP2 by RNA interference causes the Rab11 compartment to become more condensed, tubular, and peripheral. These changes correspond to a significant increase in the rate of transferrin recycling, suggesting that the RGS domains of D‐AKAP2 act as novel Rab effectors that inhibit recycling from the Rab4/Rab11 compartment. This work was funded by the NIH (DK054441) and the American Cancer Society.

Golgicide A reveals essential roles for GBF1 in Golgi assembly and function

ADP-ribosylation factor 1 (Arf1) plays a critical role in regulating secretory traffic and membrane transport within the Golgi of eukaryotic cells. Arf1 is activated by guanine nucleotide exchange factors (ArfGEFs) which confer spatial and temporal specificity to vesicular transport. We describe here the discovery and characterization of Golgicide A (GCA), a potent, highly specific, and reversible inhibitor of the cis-Golgi ArfGEF, GBF1. Inhibition of GBF1 function resulted in rapid dissociation of COPI vesicle coat from Golgi membranes and subsequent disassembly of the Golgi and trans-Golgi network (TGN). Secretion of soluble and membrane-associated proteins was arrested at the ER-Golgi intermediate compartment, whereas endocytosis and recycling of transferrin was unaffected by GBF1 inhibition. Internalized shiga toxin was arrested within the endocytic compartment and was unable to reach the dispersed TGN. Collectively, these results highlight the central role for GBF1 in coordinating bidirectional transport and maintaining structural integrity of the Golgi.

Caspase 8 Promotes Peripheral Localization and Activation of Rab5

Caspase 8 is a cysteine protease that initiates apoptotic signaling via the extrinsic pathway in a manner dependent upon association with early endosomes. Previously, we identified caspase 8 as an effector of migration, promoting motility in a manner dependent upon phosphorylation on Tyr-380 by Src family kinases and its subsequent association with Src homology 2 domain-containing proteins. Here we demonstrate the regulation of the small GTPase Rab5, which mediates early endosome formation, homotypic fusion, and maturation by caspase 8. Regulation requires the Tyr-380 phosphorylation site but not caspase proteolytic activity. Tyr-380 is essential for interaction with the Src homology 2 domains of p85α, a multifunctional adaptor for phosphatidylinositol 3-kinase, that possesses Rab-GAP activity. Interaction between caspase 8 and p85α promotes Rab5 GTP loading, alters endosomal trafficking, and results in the accumulation of Rab5-positive endosomes at the edge of the cell. Conversely, caspase 8-dependent GTP loading of Rab5 is overcome by increased expression of p85α in a Rab-GAP-dependent manner. Thus, we demonstrate a novel function for caspase 8 as a modulator of p85α Rab-GAP activity and endosomal trafficking.

Human RME-8 Is Involved in Membrane Trafficking through Early Endosomes

RME-8 is a DnaJ-domain-containing protein that was first identified in Caenorhabditis elegans as being required for uptake of yolk proteins. RME-8 has also been identified in other species, including flies and mammals, and the phenotypes of their RME-8 mutants suggest the importance of this protein in endocytosis. In the present study, we cloned human RME-8 (hRME-8) and characterized its biochemical properties and functions in endocytic pathways. hRME-8 was found to be a peripheral protein that was tightly associated with the membrane via its N-terminal region. It partially colocalized with several early endosomal markers, but not with late endosomal markers, consistent with observations by immunoelectron microscopy. When cells were transfected with a panel of dominant-active Rab proteins, hRME-8 was confined to large vacuoles induced by expression of Rab5aQ79L, but not by Rab7Q67L. Expression of C-terminally-truncated hRME-8 mutants led to the formation of large puncta and vacuoles, and compromised endocytic pathways through early endosomes, i.e., recycling of transferrin and degradation of epidermal growth factor. Taken together, these results indicate that hRME is primarily involved in membrane trafficking through early endosomes, but not through degradative organelles, such as multivesicular bodies and late endosomes.

Transferrin receptor 2 mediates uptake of transferrin-bound and non-transferrin-bound iron

Transferrin receptor 2 appears to have dual roles in iron metabolism; one is signalling, the other is iron transport. It is sensitive to high levels of diferric transferrin, which is associated with disorders of iron overload. Also present in these disorders are increased levels of plasma non-transferrin-bound iron. This study sought to clarify the role of transferrin receptor 2 in the uptake of transferrin-bound and non-transferrin-bound iron. Variant Chinese Hamster Ovary (CHO) cells, transfected with transferrin receptor 2, were incubated with radio-labelled transferrin-bound or non-transferrin-bound iron. Competition studies were performed in the presence of unlabelled dimetallic transferrin; knockdown was performed using specific siRNA. Cells expressing transferrin receptor 2 bound and internalised transferrin and transferrin-bound iron. Transferrin recycling occurred with an average cycling time of 11-15 min. Interestingly, the presence of transferrin receptor 2 was also associated with uptake of non-transferrin-bound iron which was inhibited by unlabelled transferrin-bound metals. Knockdown reduced transferrin-bound and non-transferrin-bound iron uptake by approximately 60%. Transferrin receptor 2 mediates transferrin-bound iron uptake by receptor-mediated endocytosis. It is also involved in the uptake of non-transferrin-bound iron and the inhibition of non-transferrin-bound iron uptake by diferric transferrin in CHO cells.

Tyrphostin A8 stimulates a novel trafficking pathway of apically endocytosed transferrin through Rab11-enriched compartments in Caco-2 cells

The potential application of transferrin receptors as delivery vehicles for transport of macromolecular drugs across intestinal epithelial cells is limited by several factors, including the low level of transferrin receptor-mediated transcytosis, particularly in the apical-to-basolateral direction. The GTPase inhibitor, AG10 (tyrphostin A8), has been shown previously to increase the apical-to-basolateral transcytosis of transferrin in Caco-2 cells. However, the mechanism of the increased transcytosis has not been established. In this report, the effect of AG10 on the trafficking of endocytosed transferrin among different endosomal compartments as well as the involvement of Rab11 in the intracellular trafficking of transferrin was investigated. Confocal microscopy studies showed a high level of colocalization of FITC-transferrin with Rab5 and Rab11 in Caco-2 cells pulsed at 16 degrees C and 37 degrees C, which indicated the presence of apically endocytosed FITC-transferrin in early endosomes and apical recycling endosomes at 16 degrees C and 37 degrees C, respectively. The effect of AG10 on the accumulation of transferrin within different endosomal compartment was studied, and an increase in the transcytosis and recycling of internalized (125)I-labeled transferrin, as well as a decrease in cell-associated (125)I-labeled transferrin, was observed in AG10-treated Caco-2 cells pulsed at 37 degrees C for 30 min and chased for 30 min. Moreover, confocal microscopy showed that FITC-transferrin exhibited an increased level of colocalization with Rab11, but not with Rab5, in the presence of AG10. These results suggest an effect of AG10 on the later steps of transferrin receptor trafficking, which are involved in subsequent recycling, and possibly transcytosis, of endocytosed transferrin in Caco-2 cells.

The N-Myc Down Regulated Gene1 (NDRG1) Is a Rab4a Effector Involved in Vesicular Recycling of E-Cadherin

Cell to cell adhesion is mediated by adhesion molecules present on the cell surface. Downregulation of molecules that form the adhesion complex is a characteristic of metastatic cancer cells. Downregulation of the N-myc down regulated gene1 (NDRG1) increases prostate and breast metastasis. The exact function of NDRG1 is not known. Here by using live cell confocal microscopy and in vitro reconstitution, we report that NDRG1 is involved in recycling the adhesion molecule E-cadherin thereby stabilizing it. Evidence is provided that NDRG1 recruits on recycling endosomes in the Trans Golgi network by binding to phosphotidylinositol 4-phosphate and interacts with membrane bound Rab4aGTPase. NDRG1 specifically interacts with constitutively active Rab4aQ67L mutant protein and not with GDP-bound Rab4aS22N mutant proving NDRG1 as a novel Rab4a effector. Transferrin recycling experiments reveals NDRG1 colocalizes with transferrin during the recycling phase. NDRG1 alters the kinetics of transferrin recycling in cells. NDRG1 knockdown cells show a delay in recycling transferrin, conversely NDRG1 overexpressing cells reveal an increase in rate of transferrin recycling. This novel finding of NDRG1 as a recycling protein involved with recycling of E-cadherin will aid in understanding NDRG1 role as a metastasis suppressor protein.

Dynein is required for receptor sorting and the morphogenesis of early endosomes

The early endosome is organised into domains to ensure the separation of cargo. Activated mitogenic receptors, such as epidermal growth factor (EGF) receptor, are concentrated into vacuoles enriched for the small GTPase Rab5, which progressively exclude nutrient receptors, such as transferrin receptor, into neighbouring tubules. These vacuoles become enlarged, increase their content of intralumenal vesicles as EGF receptor is sorted from the limiting membrane, and eventually mature to late endosomes. Maturation is governed by the loss of Rab5 and is accompanied by the movement of endosomes along microtubules towards the cell centre. Here, we show that EGF relocates to the cell centre in a dynein-dependent fashion, concomitant with the sorting away of transferrin receptor, although it remains in Rab5-positive early endosomes. When dynein function is acutely disrupted, efficient recycling of transferrin from EGF-containing endosomes is retarded, loss of Rab5 is slowed and endosome enlargement is reduced.

Loss of rapid transferrin receptor recycling due to a mutation in Sec15l1 in hbd mice

The hbd ( hemoglobin deficit) mutation affects iron trafficking in murine reticulocytes. It is due to a deletion that eliminates exon 8 of Sec15l1, the homolog of a gene that encodes an exocyst component in yeast. We tested the hypothesis that the mutation causes defective slow or rapid receptor recycling by measuring endocytosis and exocytosis of transferrin by hbd reticulocytes. Endocytosis and initial iron incorporation were relatively unaffected, but exocytosis was unexpectedly slowed. These data indicate that rapid transferrin recycling is defective after pSec15l1 has mutated.