BIG2, a guanine nucleotide exchange factor for ADP-ribosylation factors: its localization to recycling endosomes and implication in the endosome integrity

Small GTPases of the ADP-ribosylation factor (ARF) family play a key role in membrane trafficking by regulating coated vesicle formation, and guanine nucleotide exchange is essential for the ARF function. Brefeldin A blocks the ARF-triggered coat assembly by inhibiting the guanine nucleotide exchange on ARFs and causes disintegration of the Golgi complex and tubulation of endosomal membranes. BIG2 is one of brefeldin A-inhibited guanine nucleotide exchange factors for the ARF GTPases and is associated mainly with the trans-Golgi network. In the present study, we have revealed that another population of BIG2 is associated with the recycling endosome and found that expression of a catalytically inactive BIG2 mutant, E738K, selectively induces membrane tubules from this compartment. We also have shown that BIG2 has an exchange activity toward class I ARFs (ARF1 and ARF3) in vivo and inactivation of either ARF exaggerates the BIG2(E738K)-induced tubulation of endosomal membranes. These observations together indicate that BIG2 is implicated in the structural integrity of the recycling endosome through activating class I ARFs.     

Cargo trafficking between endosomes and the trans-Golgi network

The retrograde membrane transport pathways from endosomes to the trans-Golgi network (TGN) are now recognized as critical intracellular pathways to recycle and shuttle a selective subgroup of membrane proteins, including sorting receptors, membrane-bound enzymes, transporters, as well as providing an avenue for the intracellular transport of various bacterial toxins. Multiple pathways from endosomes to the TGN have now been defined which differ between the cargo transported and the machinery used. Here, we review advances in these pathways and the requirement for TGN organization, and also discuss the development of unbiased analytical approaches to quantitatively track cargo that use these endosome-to-TGN pathways.     

Phosphoinositides and membrane traffic at the trans-Golgi network

Cargo proteins moving along the secretory pathway are sorted at the TGN (trans-Golgi network) into distinct carriers for delivery to the plasma membrane or endosomes. Recent studies in yeast and mammals have shown that formation of these carriers is regulated by PtdIns(4)P. This phosphoinositide is abundant at the TGN and acts to recruit components required for carrier formation to the membrane. Other phosphoinositides are also present on the TGN, but the extent to which they regulate trafficking is less clear. Further characterization of phosphoinositide kinases and phosphatases together with identification of new TGN-associated phosphoinositide-binding proteins will reveal the extent to which different phosphoinositides regulate TGN trafficking, and help define the molecular mechanisms involved.     

Interactions between conserved domains within homodimers in the BIG1, BIG2, and GBF1 Arf guanine nucleotide exchange factors

Guanine nucleotide exchange factors carrying a Sec7 domain (ArfGEFs) activate the small GTP-binding protein Arf, a major regulator of membrane remodeling and protein trafficking in eukaryotic cells. Only two of the seven subfamilies of ArfGEFs (GBF and BIG) are found in all eukaryotes. In addition to the Sec7 domain, which catalyzes GDP/GTP exchange on Arf, the GBF and BIG ArfGEFs have five common homology domains. Very little is known about the functions of these noncatalytic domains, but it is likely that they serve to integrate upstream signals that define the conditions of Arf activation. Here we describe interactions between two conserved domains upstream of the Sec7 domain (DCB and HUS) that determine the architecture of the N-terminal regions of the GBF and BIG ArfGEFs using a combination of biochemical, yeast two-hybrid, and cellular assays. Our data demonstrate a strong interaction between DCB domains within GBF1, BIG1, and BIG2 to maintain homodimers and an interaction between DCB and HUS domains within each homodimer. The DCB/HUS interaction is mediated by the HUS box, the most conserved motif in large ArfGEFs after the Sec7 domain. In support of the in vitro data, we show that both the DCB and the HUS domains are necessary for GBF1 dimerization in mammalian cells and that the DCB domain is essential for yeast viability. We propose that the dimeric DCB-HUS structural unit exists in all members of the GBF and BIG ArfGEF groups and in the related Mon2p family and probably serves an important regulatory role in Arf activation.     

Dominant-negative mutant of BIG2, an ARF-guanine nucleotide exchange factor,specifically affects membrane trafficking from the trans-Golgi network through inhibiting membrane association of AP-1 and GGA coat proteins

BIG2 is one of the guanine nucleotide exchange factors (GEFs) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and AP-1 coat protein complexes and GGA proteins. Brefeldin A (BFA), an ARF-GEF inhibitor, causes redistribution of the coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). We have recently shown that BIG2 overexpression blocks BFA-induced redistribution of the AP-1 complex but not TGN membrane tubulation. In the present study, we constructed a dominant-negative BIG2 mutant and found that when expressed in cells it induced redistribution of AP-1 and GGA1 and membrane tubulation of the TGN. By contrast, the mutant did not induce COPI redistribution or Golgi membrane tubulation. These observations indicate that BIG2 is involved in trafficking from the TGN by regulating membrane association of AP-1 and GGA through activating ARF.     

ADP-ribosylation factor 1-independent protein sorting and export from the trans-Golgi network

Polarized epithelial cells efficiently sort newly synthesized apical and basolateral proteins into distinct transport carriers that emerge from the trans-Golgi network (TGN), and this sorting is recapitulated in nonpolarized cells. While the targeting signals of basolaterally destined proteins are generally cytoplasmically disposed, apical sorting signals are not typically accessible to the cytosol, and the transport machinery required for segregation and export of apical cargo remains largely unknown. Here we investigated the molecular requirements for TGN export of the apical marker influenza hemagglutinin (HA) in HeLa cells using an in vitro reconstitution assay. HA was released from the TGN in intact membrane-bound compartments, and export was dependent on addition of an ATP-regenerating system and exogenous cytosol. HA release was inhibited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS) as well as under conditions known to negatively regulate apical transport in vivo, including expression of the acid-activated proton channel influenza M2. Interestingly, release of HA was unaffected by depletion of ADP-ribosylation factor 1, a small GTPase that has been implicated in the recruitment of all known adaptors and coat proteins to the Golgi complex. Furthermore, regulation of HA release by GTPgammaS or M2 expression was unaffected by cytosolic depletion of ADP-ribosylation factor 1, suggesting that HA sorting remains functionally intact in the absence of the small GTPase. These data suggest that TGN sorting and export of influenza HA does not require classical adaptors involved in the formation of other classes of exocytic carriers and thus appears to proceed via a novel mechanism.     

The secretion inhibitor Exo2 perturbs trafficking of Shiga toxin between endosomes and the trans-Golgi network

The small-molecule inhibitor Exo2 {4-hydroxy-3-methoxy-(5,6,7,8-tetrahydrol[1]benzothieno[2,3-d]pyrimidin-4-yl)hydraz-one benzaldehyde} has been reported to disrupt the Golgi apparatus completely and to stimulate Golgi-ER (endoplasmic reticulum) fusion in mammalian cells, akin to the well-characterized fungal toxin BFA (brefeldin A). It has also been reported that Exo2 does not affect the integrity of the TGN (trans-Golgi network), or the direct retrograde trafficking of the glycolipid-binding cholera toxin from the TGN to the ER lumen. We have examined the effects of BFA and Exo2, and found that both compounds are indistinguishable in their inhibition of anterograde transport and that both reagents significantly disrupt the morphology of the TGN in HeLa and in BS-C-1 cells. However, Exo2, unlike BFA, does not induce tubulation and merging of the TGN and endosomal compartments. Furthermore, and in contrast with its effects on cholera toxin, Exo2 significantly perturbs the delivery of Shiga toxin to the ER. Together, these results suggest that the likely target(s) of Exo2 operate at the level of the TGN, the Golgi and a subset of early endosomes, and thus Exo2 provides a more selective tool than BFA for examining membrane trafficking in mammalian cells.     

Adaptor and clathrin exchange at the plasma membrane and trans-Golgi network

We previously demonstrated, using fluorescence recovery after photobleaching, that clathrin in clathrin-coated pits at the plasma membrane exchanges with free clathrin in the cytosol, suggesting that clathrin-coated pits are dynamic structures. We now investigated whether clathrin at the trans-Golgi network as well as the clathrin adaptors AP2 and AP1 in clathrin-coated pits at the plasma membrane and trans-Golgi network, respectively, also exchange with free proteins in the cytosol. We found that when the budding of clathrin-coated vesicle is blocked without significantly affecting the structure of clathrin-coated pits, both clathrin and AP2 at the plasma membrane and clathrin and AP1 at the trans-Golgi network exchange rapidly with free proteins in the cytosol. In contrast, when budding of clathrin-coated vesicles was blocked at the plasma membrane or trans-Golgi network by hypertonic sucrose or K(+) depletion, conditions that markedly affect the structure of clathrin-coated pits, clathrin exchange was blocked but AP2 at the plasma membrane and both AP1 and the GGA1 adaptor at the trans-Golgi network continue to rapidly exchange. We conclude that clathrin-coated pits are dynamic structures with rapid exchange of both clathrin and adaptors and that adaptors are able to exchange independently of clathrin when clathrin exchange is blocked.     

Phosphoinositides determine specificity of the guanine-nucleotide exchange activity of cytohesin-1 for ADP-ribosylation factors derived from a mammalian expression system.

ADP-ribosylation factors (ARFs) are small Ras-like GTPases which play important roles in intracellular vesicle transport and in the remodeling of the actin cytoskeleton. Guanine nucleotide exchange factors (GEFs) for ARFs have recently been identified. One of them, cytohesin-1, a 47-kDa cytoplasmic protein acts as an inside-out signaling molecule and regulates binding of the beta2 integrin leukocyte function antigen 1 (LFA-1) to its ligand intercellular adhesion molecule 1 (ICAM-1). In this study, we address the regulation of the GEF activity of cytohesin-1 by phosphoinositides, using mammalian expression of functional ARF-Ig chimeras. The fusion proteins, which can be quantitatively immunoprecipitated on protein A-Sepharose, target to the expected intracellular compartments, and they are readily induced to bind GTP in vitro. We show that both ARF1-Ig and ARF6-Ig chimeras are activated in vitro by cytohesin-1. However, GEF activity towards ARF6 is strongly suppressed by phosphatidylinositol-(3,4,5)-trisphosphate (PtdInsP3). In contrast, cytohesin-1-dependent GTP binding of ARF1 is significantly enhanced by PtdInsP3. We conclude that the membrane phospholipid PtdInsP3 determines the specificity of the GEF activity of cytohesin-1.     

Overexpression of an ADP-ribosylation factor-guanine nucleotide exchange factor, BIG2, uncouples brefeldin A-induced adaptor protein-1 coat dissociation and membrane tubulation.

BIG2 is a guanine nucleotide exchange factor (GEF) for the ADP-ribosylation factor (ARF) family of small GTPases, which regulate membrane association of COPI and adaptor protein (AP)-1 coat protein complexes. A fungal metabolite, brefeldin A (BFA), inhibits ARF-GEFs and leads to redistribution of coat proteins from membranes to the cytoplasm and membrane tubulation of the Golgi complex and the trans-Golgi network (TGN). To investigate the function of BIG2, we examined the effects of BIG2-overexpression on the BFA-induced redistribution of ARF, coat proteins, and organelle markers. The BIG2 overexpression blocked BFA-induced redistribution from membranes of ARF1 and the AP-1 complex but not that of the COPI complex. These observations indicate that BIG2 is implicated in membrane association of AP-1, but not that of COPI, through activating ARF. Furthermore, not only BIG2 but also ARF1 and AP-1 were found as queues of spherical swellings along the BFA-induced membrane tubules emanating from the TGN. These observations indicate that BFA-induced AP-1 dissociation from TGN membranes and tubulation of TGN membranes are not coupled events and suggest that a BFA target other than ARF-GEFs exists in the cell.     

Lowe syndrome protein OCRL1 interacts with clathrin and regulates protein trafficking between endosomes and the trans-Golgi network

Oculocerebrorenal syndrome of Lowe is caused by mutation of OCRL1, a phosphatidylinositol 4,5-bisphosphate 5-phosphatase localized at the Golgi apparatus. The cellular role of OCRL1 is unknown, and consequently the mechanism by which loss of OCRL1 function leads to disease is ill defined. Here, we show that OCRL1 is associated with clathrin-coated transport intermediates operating between the trans-Golgi network (TGN) and endosomes. OCRL1 interacts directly with clathrin heavy chain and promotes clathrin assembly in vitro. Interaction with clathrin is not, however, required for membrane association of OCRL1. Overexpression of OCRL1 results in redistribution of clathrin and the cation-independent mannose 6-phosphate receptor (CI-MPR) to enlarged endosomal structures that are defective in retrograde trafficking to the TGN. Depletion of cellular OCRL1 also causes partial redistribution of a CI-MPR reporter to early endosomes. These findings suggest a role for OCRL1 in clathrin-mediated trafficking of proteins from endosomes to the TGN and that defects in this pathway might contribute to the Lowe syndrome phenotype.     

Interaction of neuronal calcium sensor-1 and ADP-ribosylation factor 1 allows bidirectional control of phosphatidylinositol 4-kinase beta and trans-Golgi network-plasma membrane traffic

We have identified a novel Ca(2+)-dependent interaction between neuronal calcium sensor-1 (NCS-1) and the GTPase ARF1. Both of these proteins are localized to the Golgi complex, and both regulate phosphatidylinositol 4-kinase IIIbeta (PI(4)Kbeta). Spatial and temporal control of phosphatidylinositol 4-phosphate levels through activation of PI(4)Kbeta is important for the recruitment of trafficking complexes to the trans-Golgi network (TGN) and vesicular traffic from this organelle. The NCS-1-ARF1 interaction and its specificity have been demonstrated through in vitro binding assays, in vitro enzyme assay, and through functional cellular assays. We show that NCS-1 can exert bidirectional effects to activate PI(4)Kbeta on its own or inhibit the activation by ARF1. NCS-1 was shown to modulate the effects of expression of ARF mutants that disrupt Golgi morphology and to recruit GDP-loaded ARF to the Golgi complex in a Ca(2+)-dependent manner. We demonstrate antagonist effects of NCS-1 and ARF on constitutive and regulated exocytosis. The NCS-1-ARF1 interaction provides evidence for functional cross-talk between Ca(2+)-dependent and ARF-dependent pathways in TGN to plasma membrane traffic.     

Clathrin-dependent association of CVAK104 with endosomes and the trans-Golgi network

CVAK104 is a novel coated vesicle-associated protein with a serine/threonine kinase homology domain that was recently shown to phosphorylate the beta2-subunit of the adaptor protein (AP) complex AP2 in vitro. Here, we demonstrate that a C-terminal segment of CVAK104 interacts with the N-terminal domain of clathrin and with the alpha-appendage of AP2. CVAK104 localizes predominantly to the perinuclear region of HeLa and COS-7 cells, but it is also present on peripheral vesicular structures that are accessible to endocytosed transferrin. The distribution of CVAK104 overlaps extensively with that of AP1, AP3, the mannose 6-phosphate receptor, and clathrin but not at all with its putative phosphorylation target AP2. RNA interference-mediated clathrin knockdown reduced the membrane association of CVAK104. Recruitment of CVAK104 to perinuclear membranes of permeabilized cells is enhanced by guanosine 5'-O-(3-thio)triphosphate, and brefeldin A redistributes CVAK104 in cells. Both observations suggest a direct or indirect requirement for GTP-binding proteins in the membrane association of CVAK104. Live-cell imaging showed colocalization of green fluorescent protein-CVAK104 with endocytosed transferrin and with red fluorescent protein-clathrin on rapidly moving endosomes. Like AP1-depleted COS-7 cells, CVAK104-depleted cells missort the lysosomal hydrolase cathepsin D. Together, our data suggest a function for CVAK104 in clathrin-dependent pathways between the trans-Golgi network and the endosomal system.     

ADP-ribosylation factor-like GTPase ARFRP1 is required for trans-Golgi to plasma membrane trafficking of E-cadherin

ADP-ribosylation factor-related protein 1 (ARFRP1) plays a specific role in Golgi function controlling recruitment of GRIP domain proteins and ARL1 to the trans-Golgi. Deletion of the mouse Arfrp1 gene causes embryonic lethality during early gastrulation, because epiblast cells detach from the ectodermal cell layer and do not differentiate to mesodermal tissue. Here we show that in Arfrp1(-/-) embryos E-cadherin is mistargeted to intracellular compartments, whereas in control embryos it is present at the cell surface of trophectodermal and ectodermal cells. In enterocytes of intestine-specific Arfrp1 null mutants (Arfrp1(vil)(-/-)), E-cadherin is associated with intracellular membranes, partially colocalizing with the cis-Golgi marker GM130 or with punctae close to the cell surface. In contrast, in control enterocytes E-cadherin is exclusively located in the lateral membranes. In addition, ARL1 is dislocated from Golgi membranes to the cytosol of Arfrp1(vil)(-/-) enterocytes. Depletion of endogenous ARFRP1 by RNA interference leads to a dislocation of E-cadherin from the cell surface in HeLa cells and to a reduced cell aggregation in Ltk(-)Ecad cells. ARFRP1 was coimmunoprecipitated in a complex with E-cadherin, alpha-catenin, beta-catenin, gamma-catenin, and p120(ctn) from lysates of Madin-Darby canine kidney cells stably expressing myc-ARFRP1. These data indicate that knock-out of Arfrp1 disrupts the trafficking of E-cadherin through the Golgi and suggest an essential role of the GTPase in trans-Golgi network function.     

The small G protein Arl1 directs the trans-Golgi-specific targeting of the Arf1 exchange factors BIG1 and BIG2

The small G protein Arf1 regulates Golgi traffic and is activated by two related types of guanine nucleotide exchange factor (GEF). GBF1 acts at the cis-Golgi, whereas BIG1 and its close paralog BIG2 act at the trans-Golgi. Peripheral membrane proteins such as these GEFs are often recruited to membranes by small G proteins, but the basis for specific recruitment of Arf GEFs, and hence Arfs, to Golgi membranes is not understood. In this paper, we report a liposome-based affinity purification method to identify effectors for small G proteins of the Arf family. We validate this with the Drosophila melanogaster Arf1 orthologue (Arf79F) and the related class II Arf (Arf102F), which showed a similar pattern of effector binding. Applying the method to the Arf-like G protein Arl1, we found that it binds directly to Sec71, the Drosophila ortholog of BIG1 and BIG2, via an N-terminal region. We show that in mammalian cells, Arl1 is necessary for Golgi recruitment of BIG1 and BIG2 but not GBF1. Thus, Arl1 acts to direct a trans-Golgi-specific Arf1 GEF, and hence active Arf1, to the trans side of the Golgi.     

Studies of the roles of ADP-ribosylation factors and phospholipase D in phorbol ester-induced membrane ruffling

In this study, we have explored the roles of ADP-ribosylation factors (ARFs), phospholipase D (PLD) isozymes, and arfaptins in phorbol ester (PMA)-induced membrane ruffling in HeLa cells. PMA stimulation induced ruffling and translocated cortactin to the plasma membrane. The cortactin translocation was inhibited by dominant negative (DN)-ARF6, DN-ARF1, and DN-Rac1, but not by DN-RhoA and DN-Cdc42. The inability of DN-forms of ARF6, ARF1, and Rac1 to affect PLD activity in response to PMA indicated that this enzyme was not activated via these small G proteins and that its activation was not essential for the induction of ruffling. Endogenous-ARF1, -ARF6, and -Rac1 existed in the ruffling region along with cortactin after PMA stimulation. DN-ARF1 had no effect on the ruffling induced by DA-ARF6 or DA-Rac1, and DN-ARF6 had no effect on that induced by DA-ARF1 or DA-Rac1. On the other hand DN-Rac1 suppressed the effect of DA-ARF6 but not that of DA-ARF1. These results suggest that PMA causes membrane ruffling via an ARF6-Rac1 pathway and also an ARF1 pathway operating in parallel. Overexpression of PLD1 and PLD2 inhibited PMA-induced cortactin translocation and actin-cortactin complex formation, supporting the view that these enzymes are not required for ruffling, but actually suppress it. We conclude that PMA-induced membrane ruffling is caused via ARF6-Rac1 and ARF1 pathways operating in parallel and that PLD may be inhibitory.     

P4-ATPase requirement for AP-1/clathrin function in protein transport from the trans-Golgi network and early endosomes

Drs2p is a resident type 4 P-type ATPase (P4-ATPase) and potential phospholipid translocase of the trans-Golgi network (TGN) where it has been implicated in clathrin function. However, precise protein transport pathways requiring Drs2p and how it contributes to clathrin-coated vesicle budding remain unclear. Here we show a functional codependence between Drs2p and the AP-1 clathrin adaptor in protein sorting at the TGN and early endosomes of Saccharomyces cerevisiae. Genetic criteria indicate that Drs2p and AP-1 operate in the same pathway and that AP-1 requires Drs2p for function. In addition, we show that loss of AP-1 markedly increases Drs2p trafficking to the plasma membrane, but does not perturb retrieval of Drs2p from the early endosome back to the TGN. Thus AP-1 is required at the TGN to sort Drs2p out of the exocytic pathway, presumably for delivery to the early endosome. Moreover, a conditional allele that inactivates Drs2p phospholipid translocase (flippase) activity disrupts its own transport in this AP-1 pathway. Drs2p physically interacts with AP-1; however, AP-1 and clathrin are both recruited normally to the TGN in drs2Delta cells. These results imply that Drs2p acts independently of coat recruitment to facilitate AP-1/clathrin-coated vesicle budding from the TGN.     

GBF1, a guanine nucleotide exchange factor for ADP-ribosylation factors,is localized to the cis-Golgi and involved in membrane association of the COPI coat

Formation of coated carrier vesicles, such as COPI-coated vesicles from the cis -Golgi, is triggered by membrane binding of the GTP-bound form of ADP-ribosylation factors. This process is blocked by brefeldin A, which is an inhibitor of guanine nucleotide exchange factors for ADP-ribosylation factor. GBF1 is one of the guanine nucleotide-exchange factors for ADP-ribosylation factor and is localized in the Golgi region. In the present study, we have determined the detailed subcellular localization of GBF1. Immunofluorescence microscopy of cells treated with nocodazole or incubated at 15 °C has suggested that GBF1 behaves similarly to proteins recycling between the cis -Golgi and the endoplasmic reticulum. Immunoelectron microscopy has revealed that GBF1 localizes primarily to vesicular and tubular structures apposed to the cis -face of Golgi stacks and minor fractions to the Golgi stacks. GBF1 overexpressed in cells causes recruitment of class I and class II ADP-ribosylation factors onto Golgi membranes. Furthermore, overexpressed GBF1 antagonizes various effects of brefeldin A, such as inhibition of membrane recruitment of ADP-ribosylation factors and the COPI coat, and redistribution of Golgi-resident and itinerant proteins. These observations indicate that GBF1 is involved in the formation of COPI-coated vesicles from the cis -Golgi or the pre-Golgi intermediate compartment through activating ADP-ribosylation factors.