Characterization of coated vesicles that participate in endocytic recycling.

While the recycling pathway of endocytosis has been shown to participate in many cellular functions, little is known regarding the transport carriers that mediate this pathway. In this study, we overexpressed a point mutant of ADP-ribosylation factor 6 (ARF6), that perturbs its GTPase cycle, to accumulate endosome-derived coated vesicles. Characterization by their purification revealed that, upon cell homogenization, these vesicles were mostly aggregated with larger noncoated membranes, and could be released with high-salt treatment. Equilibrium centrifugation revealed that these vesicles had buoyant density similar to the COP-coated vesicles. To purify the ARF6-regulated vesicles to homogeneity, enriched fractions from equilibrium centrifugation were subjected to immunoisolation through the hemagglutinin (HA) epitope of the mutant ARF6, by using a newly developed, high-affinity, anti-HA monoclonal antibody. Surface iodination of the purified vesicles revealed multiple prominent proteins. Immunoblotting with antibodies against subunits of the currently known coat proteins suggested that these vesicles have a novel coat complex. These vesicles are carriers for endocytic recycling, because they are enriched for transferrin receptor and also the v-SNARE cellubrevin that functions in transport from the recycling endosome to the plasma membrane. Thus, we have characterized transport vesicles that participate in endocytic recycling.

The KDEL receptor mediates a retrieval mechanism that contributes to quality control at the endoplasmic reticulum.

Newly synthesized proteins in the endoplasmic reticulum (ER) must fold and assemble correctly before being transported to their final cellular destination. While some misfolded or partially assembled proteins have been shown to exit the ER, they fail to escape the early secretory system entirely, because they are retrieved from post-ER compartments to the ER. We elucidate a mechanistic basis for this retrieval and characterize its contribution to ER quality control by studying the fate of the unassembled T-cell antigen receptor (TCR) alpha chain. While the steady-state distribution of TCRalpha is in the ER, inhibition of retrograde transport by COPI induces the accumulation of TCRalpha in post-ER compartments, suggesting that TCRalpha is cycling between the ER and post-ER compartments. TCRalpha associates with BiP, a KDEL protein. Disruption of the ligand-binding function of the KDEL receptor releases TCRalpha from the early secretory system to the cell surface, so that TCRalpha is no longer subject to ER degradation. Thus, our findings suggest that retrieval by the KDEL receptor contributes to mechanisms by which the ER monitors newly synthesized proteins for their proper disposal.

ACAPs are arf6 GTPase-activating proteins that function in the cell periphery.

The GTP-binding protein ADP-ribosylation factor 6 (Arf6) regulates endosomal membrane trafficking and the actin cytoskeleton in the cell periphery. GTPase-activating proteins (GAPs) are critical regulators of Arf function, controlling the return of Arf to the inactive GDP-bound state. Here, we report the identification and characterization of two Arf6 GAPs, ACAP1 and ACAP2. Together with two previously described Arf GAPs, ASAP1 and PAP, they can be grouped into a protein family defined by several common structural motifs including coiled coil, pleckstrin homology, Arf GAP, and three complete ankyrin-repeat domains. All contain phosphoinositide-dependent GAP activity. ACAP1 and ACAP2 are widely expressed and occur together in the various cultured cell lines we examined. Similar to ASAP1, ACAP1 and ACAP2 were recruited to and, when overexpressed, inhibited the formation of platelet-derived growth factor (PDGF)-induced dorsal membrane ruffles in NIH 3T3 fibroblasts. However, in contrast with ASAP1, ACAP1 and ACAP2 functioned as Arf6 GAPs. In vitro, ACAP1 and ACAP2 preferred Arf6 as a substrate, rather than Arf1 and Arf5, more so than did ASAP1. In HeLa cells, overexpression of either ACAP blocked the formation of Arf6-dependent protrusions. In addition, ACAP1 and ACAP2 were recruited to peripheral, tubular membranes, where activation of Arf6 occurs to allow membrane recycling back to the plasma membrane. ASAP1 did not inhibit Arf6-dependent protrusions and was not recruited by Arf6 to tubular membranes. The additional effects of ASAP1 on PDGF-induced ruffling in fibroblasts suggest that multiple Arf GAPs function coordinately in the cell periphery.

The evolutionarily conserved N-terminal region of Cbl is sufficient to enhance down-regulation of the epidermal growth factor receptor.

The mammalian proto-oncoprotein Cbl and its homologues in Caenorhabditis elegans and Drosophila are evolutionarily conserved negative regulators of the epidermal growth factor receptor (EGF-R). Overexpression of wild-type Cbl enhances down-regulation of activated EGF-R from the cell surface. We report that the Cbl tyrosine kinase-binding (TKB) domain is essential for this activity. Whereas wild-type Cbl enhanced ligand-dependent EGF-R ubiquitination, down-regulation from the cell surface, accumulation in intracellular vesicles, and degradation, a Cbl TKB domain-inactivated mutant (G306E) did not. Furthermore, the transforming truncation mutant Cbl-N (residues 1-357), comprising only the Cbl TKB domain, functioned as a dominant negative protein. It colocalized with EGF-R in intracellular vesicular structures, yet it suppressed down-regulation of EGF-R from the surface of cells expressing endogenous wild-type Cbl. Therefore, Cbl-mediated down-regulation of EGF-R requires the integrity of both the N-terminal TKB domain and additional C-terminal sequences. A Cbl truncation mutant comprising amino acids 1-440 functioned like wild-type Cbl in down-regulation assays. This mutant includes the evolutionarily conserved TKB and RING finger domains but lacks the less conserved C-terminal sequences. We conclude that the evolutionarily conserved N terminus of Cbl is sufficient to effect enhancement of EGF-R ubiquitination and down-regulation from the cell surface.

Molecular aspects of the cellular activities of ADP-ribosylation factors.

Adenosine diphosphate-ribosylation factor (Arf) proteins are members of the Arf arm of the Ras superfamily of guanosine triphosphate (GTP)-binding proteins. Arfs are named for their activity as cofactors for cholera toxin-catalyzed adenosine diphosphate-ribosylation of the heterotrimeric G protein Gs. Physiologically, Arfs regulate membrane traffic and the actin cytoskeleton. Arfs function both constitutively within the secretory pathway and as targets of signal transduction in the cell periphery. In each case, the controlled binding and hydrolysis of GTP is critical to Arf function. The activities of some guanine nucleotide exchange factors (GEFs) and guanosine triphosphatase (GTPase)-activating proteins (GAPs) are stimulated by phosphoinositides, including phosphatidylinositol 3,4,5-trisphosphate (PIP3) and phosphatidylinositol 4,5-bisphosphate (PIP2), and phosphatidic acid (PA), likely providing both a means to respond to regulatory signals and a mechanism to coordinate GTP binding and hydrolysis. Arfs affect membrane traffic in part by recruiting coat proteins, including COPI and clathrin adaptor complexes, to membranes. However, Arf function likely involves many additional biochemical activities. Arf activates phospholipase D and phosphatidylinositol 4-phosphate 5-kinase with the consequent production of PA and PIP2, respectively. In addition to mediating Arf's effects on membrane traffic and the actin cytoskeleton, PA and PIP2 are involved in the regulation of Arf. Arf also works with Rho family proteins to affect the actin cytoskeleton. Several Arf-binding proteins suspected to be effectors have been identified in two-hybrid screens. Arf-dependent biochemical activities, actin cytoskeleton changes, and membrane trafficking may be integrally related. Understanding Arf's role in complex cellular functions such as protein secretion or cell movement will involve a description of the temporal and spatial coordination of these multiple Arf-dependent events.

The KDEL receptor regulates a GTPase-activating protein for ADP-ribosylation factor 1 by interacting with its non-catalytic domain.

ADP-ribosylation factor 1 (ARF1) is a key regulator of transport in the secretory system. Like all small GTPases, deactivation of ARF1 requires a GTPase-activating protein (GAP) that promotes hydrolysis of GTP to GDP on ARF1. Structure-function analysis of a GAP for ARF1 revealed that its activity in vivo requires not only a domain that catalyzes hydrolysis of GTP on ARF1 but also a non-catalytic domain. In this study, we show that the non-catalytic domain of GAP is required for its recruitment from cytosol to membranes and that this domain mediates the interaction of GAP with the transmembrane KDEL receptor. Blocking its interaction with the KDEL receptor leaves the GAP cytosolic and prevents the deactivation in vivo of Golgi-localized ARF1. Thus, these findings suggest that the KDEL receptor plays a critical role in the function of GAP by regulating its recruitment from cytosol to membranes, where it can then act on its membrane-restricted target, the GTP-bound form of ARF1.

Distribution of ARF6 between membrane and cytosol is regulated by its GTPase cycle.

The ADP-ribosylation factor (ARF) subfamily of small GTPases regulates intracellular transport. Although much is known about how ARF1 regulates transport in the secretory pathways, regulation of the endocytic pathways by ARF6 remains less understood. In particular, whereas cycling of ARF1 between membrane and cytosol represents a major mechanism of regulating its function, this regulation has been questioned for ARF6. In this study, we found that ARF6 is distributed both on membranes and in the cytosol. Cytosolic ARF6 is recruited to membranes in a GTP-dependent manner that is fundamentally similar to ARF1. However, unlike ARF1, release of membrane-bound ARF6 to the cytosol requires hydrolysis of GTP that is sensitive to the level of magnesium. These findings suggest that the GTPase cycle of ARF6 also regulates its distribution between membrane and cytosol and that this form of regulation will also likely be important for the function of ARF6. Moreover, as ARF6 has little intrinsic ability to hydrolyze GTP, magnesium concentration most likely affects the release of membrane-bound ARF6 by altering the activity of its GTPase-activating protein.

Separate pathways for antigen presentation by CD1 molecules.

The ability to sample relevant intracellular compartments is necessary for effective antigen presentation. To detect peptide antigens, MHC class I and II molecules differentially sample cytosolic and endosomal compartments. CD1 constitutes another lineage of lipid antigen-presenting molecules. We show that CD1b traffics deeply into late endosomal compartments, while CD1a is excluded from these compartments and instead traffics independently in the recycling pathway of the early endocytic system. Further, CD1b but not CD1a antigen presentation is dependent upon vesicular acidification. Since lipids and various bacteria are known to traffic differentially, either penetrating deeply into the endocytic system or following the route of recycling endosomes, these findings elucidate efficient monitoring of distinct components of the endocytic compartment by CD1 lipid antigen-presenting molecules.

Requirement for both the amino-terminal catalytic domain and a noncatalytic domain for in vivo activity of ADP-ribosylation factor GTPase-activating protein.

The small GTP-binding protein ADP-ribosylation factor-1 (ARF1) regulates intracellular transport by modulating the interaction of coat proteins with the Golgi complex. Coat protein association with Golgi membranes requires activated, GTP-bound ARF1, whereas GTP hydrolysis catalyzed by an ARF1-directed GTPase-activating protein (GAP) deactivates ARF1 and results in coat protein dissociation. We have recently cloned a Golgi-associated ARF GAP. Overexpression of GAP was found to result in a phenotype that reflects ARF1 deactivation (Aoe, T., Cukierman, E., Lee, A., Cassel, D., Peters, P. J., and Hsu, V. W. (1997) EMBO J. 16, 7305-7316). In this study, we used this phenotype to define domains in GAP that are required for its function in vivo. As expected, mutations in the amino-terminal part of GAP that were previously found to abolish ARF GAP catalytic activity in vitro abrogated ARF1 deactivation in vivo. Significantly, truncations at the carboxyl-terminal part of GAP that did not affect GAP catalytic activity in vitro also diminished ARF1 deactivation. Thus, a noncatalytic domain is required for GAP activity in vivo. This domain may be involved in the targeting of GAP to the Golgi membrane.

Modulation of intracellular transport by transported proteins: insight from regulation of COPI-mediated transport.

Intracellular transport is best understood for how proteins are shuttled among different compartments of the secretory pathway by membrane-bound transport carriers. However, it remains unclear whether regulation of this transport is modulated by the transported (cargo) proteins in the lumen of transport pathways. In the early secretory pathways that connect the endoplasmic reticulum (ER) and the Golgi complex, the small GTPase ADP-ribosylation factor 1 (ARF1) recruits a cytosolic coat protein complex named COPI onto membranes as a key step in the formation of transport vesicles. Transport of newly synthesized proteins that leave the ER includes a class of cargo proteins with a sequence motif of KDEL. When these KDEL proteins leave the ER to reach the Golgi complex, they are recognized by their receptor and transported retrograde in COPI-coated vesicles back to the ER. We now demonstrate that stimulation of the KDEL receptor by a KDEL protein enhances an interaction between the KDEL receptor and a GTPase-activating protein for ARF1. As a result, more cytosolic GTPase-activating protein is recruited to membranes to inactivate ARF1. Thus, the KDEL proteins are examples of luminal cargo proteins that regulate transport by activating their receptor. Most likely, this regulation affects retrograde transport from the Golgi complex to the ER, as activated KDEL receptor appears to reside only in retrograde COPI-coated vesicles.

ARF6 targets recycling vesicles to the plasma membrane: insights from an ultrastructural investigation.

We have shown previously that the ADP-ribosylation factor (ARF)-6 GTPase localizes to the plasma membrane and intracellular endosomal compartments. Expression of ARF6 mutants perturbs endosomal trafficking and the morphology of the peripheral membrane system. However, another study on the distribution of ARF6 in subcellular fractions of Chinese hamster ovary (CHO) cells suggested that ARF6 did not localize to endosomes labeled after 10 min of horseradish peroxidase (HRP) uptake, but instead was uniquely localized to the plasma membrane, and that its reported endosomal localization may have been a result of overexpression. Here we demonstrate that at the lowest detectable levels of protein expression by cryoimmunogold electron microscopy, ARF6 localized predominantly to an intracellular compartment at the pericentriolar region of the cell. The ARF6-labeled vesicles were partially accessible to HRP only on prolonged exposure to the endocytic tracer but did not localize to early endocytic structures that labeled with HRP shortly after uptake. Furthermore, we have shown that the ARF6-containing intracellular compartment partially colocalized with transferrin receptors and cellubrevin and morphologically resembled the recycling endocytic compartment previously described in CHO cells. HRP labeling in cells expressing ARF6(Q67L), a GTP-bound mutant of ARF6, was restricted to small peripheral vesicles, whereas the mutant protein was enriched on plasma membrane invaginations. On the other hand, expression of ARF6(T27N), a mutant of ARF6 defective in GDP binding, resulted in an accumulation of perinuclear ARF6-positive vesicles that partially colocalized with HRP on prolonged exposure to the tracer. Taken together, our findings suggest that ARF activation is required for the targeted delivery of ARF6-positive, recycling endosomal vesicles to the plasma membrane.

The KDEL receptor, ERD2, regulates intracellular traffic by recruiting a GTPase-activating protein for ARF1.

The small GTPase ADP-ribosylation factor 1 (ARF1) is a key regulator of intracellular membrane traffic. Regulators of ARF1, its GTPase-activating protein (GAP) and its guanine nucleotide exchange factor have been identified recently. However, it remains uncertain whether these regulators drive the GTPase cycle of ARF1 autonomously or whether their activities can be regulated by other proteins. Here, we demonstrate that the intracellular KDEL receptor, ERD2, self-oligomerizes and interacts with ARF1 GAP, and thereby regulates the recruitment of cytosolic ARF1 GAP to membranes. Because ERD2 overexpression enhances the recruitment of GAP to membranes and results in a phenotype that reflects ARF1 inactivation, our findings suggest that ERD2 regulates ARF1 GAP, and thus regulates ARF1-mediated transport.

Overexpression of wild-type and mutant ARF1 and ARF6: distinct perturbations of nonoverlapping membrane compartments.

The ARF GTP binding proteins are believed to function as regulators of membrane traffic in the secretory pathway. While the ARF1 protein has been shown in vitro to mediate the membrane interaction of the cytosolic coat proteins coatomer (COP1) and gamma-adaptin with the Golgi complex, the functions of the other ARF proteins have not been defined. Here, we show by transient transfection with epitope-tagged ARFs, that whereas ARF1 is localized to the Golgi complex and can be shown to affect predictably the assembly of COP1 and gamma-adaptin with Golgi membranes in cells, ARF6 is localized to the endosomal/plasma membrane system and has no effect on these Golgi-associated coat proteins. By immuno-electron microscopy, the wild-type ARF6 protein is observed along the plasma membrane and associated with endosomes, and overexpression of ARF6 does not appear to alter the morphology of the peripheral membrane system. In contrast, overexpression of ARF6 mutants predicted either to hydrolyze or bind GTP poorly shifts the distribution of ARF6 and affects the structure of the endocytic pathway. The GTP hydrolysis-defective mutant is localized to the plasma membrane and its overexpression results in a profound induction of extensive plasma membrane vaginations and a depletion of endosomes. Conversely, the GTP binding-defective ARF6 mutant is present exclusively in endosomal structures, and its overexpression results in a massive accumulation of coated endocytic structures.

An activating mutation in ARF1 stabilizes coatomer binding to Golgi membranes.

The Ras-related protein ADP-ribosylation factor 1 (ARF1) is a low molecular weight GTP binding protein, which in its GTP state supports the binding of coatomer, a cytosolic coat protein complex, to Golgi membranes. To create an "active" ARF, we constructed a point mutation in ARF1, Q71I, which was predicted to slow the rate of GTP hydrolysis. We demonstrate that Q71I, in contrast to wild type ARF1, exhibits a 2-3-fold increase in the half-life of ARF-GTP and is able to promote stable coatomer binding to Golgi membranes in the presence of GTP in vitro. Additionally, Q71I is able to support the binding of a significant amount of coatomer to membranes in the absence of added nucleotides, effectively bypassing the brefeldin A (BFA)-sensitive exchange activity. Furthermore, transfection of cells with Q71I, but not ARF1, renders the Golgi association of coatomer resistant to the effects of BFA in vivo. These observations provide compelling evidence that ARF1 is a necessary GTP binding protein that regulates the reversible binding of coat proteins to Golgi membranes and that the effects of BFA on this process in living cells must be a consequence of BFA's inhibition of guanine nucleotide exchange onto ARF1.

A brefeldin A-like phenotype is induced by the overexpression of a human ERD-2-like protein, ELP-1.

Brefeldin A (BFA) is a unique drug affecting the molecular mechanisms that regulate membrane traffic and organelle structure. BFA's ability to alter retrograde traffic from the Golgi to the endoplasmic reticulum (ER) led us to ask whether the ERD-2 retrieval receptor, proposed to return escaped ER resident proteins from the Golgi, might either interfere with or mimic the effects of the drug. When either human ERD-2 or a novel human homolog (referred to as ELP-1) is overexpressed in a variety of cell types, the effects are phenotypically indistinguishable from the addition of BFA. These include the redistribution of the Golgi coat protein, beta-COP, to the cytosol, the loss of the Golgi apparatus as a distinct organelle, the mixing of this organelle with the ER, the addition of complex oligosaccharides to resident ER glycoproteins, and the block of anterograde traffic. Thus, these receptors may provide signals that regulate retrograde traffic between the Golgi and the ER.

Changes in the methylation pattern of the TCR zeta-chain gene correlate with its expression in T cells and developing thymocytes.

The tight regulation of T-cell gene expression during thymic ontogeny is essential to the development of a normal immune system. One set of developmentally regulated genes encodes the multicomponent T-cell antigen receptor (TCR). The zeta chain, a component of the TCR, has been shown to play important roles in signal transduction from antigen binding to T-cell activation and in transport of the TCR complex to the cell surface. In this study, we examine the regulation of zeta gene expression in murine T-cell hybridomas. In these cells, zeta expression is correlated with complex, but predictable, changes in the pattern of cytosine methylation of its gene. Some of these structural changes are identical to those observed in murine fetal thymocytes and correlate with the rapid alteration of zeta message seen in the thymus between days 15 and 18 of gestation.

A recycling pathway between the endoplasmic reticulum and the Golgi apparatus for retention of unassembled MHC class I molecules.

Assembly of class I major histocompatibility complex (MHC) molecules involves the interaction of two distinct polypeptides (the heavy and light chains) with peptide antigen. Cell lines synthesizing both chains but expressing low levels of MHC class I molecules on their surface as a result of a failure in assembly and transport have been identified. We now report that although the apparent steady-state distribution in these cells of class I molecules is in the endoplasmic reticulum (ER), the molecules in fact are recycled between the ER and Golgi, rather than retained in the ER. This explains the failure of class I molecules to negotiate the secretory pathway. Class I molecules do not seem to be modified by Golgi enzymes, suggesting that the proteins do not reach the Golgi apparatus during recycling. But morphological and subcellular fractionation evidence indicates that they pass through the cis Golgi or a Golgi-associated organelle, which we postulate to be the recycling organelle. This compartment, which we call the 'cis-Golgi network', would thereby be a sorting organelle that selects proteins for return to the ER.

The isolation and characterization of the murine T cell antigen receptor zeta chain gene.

The T cell antigen receptor (TCR) is a multisubunit complex which has a dual function of antigen recognition and signal transduction. One of its invariant subunits, the zeta chain, has been shown to have a significant role in the expression and function of the TCR on the cell surface. The mouse and human zeta cDNAs share significant homologies to each other but are distinct from all of the previously characterized TCR components. We now report the isolation and structural analysis of the complete murine zeta gene. This gene spans at least 31 kilobases and divides into eight exons. The first exon, which is located at least 20 kilobases upstream from the second exon, codes for the 5'-untranslated region and most of the signal peptide. The second exon codes for the remainder of the signal peptide, the extracellular domain, the transmembrane domain, and the first three amino acids of the intracytoplasmic domain. Exons 3-7 encode the majority of the intracytoplasmic domain. The eight exon encodes the carboxyl-terminal 21 amino acids and the 3'-untranslated region. Four groups of mRNA initiation sites have been identified at approximately 140 base pairs upstream to the AUG codon. No TATA-like box has been detected. The gene is localized to the distal part of chromosome 1 in a linkage group highly conserved between man and mouse.

Pyomyositis and polyarticular septic arthritis from Hemophilus influenzae in a nonimmunocompromised adult.

We describe a healthy woman in whom pyomyositis of the left buttock, polyarticular septic arthritis, and meningitis due to Hemophilus influenzae type B developed after pneumonia. Systemic antibiotic therapy and local drainage provided a good result. This is the first case of pyomyositis and the 30th case of septic arthritis from Hemophilus influenzae described in an adult.