Cell-free reconstitution of transport from the trans-golgi network to the late endosome/prevacuolar compartment

Vesicle-mediated transport between the trans-Golgi network (TGN) and the late endosome/prevacuolar compartment (PVC) is an essential step in lysosomal/vacuolar biogenesis. In addition, localization of integral membrane proteins to the TGN requires continual cycles of vesicular transport between the TGN and endosomal compartments. Genetic and biochemical analyses in yeast have identified a variety of proteins required for TGN-to-PVC transport. However, the precise mechanisms of vesicle formation, transport, and fusion have not been fully elucidated. To study the steps of TGN-to-PVC transport in mechanistic detail, we have developed a cell-free assay to monitor delivery of the processing protease Kex2p from the TGN to PVC compartments containing a Kex2p substrate. Transport is time-, temperature-, and ATP-dependent and requires the t-SNARE Pep12p. Moreover, cell-free delivery of Kex2p to the PVC results in the co-integration of Kex2p into PVC membranes containing the Kex2p substrate as determined by co-immunoisolation of Kex2p and the substrate using antibody against the Kex2p cytosolic tail. This work represents the first cell-free reconstitution and biochemical analysis of the essential vacuolar/lysosomal sorting step TGN to late endosome transport.     

Vps10p transport from the trans-Golgi network to the endosome is mediated by clathrin-coated vesicles

A native immunoisolation procedure has been used to investigate the role of clathrin-coated vesicles (CCVs) in the transport of vacuolar proteins between the trans-Golgi network (TGN) and the prevacuolar/endosome compartments in the yeast Saccharomyces cerevisiae. We find that Apl2p, one large subunit of the adaptor protein-1 complex, and Vps10p, the carboxypeptidase Y vacuolar protein receptor, are associated with clathrin molecules. Vps10p packaging in CCVs is reduced in pep12 Delta and vps34 Delta, two mutants that block Vps10p transport from the TGN to the endosome. However, Vps10p sorting is independent of Apl2p. Interestingly, a Vps10C(t) Delta p mutant lacking its C-terminal cytoplasmic domain, the portion of the receptor responsible for carboxypeptidase Y sorting, is also coimmunoprecipitated with clathrin. Our results suggest that CCVs mediate Vps10p transport from the TGN to the endosome independent of direct interactions between Vps10p and clathrin coats. The Vps10p C-terminal domain appears to play a principal role in retrieval of Vps10p from the prevacuolar compartment rather than in sorting from the TGN.     

Topological mechanisms involved in the formation of clathrin-coated vesicles.

By examining the basic characteristics of clathrin lattices, we discover that simple topological rules impose strict constraints on clathrin lattice transformations. These constraints require that internal bond rearrangements take place in conjunction with the addition or removal of pairs of clathrin triskelions within the interior of existing clathrin lattice patches. Similar constraints also are relevant to coated-vesicle shape changes and their budding-off from pit lattices. Via specific illustrations, successive vesicles with hexagonal-barrel and other coats are shown to grow out from the interior of a initially flat clathrin-coated pit so long as free triskelions are available from cytoplasm. Concomitantly, we present mathematical derivations of several simple and useful topological equations. These equations govern the numbers of nonhexagonal clathrin lattice facets and their variations during internal shape transformations and justify the proposed mechanisms of triskelion pair insertion and removal.     

Rhythmic cycle of clathrin-coated pit formation at the trans-golgi network in human MDA-MB-435 cells

We studied the in vivo dynamics of enhanced green fluorescent protein-tagged clathrin light chain a (GFP-CLCa) at the trans-Golgi network (TGN) in MDA-MB-435 cells. The intensity of fluorescence signals of GFP-CLCa periodically increased and decreased at the TGN approximately every 100 s. This suggests that the formation of clathrin-coated pits occurs synchronously and periodically at the TGN.     

Dynamin II is involved in endocytosis but not in the formation of transport vesicles from the trans-Golgi network

Dynamins are a family of approximately 100-kDa GTPases that are thought to play a pivotal role in the formation of endocytic coated vesicles. There are three dynamin genes in mammals: dynamin I is neuron-specific, dynamin II shows ubiquitous expression, and dynamin III is expressed in testis, brain, and lung. However, most studies on the functions of dynamins to date have been restricted to dynamin I. In the present study, we show that, like dynamin I, dynamin II is involved in receptor-mediated endocytosis. While this study was in progress, Jones et al. [Jones, S.M., Howell, K.E., Henley, J.R., Cao, H., and McNiven, M.A. (1998) Science 279, 573-577] reported that dynamin II is localized in the trans-Golgi network (TGN) and involved in the formation of constitutive transport vesicles and clathrin-coated vesicles from this compartment. However, immunofluorescence analyses and experiments using cells transfected with dominant-negative dynamin II failed to show any evidence for localization of dynamin II in the TGN or for its involvement in vesicle formation from this compartment. Our data thus indicate that dynamin II is involved in endocytosis but not in the formation of transport vesicles from the TGN.     

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.     

Autoantigen Golgin-97, an effector of Arl1 GTPase, participates in traffic from the endosome to the trans-golgi network

The precise cellular function of Arl1 and its effectors, the GRIP domain Golgins, is not resolved, despite our recent understanding that Arl1 regulates the membrane recruitment of these Golgins. In this report, we describe our functional study of Golgin-97. Using a Shiga toxin B fragment (STxB)-based in vitro transport assay, we demonstrated that Golgin-97 plays a role in transport from the endosome to the trans-Golgi network (TGN). The recombinant GRIP domain of Golgin-97 as well as antibodies against Golgin-97 inhibited the transport of STxB in vitro. Membrane-associated Golgin-97, but not its cytosolic pool, was required in the in vitro transport assay. The kinetic characterization of inhibition by anti-Golgin-97 antibody in comparison with anti-Syntaxin 16 antibody established that Golgin-97 acts before Syntaxin 16 in endosome-to-TGN transport. Knock down of Golgin-97 or Arl1 by their respective small interference RNAs (siRNAs) also significantly inhibited the transport of STxB to the Golgi in vivo. In siRNA-treated cells with reduced levels of Arl1, internalized STxB was instead distributed peripherally. Microinjection of Golgin-97 antibody led to the fragmentation of Golgi apparatus and the arrested transport to the Golgi of internalized Cholera toxin B fragment. We suggest that Golgin-97 may function as a tethering molecule in endosome-to-TGN retrograde traffic.     

Drs2p-dependent formation of exocytic clathrin-coated vesicles in vivo

The small GTP binding protein ARF has been implicated in budding clathrin-coated vesicles (CCVs) from Golgi and endosomal membranes. An arf1 synthetic lethal screen identified DRS2/SWA3 along with a clathrin heavy-chain conditional allele (chc1-5/swa5-1) and SWA2, encoding the yeast auxilin-like protein involved in uncoating CCVs. Drs2p/Swa3p is a P-type ATPase and a potential aminophospholipid translocase that localizes to the trans-Golgi network (TGN) in yeast. Genetic and phenotypic analyses of drs2Delta mutants suggested that Drs2p was required for clathrin function. To address a potential role for Drs2p in CCV formation from the TGN in vivo, we have performed epistasis analyses between drs2 and mutations that cause accumulation of distinct populations of post-Golgi vesicles. We find that Drs2p is required to form a specific class of secretory vesicles that accumulate when the actin cytoskeleton is disrupted. Accumulation of these vesicles also requires clathrin and is perturbed by mutation of AP-1, but not AP-2, AP-3, or GGA adaptins. Most of the accumulated vesicles are uncoated; however, clathrin coats can be partially stabilized on these vesicles by deletion of SWA2. These data provide the first in vivo evidence for an integral membrane protein requirement in forming CCVs.     

Purification and characterization of clathrin-coated vesicles from Chlamydomonas

Clathrin-coated vesicles, identified by negative staining with uranyl acetate, were purified from Chlamydomonas reinhardtii. Isolated coated vesicles had diameters ranging from 70 to 140 nm (mean diameter +/- SD of 95 +/- 17 nm, n = 300). These vesicles were markedly heterogeneous in both density and surface charge, as indicated by equilibrium density sedimentation and elution from anion-exchange columns. Highly-purified coated-vesicle fractions contained 2 major polypeptides, identified as the clathrin heavy chain (185 kDa) and the clathrin light chain (40 kDa). Chlamydomonas clathrin heavy chain cross-reacts weakly with an antibody against bovine brain clathrin heavy chain. Coat stability in several buffers was compared to that of bovine brain coated vesicles. Stability was similar, except for a greater stability of Chlamydomonas coated vesicles in 0.5 M Tris at pH 7.0.     

Actin dynamics coupled to clathrin-coated vesicle formation at the trans-Golgi network

In diverse species, actin assembly facilitates clathrin-coated vesicle (CCV) formation during endocytosis. This role might be an adaptation specific to the unique environment at the cell cortex, or it might be fundamental, facilitating CCV formation on different membranes. Proteins of the Sla2p/Hip1R family bind to actin and clathrin at endocytic sites in yeast and mammals. We hypothesized that Hip1R might also coordinate actin assembly with clathrin budding at the trans-Golgi network (TGN). Using deconvolution and time-lapse microscopy, we showed that Hip1R is present on CCVs emerging from the TGN. These vesicles contain the mannose 6-phosphate receptor involved in targeting proteins to the lysosome, and the actin nucleating Arp2/3 complex. Silencing of Hip1R expression by RNAi resulted in disruption of Golgi organization and accumulation of F-actin structures associated with CCVs on the TGN. Hip1R silencing and actin poisons slowed cathepsin D exit from the TGN. These studies establish roles for Hip1R and actin in CCV budding from the TGN for lysosome biogenesis.     

Rab11 regulates the compartmentalization of early endosomes required for efficient transport from early endosomes to the trans-golgi network

Several GTPases of the Rab family, known to be regulators of membrane traffic between organelles, have been described and localized to various intracellular compartments. Rab11 has previously been reported to be associated with the pericentriolar recycling compartment, post-Golgi vesicles, and the trans-Golgi network (TGN). We compared the effect of overexpression of wild-type and mutant forms of Rab11 on the different intracellular transport steps in the endocytic/degradative and the biosynthetic/exocytic pathways in HeLa cells. We also studied transport from endosomes to the Golgi apparatus using the Shiga toxin B subunit (STxB) and TGN38 as reporter molecules. Overexpression of both Rab11 wild-type (Rab11wt) and mutants altered the localization of the transferrrin receptor (TfR), internalized Tf, the STxB, and TGN38. In cells overexpressing Rab11wt and in a GTPase-deficient Rab11 mutant (Rab11Q70L), these proteins were found in vesicles showing characteristics of sorting endosomes lacking cellubrevin (Cb). In contrast, they were redistributed into an extended tubular network, together with Cb, in cells overexpressing a dominant negative mutant of Rab11 (Rab11S25N). This tubularized compartment was not accessible to Tf internalized at temperatures <20 degrees C, suggesting that it is of recycling endosomal origin. Overexpression of Rab11wt, Rab11Q70L, and Rab11S25N also inhibited STxB and TGN38 transport from endosomes to the TGN. These results suggest that Rab11 influences endosome to TGN trafficking primarily by regulating membrane distribution inside the early endosomal pathway.     

Hepatitis C virus entry requires a critical postinternalization step and delivery to early endosomes via clathrin-coated vesicles

Hepatitis C virus (HCV) is a major human pathogen associated with life-threatening liver disease. Entry into hepatocytes requires CD81 and a putative second receptor. In this study, we elucidated the postreceptor attachment stages of HCV entry using HCV pseudoparticles (HCVpp) as a model system. By means of dominant-negative mutants and short interfering RNAs of various cellular proteins, we showed that HCVpp enter via clathrin-coated vesicles and require delivery to early but not to late endosomes. However, the kinetics of HCV envelope glycoprotein-mediated fusion are delayed compared to those of other viruses that enter in early endosomes. Entry of HCVpp can be efficiently blocked by bafilomycin A1, which neutralizes the pH in early endosomes and impairs progression of endocytosis beyond this stage. However, low-pH exposure of bafilomycin A1-treated target cells does not induce entry of HCVpp at the plasma membrane or in the early stages of endocytosis. These observations indicate that, subsequent to internalization, HCVpp entry necessitates additional, low-pH-dependent interactions, modifications, or trafficking, and that these events are irreversibly disrupted by bafilomycin A1 treatment.     

Sizes and mass distributions of clathrin-coated vesicles from bovine brain

Clathrin-coated vesicles obtained from bovine brain have been studied by ultracentrifugation and dynamic light scattering techniques to provide information on their sedimentation and mass distributions and their average diffusion coefficients. "Uncoated" vesicles, obtained by removing the protein coat from coated vesicles, have been similarly characterized. For typical preparations, maximal values of approximately 210 and 95 S are observed for the sedimentation coefficients of coated and uncoated vesicles, respectively. Corresponding values for the average molecular weights, determined from values of average sedimentation and diffusion coefficients, are 49 X 10(6) and 13 X 10(6); values obtained by equilibrium sedimentation are 37.2 X 10(6) and 10.6 X 10(6). In order to obtain these results, some minor modifications of sedimentation and light-scattering techniques have been devised which may have application to other studies of size distributions of large particles.     

A selective transport route from Golgi to late endosomes that requires the yeast GGA proteins

Pep12p is a yeast syntaxin located primarily in late endosomes. Using mutagenesis of a green fluorescent protein chimera we have identified a sorting signal FSDSPEF, which is required for transport of Pep12p from the exocytic pathway to late endosomes, from which it can, when overexpressed, reach the vacuole. When this signal is mutated, Pep12p instead passes to early endosomes, a step that is determined by its transmembrane domain. Surprisingly, Pep12p is then specifically retained in early endosomes and does not go on to late endosomes.By testing appropriate chimeras in mutant strains, we found that FSDSPEF-dependent sorting was abolished in strains lacking Gga1p and Gga2p, Golgi-associated coat proteins with homology to gamma adaptin. In the gga1 gga2 double mutant endogenous Pep12p cofractionated with the early endosome marker Tlg1p, and recycling of Snc1p through early endosomes was defective. Pep12p sorting was also defective in cells lacking the clathrin heavy or light chain. We suggest that specific and direct delivery of proteins to early and late endosomes is required to maintain the functional heterogeneity of the endocytic pathway and that the GGA proteins, probably in association with clathrin, help create vesicles destined for late endosomes.     

VAN3 ARF-GAP-mediated vesicle transport is involved in leaf vascular network formation

Within the leaf of an angiosperm, the vascular system is constructed in a complex network pattern called venation. The formation of this vein pattern has been widely studied as a paradigm of tissue pattern formation in plants. To elucidate the molecular mechanism controlling the vein patterning process, we previously isolated Arabidopsis mutants van1 to van7, which show a discontinuous vein pattern. Here we report the phenotypic analysis of the van3 mutant in relation to auxin signaling and polar transport, and the molecular characterization of the VAN3 gene and protein. Double mutant analyses with pin1, emb30-7/gn and mp, and physiological analyses using the auxin-inducible marker DR5::GUS and an auxin transport inhibitor indicated that VAN3 may be involved in auxin signal transduction, but not in polar auxin transport. Positional cloning identified VAN3 as a gene that encodes an adenosine diphosphate (ADP)-ribosylation factor-guanosine triphosphatase (GTPase) activating protein (ARF-GAP). It resembles animal ACAPs and contains four domains: a BAR (BIN/amphiphysin/RVS) domain, a pleckstrin homology (PH) domain, an ARF-GAP domain and an ankyrin (ANK)-repeat domain. Recombinant VAN3 protein showed GTPase-activating activity and a specific affinity for phosphatidylinositols. This protein can self-associate through the N-terminal BAR domain in the yeast two-hybrid system. Subcellular localization analysis by double staining for Venus-tagged VAN3 and several green-fluorescent-protein-tagged intracellular markers indicated that VAN3 is located in a subpopulation of the trans-Golgi network (TGN). Our results indicate that the expression of this gene is induced by auxin and positively regulated by VAN3 itself, and that a specific ACAP type of ARF-GAP functions in vein pattern formation by regulating auxin signaling via a TGN-mediated vesicle transport system.     

Selective membrane recruitment of EEA1 suggests a role in directional transport of clathrin-coated vesicles to early endosomes

The molecular mechanisms ensuring directionality of endocytic membrane trafficking between transport vesicles and target organelles still remain poorly characterized. We have been investigating the function of the small GTPase Rab5 in early endocytic transport. In vitro studies have demonstrated a role of Rab5 in two membrane fusion events: the heterotypic fusion between plasma membrane-derived clathrin-coated vesicles (CCVs) and early endosomes and in the homotypic fusion between early endosomes. Several Rab5 effectors are required in homotypic endosome fusion, including EEA1, which mediates endosome membrane docking, as well as Rabaptin-5 x Rabex-5 complex and phosphatidylinositol 3-kinase hVPS34. In this study we have examined the localization and function of Rab5 and its effectors in heterotypic fusion in vitro. We report that the presence of active Rab5 is necessary on both CCVs and early endosomes for a heterotypic fusion event to occur. This process requires EEA1 in addition to the Rabaptin-5 complex. However, whereas Rab5 and Rabaptin-5 are symmetrically distributed between CCVs and early endosomes, EEA1 is recruited selectively onto the membrane of early endosomes. Our results suggest that EEA1 is a tethering molecule that provides directionality to vesicular transport from the plasma membrane to the early endosomes.     

Mutants in trs120 disrupt traffic from the early endosome to the late Golgi

Transport protein particle (TRAPP), a large complex that mediates membrane traffic, is found in two forms (TRAPPI and -II). Both complexes share seven subunits, whereas three subunits (Trs130p, -120p, and -65p) are specific to TRAPPII. Previous studies have shown that mutations in the TRAPPII-specific gene trs130 block traffic through or from the Golgi. Surprisingly, we report that mutations in trs120 do not block general secretion. Instead, trs120 mutants accumulate aberrant membrane structures that resemble Berkeley bodies and disrupt the traffic of proteins that recycle through the early endosome. Mutants defective in recycling also display a defect in the localization of coat protein I (COPI) subunits, implying that Trs120p may participate in a COPI-dependent trafficking step on the early endosomal pathway. Furthermore, we demonstrate that Trs120p largely colocalizes with the late Golgi marker Sec7p. Our findings imply that Trs120p is required for vesicle traffic from the early endosome to the late Golgi.     

Profilin I attached to the Golgi is required for the formation of constitutive transport vesicles at the trans-Golgi network

Profilin I was identified, by mass spectrometric sequencing and immunoblotting, as a component of purified Golgi cisternae from HepG2 cells. Binding to the Golgi was verified by indirect immunofluorescence in MT-1 cells showing that a fraction of profilin I colocalizes with TGN38, a marker of the trans-Golgi network (TGN). Studying the formation of constitutive exocytic vesicles at the TGN in a cell-free system demonstrated that cytosolic profilin I has no effect, while incubation of Golgi cisternae with a profilin I-specific antibody reduced vesicle formation by about 50%. Notably, the antibody displaces a fraction of the Golgi-bound dynamin II indicating that profilin I may indirectly promote vesicle formation by supporting the binding of dynamin II to the Golgi membrane. The impact of dynamin II on vesicle formation is demonstrated by incubating the Golgi with the proline-rich domain of dynamin II which concomitantly displaces dynamin II and inhibits vesicle formation. The data provide evidence that profilin I attaches to the Golgi apparatus and is required for the formation of constitutive transport vesicles.     

Immunolocalization of the vacuolar-type (H+)-ATPase from clathrin-coated vesicles.

Proton-translocating ATPases of the vacuolar class (V-ATPases) are found in a variety of animal cell compartments that participate in vesicular membrane transport, including clathrin-coated vesicles, endosomes, the Golgi apparatus, and lysosomes. The exact structural relationship that exists among the V-ATPases of these intracellular compartments is not currently known. In the present study, we have localized the V-ATPase by light and electron microscopy, using monoclonal antibodies that recognize the V-ATPase present in clathrin-coated vesicles. Localization using light microscopy and fluorescently labeled antibodies reveals that the V-ATPase is concentrated in the juxtanuclear region, where extensive colocalization with the Golgi marker wheat germ agglutinin is observed. The V-ATPase is also present in approximately 60% of endosomes and lysosomes fluorescently labeled using alpha 2-macroglobulin as a marker for the receptor-mediated endocytic pathway. Localization using transmission electron microscopy and colloidal gold-labeled antibodies reveals that the V-ATPase is present at and near the plasma membrane, alone or in association with clathrin. These results provide evidence that the V-ATPases of plasma membrane, endosomes, lysosomes, and the Golgi apparatus are immunologically related to the V-ATPase of clathrin-coated vesicles.