post-Golgi Carriers Research Articles

A 14-3-3γ dimer-based scaffold bridges CtBP1-S/BARS to PI(4)KIIIβ to regulate post-Golgi carrier formation

Large pleiomorphic carriers leave the Golgi complex for the plasma membrane by en bloc extrusion of specialized tubular domains, which then undergo fission. Several components of the underlying molecular machinery have been identified, including those involved in the budding/initiation of tubular carrier precursors (for example, the phosphoinositide kinase PI(4)KIIIβ, the GTPase ARF, and FAPP2), and in the fission of these precursors (for example, PKD, CtBP1-S/BARS). However, how these proteins interact to bring about carrier formation is poorly understood. Here, we describe a protein complex that mediates carrier formation and contains budding and fission molecules, as well as other molecules, such as the adaptor protein 14-3-3γ. Specifically, we show that 14-3-3γ dimers bridge CtBP1-S/BARS with PI(4)KIIIβ, and that the resulting complex is stabilized by phosphorylation by PKD and PAK. Disrupting the association of these proteins inhibits the fission of elongating carrier precursors, indicating that this complex couples the carrier budding and fission processes.

Myosin 1b promotes the formation of post-Golgi carriers by regulating actin assembly and membrane remodelling at the trans-Golgi network

The function of organelles is intimately associated with rapid changes in membrane shape. By exerting force on membranes, the cytoskeleton and its associated motors have an important role in membrane remodelling. Actin and myosin 1 have been implicated in the invagination of the plasma membrane during endocytosis. However, whether myosin 1 and actin contribute to the membrane deformation that gives rise to the formation of post-Golgi carriers is unknown. Here we report that myosin 1b regulates the actin-dependent post-Golgi traffic of cargo, generates force that controls the assembly of F-actin foci and, together with the actin cytoskeleton, promotes the formation of tubules at the TGN. Our results provide evidence that actin and myosin 1 regulate organelle shape and uncover an important function for myosin 1b in the initiation of post-Golgi carrier formation by regulating actin assembly and remodelling TGN membranes.

Mobile ER-to-Golgi but not post-Golgi membrane transport carriers disappear during the terminal myogenic differentiation

The organelles of the exocytic pathway undergo a profound reorganization during the myogenic differentiation. Here, we have investigated the dynamics of the membrane trafficking at various stages of the differentiation process by using the green fluorescent protein-tagged, temperature-sensitive vesicular stomatitis virus G protein (tsG-GFP) as a marker. At the restrictive temperature of 39°C, the tsG-GFP located to the endoplasmic reticulum (ER) at each stage of differentiation. Mobile membrane containers moving from the ER to the Golgi elements were seen in myoblasts and myotubes upon shifting the temperature to 20°C. In adult myofibers, in contrast, such containers were not seen although the tsG-GFP rapidly shifted from the ER to the Golgi elements. The mobility of tsG-GFP in the myofiber ER was restricted, suggesting localization in an ER sub-compartment. Contrasting with the ER-to-Golgi trafficking, transport from the Golgi elements to the plasma membrane involved mobile transport containers in all differentiation stages. These findings indicate that ER-to-Golgi trafficking in adult skeletal myofibers does not involve long-distance moving membrane carriers as occurs in other mammalian cell types.

Enhancement of Procollagen Biosynthesis by p180 through Augmented Ribosome Association on the Endoplasmic Reticulum in Response to Stimulated Secretion

A coiled-coil microtubule-bundling protein, p180, was originally reported as a ribosome-binding protein on the rough endoplasmic reticulum (ER) and is highly expressed in secretory tissues. Recently, we reported a novel role for p180 in the trans-Golgi network (TGN) expansion following stimulated collagen secretion. Here, we show that p180 plays a key role in procollagen biosynthesis and secretion in diploid fibroblasts. Depletion of p180 caused marked reductions of secreted collagens without significant loss of the ER membrane or mRNA. Metabolic labeling experiments revealed that the procollagen biosynthetic activity was markedly affected following p180 depletion. Moreover, loss of p180 perturbs ascorbate-stimulated de novo biosynthesis mainly in the membrane fraction with a preferential secretion defect of large proteins. At the EM level, one of the most prominent morphological features of p180-depleted cells was insufficient ribosome association on the ER membranes. In contrast, the ER of control cells was studded with numerous ribosomes, which were further enhanced by ascorbate. Similarly biochemical analysis confirmed that levels of membrane-bound ribosomes were altered in a p180-dependent manner. Taken together, our data suggest that p180 plays crucial roles in enhancing collagen biosynthesis at the entry site of the secretory compartments by a novel mechanism that mainly involves facilitating ribosome association on the ER.

Exocytosis of Post-Golgi Vesicles Is Regulated by Components of the Endocytic Machinery

Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

Calsyntenins Mediate TGN Exit of APP in a Kinesin‐1‐Dependent Manner

Kinesin motors are required for the export of membranous cargo from the trans-Golgi network (TGN), yet information about how kinesins are recruited to forming transport intermediates is sparse. Here we show that the Kinesin-1 docking protein calsyntenin-1 localizes to the TGN in vivo and directly and specifically recruits Kinesin-1 to Golgi/TGN membranes as well as to dynamic post-Golgi carriers. Overexpression of various calsyntenin chimeras and kinesin light chain 1 (KLC1) at high levels caused the formation of aberrant membrane stacks at the endoplasmic reticulum (ER) or the Golgi, disrupted overall Golgi structure and blocked exit of calsyntenin from the TGN. Intriguingly, this blockade of calsyntenin exit strongly and selectively impeded TGN exit of amyloid precursor protein (APP). Using live cell microscopy we found that calsyntenins exit the TGN in Kinesin-1-decorated tubular structures which may serve as carriers for calsyntenin-1-mediated post-TGN transport of APP. Abrogation of this pathway via virus-mediated knockdown of calsyntenin-1 expression in primary cultured neurons caused a marked elevation of APP C-terminal fragments. Together, these results indicate a role for calsyntenin-1 in Kinesin-1-dependent TGN exit and post-Golgi transport of APP-containing organelles and further suggest that distinct intracellular routes may exhibit different capacities for proteolytic processing of APP.

CtBP1/BARS is an activator of phospholipase D1 necessary for agonist-induced macropinocytosis

Vesicular trafficking such as macropinocytosis is a dynamic process that requires coordinated interactions between specialized proteins and lipids. A recent report suggests the involvement of CtBP1/BARS in epidermal growth factor (EGF)-induced macropinocytosis. Detailed mechanisms as to how lipid remodelling is regulated during macropinocytosis are still undefined. Here, we show that CtBP1/BARS is a physiological activator of PLD1 required in agonist-induced macropinocytosis. EGF-induced macropinocytosis was specifically blocked by 1-butanol but not by 2-butanol. In addition, stimulation of cells by serum or EGF resulted in the association of CtBP1/BARS with PLD1. Finally, CtBP1/BARS activated PLD1 in a synergistic manner with other PLD activators, including ADP-ribosylation factors as demonstrated by in vitro and intact cell systems. The present results shed light on the molecular basis of how the ‘fission protein' CtBP1/BARS controls vesicular trafficking events including macropinocytosis.

Morphogenesis of post-Golgi transport carriers

The trans-Golgi network (TGN) is one of the main, if not the main, sorting stations in the process of intracellular protein trafficking. It is therefore of central importance to understand how the key players in the TGN-based sorting and delivery process, the post-Golgi carriers (PGCs), form and function. Over the last few years, modern morphological approaches have generated new insights into the questions of PGC biogenesis, structure and dynamics. Here, we present a view by which the “lifecycle” of a PGC consists of several distinct stages: the formation of TGN tubular export domains (where different cargoes are segregated from each other and from the Golgi enzymes); the docking of these tubular domains onto molecular motors and their extrusion towards the cell periphery along microtubules; the fission of the forming PGC from the donor membrane; and the delivery of the newly formed PGC to its specific acceptor organelle. It is now important to add the many molecular machineries that have been described as operating at the TGN to this “morphofunctional map” of the TGN export process.

Ypt32p and Mlc1p bind within the vesicle binding region of the class V myosin Myo2p globular tail domain

Myosin V is an actin-based motor essential for a variety of cellular processes including skin pigmentation, cell separation and synaptic transmission. Myosin V transports organelles, vesicles and mRNA by binding, directly or indirectly, to cargo-bound receptors via its C-terminal globular tail domain (GTD). We have used the budding yeast myosin V Myo2p to shed light on the mechanism of how Myo2p interacts with post-Golgi carriers. We show that the Rab/Ypt protein Ypt32p, which associates with membranes of the trans-Golgi network, secretory vesicles and endosomes and is related to the mammalian Rab11, interacts with the Myo2p GTD within a region previously identified as the 'vesicle binding region'. Furthermore, we show that the essential myosin light chain 1 (Mlc1p), required for vesicle delivery at the mother-bud neck during cytokinesis, binds to the Myo2p GTD in a region overlapping that of Ypt32p. Our data are consistent with a role of Ypt32p and Mlc1p in regulating the interaction of post-Golgi carriers with Myo2p subdomain II.

A role for BARS at the fission step of COPI vesicle formation from Golgi membrane

The core complex of Coat Protein I (COPI), known as coatomer, is sufficient to induce coated vesicular-like structures from liposomal membrane. In the context of biological Golgi membrane, both palmitoyl-coenzyme A (p-coA) and ARFGAP1, a GTPase-activating protein (GAP) for ADP-Ribosylation Factor 1, also participate in vesicle formation, but how their roles may be linked remains unknown. Moreover, whether COPI vesicle formation from Golgi membrane requires additional factors also remains unclear. We now show that Brefeldin-A ADP-Ribosylated Substrate (BARS) plays a critical role in the fission step of COPI vesicle formation from Golgi membrane. This role of BARS requires its interaction with ARFGAP1, which is in turn regulated oppositely by p-coA and nicotinamide adenine dinucleotide, which act as cofactors of BARS. Our findings not only identify a new factor needed for COPI vesicle formation from Golgi membrane but also reveal a surprising mechanism by which the roles of p-coA and GAP are linked in this process.

Mechanisms of Cx43 and Cx26 transport to the plasma membrane and gap junction regeneration

Previous reports have suggested that Cx26 exhibits unique intracellular transport pathways en route to the cell surface compared with other members of the connexin family. To directly examine and compare nascent and steady-state delivery of Cx43 and Cx26 to the plasma membrane and gap junction biogenesis we expressed fluorescent-protein-tagged Cx43 and Cx26 in BICR-M1Rk and NRK cells. Static and time-lapse imaging revealed that both connexins were routed through the Golgi apparatus prior to being transported to the cell surface, a process inhibited in the presence of brefeldin A (BFA) or the expression of a dominant-negative form of Sar1 GTPase. During recovery from BFA, time-lapse imaging of nascent connexin Golgi-to-plasma membrane delivery revealed many dynamic post-Golgi carriers (PGCs) originating from the distal side of the Golgi apparatus consisting of heterogeneous vesicles and long, tubular-like extensions. Vesicles and tubular extensions were also observed in HBL-100 cells expressing a human, disease-linked, Golgi-localized Cx26 mutant, D66H-GFP. A diffuse cell surface rim of fluorescent-protein-tagged wild-type connexins was observed prior to the appearance of punctate gap junctions, which suggests that random fusion of PGCs occurred with the plasma membrane followed by lateral diffusion of connexins into clusters. Fluorescence recovery after photobleaching studies revealed that Cx26-YFP was more mobile within gap junction plaques compared with Cx43-GFP. Intriguingly, Cx43-GFP delivery and gap junction regeneration was inhibited by BFA and nocodazole, whereas Cx26-GFP delivery was prevented by BFA but not nocodazole. Collectively, these studies suggest that during gap junction biogenesis two phylogenetically distinct members of the connexin family, Cx43 and Cx26, share common secretory pathways, types of transport intermediates and turnover dynamics but differ in their microtubule-dependence and mobility within the plasma membrane, which might reflect differences in binding to protein scaffolds.

Galectin-4 and sulfatides in apical membrane trafficking in enterocyte-like cells

We have previously reported that 1-benzyl-2-acetamido-2-deoxy-α-d-galactopyranoside (GalNAcα-O-bn), an inhibitor of glycosylation, perturbed apical biosynthetic trafficking in polarized HT-29 cells suggesting an involvement of a lectin-based mechanism. Here, we have identified galectin-4 as one of the major components of detergent-resistant membranes (DRMs) isolated from HT-29 5M12 cells. Galectin-4 was also found in post-Golgi carrier vesicles. The functional role of galectin-4 in polarized trafficking in HT-29 5M12 cells was studied by using a retrovirus-mediated RNA interference. In galectin-4–depleted HT-29 5M12 cells apical membrane markers accumulated intracellularly. In contrast, basolateral membrane markers were not affected. Moreover, galectin-4 depletion altered the DRM association characteristics of apical proteins. Sulfatides with long chain-hydroxylated fatty acids, which were also enriched in DRMs, were identified as high-affinity ligands for galectin-4. Together, our data propose that interaction between galectin-4 and sulfatides plays a functional role in the clustering of lipid rafts for apical delivery.

Rhodopsin C terminus, the site of mutations causing retinal disease, regulates trafficking by binding to ADP-ribosylation factor 4 (ARF4)

The maintenance of photoreceptor cell polarity is compromised by the rhodopsin mutations causing the human disease autosomal dominant retinitis pigmentosa. The severe form mutations occur in the C-terminal sorting signal of rhodopsin, VXPX-COOH. Here, we report that this sorting motif binds specifically to the small GTPase ARF4, a member of the ARF family of membrane budding and protein sorting regulators. The effects of blocking ARF4 action were functionally equivalent to the effects of blocking the rhodopsin C-terminal sorting signal. ARF4 was essential for the generation of post-Golgi carriers targeted to the rod outer segments of retinal photoreceptors. Thus, the severe retinitis pigmentosa alleles that affect the rhodopsin sorting signal interfere with interactions between ARF4 and rhodopsin, leading to aberrant trafficking and initiation of retinal degeneration.

Ca 2+-independent phospholipase A2 participates in the vesicular transport of milk proteins

Changes in the lipid composition of intracellular membranes are believed to take part in the molecular processes that sustain traffic between organelles of the endocytic and exocytic transport pathways. Here, we investigated the participation of the calcium-independent phospholipase A2 in the secretory pathway of mammary epithelial cells. Treatment with bromoenol lactone, a suicide substrate which interferes with the production of lysophospholipids by the calcium-independent phospholipase A2, resulted in the reduction of milk proteins secretion. The inhibitor slowed down transport of the caseins from the endoplasmic reticulum to the Golgi apparatus and affected the distribution of p58 and p23, indicating that the optimal process of transport of these proteins between the endoplasmic reticulum, the endoplasmic reticulum/Golgi intermediate compartment and/or the cis-side of the Golgi was dependent upon the production of lysolipids. Moreover, bromoenol lactone was found to delay the rate of protein transport from the trans-Golgi network to the plasma membrane. Concomitantly, membrane-bound structures containing casein accumulated in the juxtanuclear Golgi region. We concluded from these results that efficient formation of post-Golgi carriers also requires the phospholipase activity. These data further support the participation of calcium-independent phospholipase A2 in membrane trafficking and shed a new light on the tubulo/vesicular transport of milk protein through the secretory pathway.

Different ways to reach the top of a cell. Analysis of rotavirus assembly and targeting in human intestinal cells reveals an original raft-dependent, Golgi-independent apical targeting pathway

IntroductionSince the pioneering works of David Sabatini’s group,viruses have demonstrated their high usefulness to unravelproperties of eukaryotic cells. This holds especially true forstudies on polarized protein targeting in epithelial cells inwhich influenza and vesicular stomatitis virus proteins havebeen widely used to characterize the mechanisms of proteinsorting (Rodriguez-Boulan and Sabatini, 1978). Since then,the study of polarized targeting mechanisms in epithelialcellshasremainedahighlyactivefieldofresearch.Althoughseveral signals and pathways have been described, there isuntil now no clear consensus on how molecules are sortedand transported from the Golgi apparatus to the cell surface.Simons and Ikonen (1997) suggested that specializedmembrane microdomains termed braftsQ, which are enrichedincholesteroland(glyco)sphingolipidsandformedbylateralsegregation of membrane lipids, may play a crucial role insorting mechanisms of subsets of proteins that will betargeted to the apical membrane. This braft hypothesisQ fitswith numerous observations indicating that apically targetedmolecules associate with lipid rafts in the Golgi apparatusbefore being incorporated into specific post-Golgi carriers(Simons and Ikonen, 1997). However, it has also beenobserved that raft association is not sufficient to confer astrict apical targeting, because several raft-associated pro-teins are targeted to the basolateral membrane in various celltypes (Kreitzer et al., 2003; Lipardi et al. 2000). In a recentreview,ithasalsobeensuggestedthatformationofpolymersor aggregates that are favored by the presence of membranemicrodomains may help the sorting process between apicaland basolateral proteins (Helms and Zurzolo, 2004).A few years ago, we started to study the morphogenesisand targeting of another virus, namely rotavirus, whichappeared to behave very differently as compared topreviously studied virus. As outlined below, this virus andits main structural proteins do not seem to follow a classicalexocytic route, although the virus was specifically deliveredto the apical pole of intestinal cells (Jourdan et al., 1997).We realized that most of the proteins of this virus werecytosolic proteins that have no specific signal to enter theexocytic pathway. These proteins are synthesized on freeribosomes and directly released within the cytosol. Little isknown on the mechanisms that control their sorting andtargeting. In a recent review, Walter Nickel summarized thedata obtained on what was called bthe nonclassical proteinsecretionQ, a pathway that by-passes ER and Golgi compart-ments (Nickel, 2003). Four mechanisms have been sug-gested for this atypical plasma membrane targeting: (1) a re-entry in the endosomal compartment (used for example byIL1h); (2) the use of specific transporters at the cell surface(used by FGF1 and FGF2); (3) a translocation at the

FAPPs control Golgi-to-cell-surface membrane traffic by binding to ARF and PtdIns(4)P.

The molecular mechanisms underlying the formation of carriers trafficking from the Golgi complex to the cell surface are still ill-defined; nevertheless, the involvement of a lipid-based machinery is well established. This includes phosphatidylinositol 4-phosphate (PtdIns(4)P), the precursor for phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)). In yeast, PtdIns(4)P exerts a direct role, however, its mechanism of action and its targets in mammalian cells remain uncharacterized. We have identified two effectors of PtdIns(4)P, the four-phosphate-adaptor protein 1 and 2 (FAPP1 and FAPP2). Both proteins localize to the trans-Golgi network (TGN) on nascent carriers, and interact with PtdIns(4)P and the small GTPase ADP-ribosylation factor (ARF) through their plekstrin homology (PH) domain. Displacement or knockdown of FAPPs inhibits cargo transfer to the plasma membrane. Moreover, overexpression of FAPP-PH impairs carrier fission. Therefore, FAPPs are essential components of a PtdIns(4)P- and ARF-regulated machinery that controls generation of constitutive post-Golgi carriers.

Microtubules provide directional cues for polarized axonal transport through interaction with kinesin motor head.

Post-Golgi carriers of various newly synthesized axonal membrane proteins, which possess kinesin (KIF5)-driven highly processive motility, were transported from the TGN directly to axons. We found that KIF5 has a preference to the microtubules in the initial segment of axon. Low dose paclitaxel treatment caused missorting of KIF5, as well as axonal membrane proteins to the tips of dendrites. Microtubules in the initial segment of axons showed a remarkably high affinity to EB1–YFP, which was known to bind the tips of growing microtubules. These findings revealed unique features of the microtubule cytoskeletons in the initial segment, and suggested that they provide directional information for polarized axonal transport.

GAIP participates in budding of membrane carriers at the trans-Golgi network.

Galpha interacting protein (GAIP) is a regulator of G protein signaling protein that associates dynamically with vesicles and has been implicated in membrane trafficking, although its specific role is not yet known. Using an in vitro budding assay, we show that GAIP is recruited to a specific population of trans-Golgi network-derived vesicles and that these are distinct from coatomer or clathrin-coated vesicles. A truncation mutant (NT-GAIP) encoding only the N-terminal half of GAIP is recruited to trans-Golgi network membranes during the formation of vesicle carriers. Overexpression of NT-GAIP induces the formation of long, coated tubules, which are stabilized by microtubules. Results from the budding assay and from imaging in live cells show that these tubules remain attached to the Golgi stack rather than being released as carrier vesicles. NT-GAIP expression blocks membrane budding and results in the accumulation of tubular carrier intermediates. NT-GAIP-decorated tubules are competent to load vesicular stomatitis virus protein G-green fluorescent protein as post-Golgi, exocytic cargo and in cells expressing NT-GAIP there is reduced surface delivery of vesicular stomatitis virus protein G-green fluorescent protein. We conclude that GAIP functions as an essential part of the membrane budding machinery for a subset of post-Golgi exocytic carriers derived from the trans-Golgi network.

Three-dimensional analysis of post-Golgi carrier exocytosis in epithelial cells.

Targeted delivery of proteins to distinct plasma membrane domains is critical to the development and maintenance of polarity in epithelial cells. We used confocal and time-lapse total internal reflection fluorescence microscopy (TIR-FM) to study changes in localization and exocytic sites of post-Golgi transport intermediates (PGTIs) carrying GFP-tagged apical or basolateral membrane proteins during epithelial polarization. In non-polarized Madin Darby Canine Kidney (MDCK) cells, apical and basolateral PGTIs were present throughout the cytoplasm and were observed to fuse with the basal domain of the plasma membrane. During polarization, apical and basolateral PGTIs were restricted to different regions of the cytoplasm and their fusion with the basal membrane was completely abrogated. Quantitative analysis suggested that basolateral, but not apical, PGTIs fused with the lateral membrane in polarized cells, correlating with the restricted localization of Syntaxins 4 and 3 to lateral and apical membrane domains, respectively. Microtubule disruption induced Syntaxin 3 depolarization and fusion of apical PGTIs with the basal membrane, but affected neither the lateral localization of Syntaxin 4 or Sec6, nor promoted fusion of basolateral PGTIs with the basal membrane.

Furin initiates gelsolin familial amyloidosis in the Golgi through a defect in Ca(2+) stabilization.

Hereditary familial amyloidosis of Finnish type (FAF) leading to amyloid in the peripheral and central nervous systems stems from deposition of a 71 residue fragment generated from the D187N/Y variants of plasma gelsolin by two sequential endoproteolytic events. We identify the protease accomplishing the first cleavage as furin, a proprotein convertase. Endoproteolysis of plasma gelsolin occurs in the trans-Golgi network due to the inability of the FAF variants to bind and be stabilized by Ca(2+). Secretion and processing of the FAF variants by furin can be uncoupled by blocking the convergence of the exocytic pathway transporting plasma gelsolin and the endocytic recycling of furin. We propose that coincidence of membrane trafficking pathways contributes to the development of proteolysis-initiated amyloid disease.