Integration of non-vesicular and vesicular transport processes at the Golgi complex by the PKD–CERT network

Non-vesicular transport of ceramide from endoplasmic reticulum to Golgi membranes is essential for cellular lipid homeostasis. Protein kinase D (PKD) is a serine–threonine kinase that controls vesicle fission at Golgi membranes. Here we highlight the intimate connections between non-vesicular and vesicular transport at the level of the Golgi complex, and suggest that PKD and its substrate CERT, the ceramide transfer protein, play central roles in coordinating these processes by fine-tuning the local membrane lipid composition to maintain Golgi secretory function. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Lipid peroxidation and covalent binding in the early functional impairment of liver Golgi apparatus by carbon tetrachloride

The onset of the lipoprotein secretory block provoked by CCl4 in the whole animal was monitored after purification of liver Golgi membranes. Both lipid transit through the apparatus and hexosylation of the lipoprotein are markedly inhibited 5-15 min after poisoning. Pre-treating the animal with alpha-tocopherol, shown to prevent lipid peroxidation without modifying the covalent binding due to CCl4 metabolites, affords little protection against lipid accumulation in the Golgi, but total preservation of galactosyl transferase activity. While haloalkylation therefore appears to be the major mechanism of damage in the early phases of CCl4-induced derangement of lipid secretion, lipid peroxidation is probably more involved later; this is indicated by the marked, though never complete, protection against fatty liver afforded at 24 h after CCl4 poisoning by supplementation of the membrane with alpha-tocopherol.     

Golgi Tubule Traffic and the Effects of Brefeldin A Visualized in Living Cells

The Golgi complex is a dynamic organelle engaged in both secretory and retrograde membrane traffic. Here, we use green fluorescent protein–Golgi protein chimeras to study Golgi morphology in vivo. In untreated cells, membrane tubules were a ubiquitous, prominent feature of the Golgi complex, serving both to interconnect adjacent Golgi elements and to carry membrane outward along microtubules after detaching from stable Golgi structures. Brefeldin A treatment, which reversibly disassembles the Golgi complex, accentuated tubule formation without tubule detachment. A tubule network extending throughout the cytoplasm was quickly generated and persisted for 5–10 min until rapidly emptying Golgi contents into the ER within 15–30 s. Both lipid and protein emptied from the Golgi at similar rapid rates, leaving no Golgi structure behind, indicating that Golgi membranes do not simply mix but are absorbed into the ER in BFA-treated cells. The directionality of redistribution implied Golgi membranes are at a higher free energy state than ER membranes. Analysis of its kinetics suggested a mechanism that is analogous to wetting or adsorptive phenomena in which a tension-driven membrane flow supplements diffusive transfer of Golgi membrane into the ER. Such nonselective, flow-assisted transport of Golgi membranes into ER suggests that mechanisms that regulate retrograde tubule formation and detachment from the Golgi complex are integral to the existence and maintenance of this organelle.     

Growth and metabolic control of lipid signalling at the Golgi

PtdIns4P is a key regulator of the secretory pathway and plays an essential role in trafficking from the Golgi. Our recent work demonstrated that spatial control of PtdIns4P at the ER (endoplasmic reticulum) and Golgi co-ordinates secretion with cell growth. The central elements of this regulation are specific phosphoinositide 4-kinases and the phosphoinositide phosphatase Sac1. Growth-dependent translocation of Sac1 between the ER and Golgi modulates the levels of PtdIns4P and anterograde traffic at the Golgi. In yeast, this mechanism is largely dependent on the availability of glucose, but our recent results in mammalian cells suggest that Sac1 phosphatases play evolutionarily conserved roles in the growth control of secretion. Sac1 lipid phosphatase plays also an essential role in the spatial control of PtdIns4P at the Golgi complex. A restricted pool of PtdIns4P at the TGN (trans-Golgi network) is required for Golgi integrity and for proper lipid and protein sorting. In mammalian cells, the stress-activated MAPK (mitogen-activated protein kinase) p38 appears to play a critical role in transmitting nutrient signals to the phosphoinositide signalling machinery at the ER and Golgi. These results suggest that temporal and spatial integration of metabolic and lipid signalling networks at the Golgi is required for controlling the secretory pathway.     

Inhibition of Liver Golgi Glycosylation Activities by Carbonyl Products of Lipid

The present report deals with the investigation of the effect of 4-hydroxy-/ram 2,3-nonenal (HNE), hexanal (HEX) and malondialdehyde (MDA), the major products of lipid peroxidation, on the glycosylation pathway of rat liver Golgi apparatus. Defined concentrations of the aldehydes were added to isolated fractions of formative (F,) and secretory (F, + F,) Golgi compartments, then incubated at 37°C for lOmin. At the end of the incubation the activity of galactosyl-(GT) and sialyl-(ST)transferases, the main enzymes of the terminal protein and lipoprotein glycosylation, was evalued. A significant impairment of both these activities was observed with HNE and HEX but not with MDA.

These data suggest that aldehydes generated during peroxidation reactions are able to impair the protein and lipoprotein maturation mechanism which is normally achieved through a complete glycosylation.     

Integration of non-vesicular and vesicular transport processes at the Golgi complex by the PKD-CERT network

Non-vesicular transport of ceramide from endoplasmic reticulum to Golgi membranes is essential for cellular lipid homeostasis. Protein kinase D (PKD) is a serine-threonine kinase that controls vesicle fission at Golgi membranes. Here we highlight the intimate connections between non-vesicular and vesicular transport at the level of the Golgi complex, and suggest that PKD and its substrate CERT, the ceramide transfer protein, play central roles in coordinating these processes by fine-tuning the local membrane lipid composition to maintain Golgi secretory function. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Regulation of secretory transport by protein kinase D-mediated phosphorylation of the ceramide transfer protein

Protein kinase D (PKD) has been identified as a crucial regulator of secretory transport at the trans-Golgi network (TGN). Recruitment and activation of PKD at the TGN is mediated by the lipid diacylglycerol, a pool of which is generated by sphingomyelin synthase from ceramide and phosphatidylcholine. The nonvesicular transfer of ceramide from the endoplasmic reticulum to the Golgi complex is mediated by the lipid transfer protein CERT (ceramide transport). In this study, we identify CERT as a novel in vivo PKD substrate. Phosphorylation on serine 132 by PKD decreases the affinity of CERT toward its lipid target phosphatidylinositol 4-phosphate at Golgi membranes and reduces ceramide transfer activity, identifying PKD as a regulator of lipid homeostasis. We also show that CERT, in turn, is critical for PKD activation and PKD-dependent protein cargo transport to the plasma membrane. Thus, the interdependence of PKD and CERT is key to the maintenance of Golgi membrane integrity and secretory transport.     

Molecular determinants and dynamics of hepatitis C virus secretion

The current model of hepatitis C virus (HCV) production involves the assembly of virions on or near the surface of lipid droplets, envelopment at the ER in association with components of VLDL synthesis, and egress via the secretory pathway. However, the cellular requirements for and a mechanistic understanding of HCV secretion are incomplete at best. We combined an RNA interference (RNAi) analysis of host factors for infectious HCV secretion with the development of live cell imaging of HCV core trafficking to gain a detailed understanding of HCV egress. RNAi studies identified multiple components of the secretory pathway, including ER to Golgi trafficking, lipid and protein kinases that regulate budding from the trans-Golgi network (TGN), VAMP1 vesicles and adaptor proteins, and the recycling endosome. Our results support a model wherein HCV is infectious upon envelopment at the ER and exits the cell via the secretory pathway. We next constructed infectious HCV with a tetracysteine (TC) tag insertion in core (TC-core) to monitor the dynamics of HCV core trafficking in association with its cellular cofactors. In order to isolate core protein movements associated with infectious HCV secretion, only trafficking events that required the essential HCV assembly factor NS2 were quantified. TC-core traffics to the cell periphery along microtubules and this movement can be inhibited by nocodazole. Sub-populations of TC-core localize to the Golgi and co-traffic with components of the recycling endosome. Silencing of the recycling endosome component Rab11a results in the accumulation of HCV core at the Golgi. The majority of dynamic core traffics in association with apolipoprotein E (ApoE) and VAMP1 vesicles. This study identifies many new host cofactors of HCV egress, while presenting dynamic studies of HCV core trafficking in infected cells.     

Evidence for segregation of sphingomyelin and cholesterol during formation of COPI-coated vesicles

In higher eukaryotes, phospholipid and cholesterol synthesis occurs mainly in the endoplasmic reticulum, whereas sphingomyelin and higher glycosphingolipids are synthesized in the Golgi apparatus. Lipids like cholesterol and sphingomyelin are gradually enriched along the secretory pathway, with their highest concentration at the plasma membrane. How a cell succeeds in maintaining organelle-specific lipid compositions, despite a steady flow of incoming and outgoing transport carriers along the secretory pathway, is not yet clear. Transport and sorting along the secretory pathway of both proteins and most lipids are thought to be mediated by vesicular transport, with coat protein I (COPI) vesicles operating in the early secretory pathway. Although the protein constituents of these transport intermediates are characterized in great detail, much less is known about their lipid content. Using nano-electrospray ionization tandem mass spectrometry for quantitative lipid analysis of COPI-coated vesicles and their parental Golgi membranes, we find only low amounts of sphingomyelin and cholesterol in COPI-coated vesicles compared with their donor Golgi membranes, providing evidence for a significant segregation from COPI vesicles of these lipids. In addition, our data indicate a sorting of individual sphingomyelin molecular species. The possible molecular mechanisms underlying this segregation, as well as implications on COPI function, are discussed.     

Multi‐step down‐regulation of the secretory pathway in mitosis: A fresh perspective on protein trafficking

The secretory pathway delivers proteins synthesized at the rough endoplasmic reticulum (RER) to various subcellular locations via the Golgi apparatus. Currently, efforts are focused on understanding the molecular machineries driving individual processes at the RER and Golgi that package, modify and transport proteins. However, studies are routinely performed using non‐dividing cells. This obscures the critical issue of how the secretory pathway is affected by cell division. Indeed, several studies have indicated that protein trafficking is down‐regulated during mitosis. Moreover, the RER and Golgi apparatus exhibit gross reorganization in mitosis. Here I provide a relatively neglected perspective of how the mitotic cyclin‐dependent kinase (CDK1) could regulate various stages of the secretory pathway. I highlight several aspects of the mitotic control of protein trafficking that remain unresolved and suggest that further studies on how the mitotic CDK1 influences the secretory pathway are necessary to obtain a deeper understanding of protein transport.     

Synthetic genetic array analysis of the PtdIns 4-kinase Pik1p identifies components in a Golgi-specific Ypt31/rab-GTPase signaling pathway

Phosphorylated derivatives of phosphatidylinositol are essential regulators of both endocytic and exocytic trafficking in eukaryotic cells. In Saccharomyces cerevisiae, the phosphatidylinositol 4-kinase, Pik1p generates a distinct pool of PtdIns(4)P that is required for normal Golgi structure and secretory function. Here, we utilize a synthetic genetic array analysis of a conditional pik1 mutant to identify candidate components of the Pik1p/PtdIns(4)P signaling pathway at the Golgi. Our data suggest a mechanistic involvement for Pik1p with a specific subset of Golgi-associated proteins, including the Ypt31p rab-GTPase and the TRAPPII protein complex, to regulate protein trafficking through the secretory pathway. We further demonstrate that TRAPPII specifically functions in a Ypt31p-dependent pathway and identify Gyp2p as the first biologically relevant GTPase activating protein for Ypt31p. We propose that multiple stage-specific signals, which may include Pik1p/PtdIns(4)P, TRAPPII and Gyp2p, impinge upon Ypt31 signaling to regulate Golgi secretory function.     

Characterization of a secretory vesicle-rich fraction from lactating bovine mammary gland.

Fractions enriched in secretory vesicles were obtained from lactating bovine mammary tissue by a straightforward procedure involving gentle homogenization and centrifugation in isotonic milk salt solution containing Ficoll. Secretory vesicle-rich fractions could also be obtained from lactating rat mammary gland by this procedure. With rats, yields of vesicles were substantially increased by administration of colchicine or thioglucose to animals several hours before sacrifice. Isolated fractions were enriched in lactose and consisted predominantly of 0.2–1.2 μm diameter vesicles, many of which contained casein micelles. Enzymatic, compositional and morphological examination revealed vesicle preparations to be largely free of contamination by rough endoplasmic reticulum, mitochondria, nuclei, peroxisomes and lysosomes. Specific activity of several marker enzymes of the secretory vesicle fraction were similar to, or intermediate between, Golgi apparatus and milk lipid globule membranes. Amounts of cholesterol and gangliosides in vesicle fractions approached levels found in plasma membranes. In distribution of major phospholipids, secretory vesicles were intermediate between Golgi apparatus and milk lipid globule membranes. The pattern of polypeptides of secretory vesicle membrane was qualitatively similar to that of Golgi apparatus membranes. While there were similarities between these polypeptide patterns and that of lipid globule membranes, the latter contained relatively more of certain polypeptides, particularly the internal coat-associated polypeptides of the globule membrane. These observations are discussed in relation to the endomembrane hypothesis and the origin of the membrane of milk lipid globules.     

Calcium gradients and the Golgi

Changes in intracellular free calcium regulate many intracellular processes. With respect to the secretory pathway and the Golgi apparatus, changes in calcium concentration occurring either in the adjacent cytosol or within the lumen of the Golgi act to regulate Golgi function. Conversely, the Golgi sequesters calcium to shape cytosolic calcium signals as well as initiate them by releasing calcium via inositol-1,4,5-triphosphate (IP(3)) receptors, located on Golgi membranes. Local calcium transients juxtaposed to the Golgi (arising from release by the Golgi or other organelles) can activate calcium dependent signalling molecules located on or around the Golgi. This review focuses on the reciprocal relationship between the cell biology of the Golgi apparatus and intracellular calcium homeostasis.     

Identification of class II ADP‐ribosylation factors as cellular factors required for hepatitis C virus replication

GBF1 is a host factor required for hepatitis C virus (HCV) replication. GBF1 functions as a guanine nucleotide exchange factor for G‐proteins of the Arf family, which regulate membrane dynamics in the early secretory pathway and the metabolism of cytoplasmic lipid droplets. Here we established that the Arf‐guanine nucleotide exchange factor activity of GBF1 is critical for its function in HCV replication, indicating that it promotes viral replication by activating one or more Arf family members. Arf involvement was confirmed with the use of two dominant negative Arf1 mutants. However, siRNA‐mediated depletion of Arf1, Arf3 (class I Arfs), Arf4 or Arf5 (class II Arfs), which potentially interact with GBF1, did not significantly inhibit HCV infection. In contrast, the simultaneous depletion of both Arf4 and Arf5, but not of any other Arf pair, imposed a significant inhibition of HCV infection. Interestingly, the simultaneous depletion of both Arf4 and Arf5 had no impact on the activity of the secretory pathway and induced a compaction of the Golgi and an accumulation of lipid droplets. A similar phenotype of lipid droplet accumulation was also observed when GBF1 was inhibited by brefeldin A. In contrast, the simultaneous depletion of both Arf1 and Arf4 resulted in secretion inhibition and Golgi scattering, two actions reminiscent of GBF1 inhibition. We conclude that GBF1 could regulate different metabolic pathways through the activation of different pairs of Arf proteins.     

Phospholipid synthesis participates in the regulation of diacylglycerol required for membrane trafficking at the Golgi complex

The lipid metabolite diacylglycerol (DAG) is required for transport carrier biogenesis at the Golgi, although how cells regulate its levels is not well understood. Phospholipid synthesis involves highly regulated pathways that consume DAG and can contribute to its regulation. Here we altered phosphatidylcholine (PC) and phosphatidylinositol synthesis for a short period of time in CHO cells to evaluate the changes in DAG and its effects in membrane trafficking at the Golgi. We found that cellular DAG rapidly increased when PC synthesis was inhibited at the non-permissive temperature for the rate-limiting step of PC synthesis in CHO-MT58 cells. DAG also increased when choline and inositol were not supplied. The major phospholipid classes and triacylglycerol remained unaltered for both experimental approaches. The analysis of Golgi ultrastructure and membrane trafficking showed that 1) the accumulation of the budding vesicular profiles induced by propanolol was prevented by inhibition of PC synthesis, 2) the density of KDEL receptor-containing punctated structures at the endoplasmic reticulum-Golgi interface correlated with the amount of DAG, and 3) the post-Golgi transport of the yellow fluorescent temperature-sensitive G protein of stomatitis virus and the secretion of a secretory form of HRP were both reduced when DAG was lowered. We confirmed that DAG-consuming reactions of lipid synthesis were present in Golgi-enriched fractions. We conclude that phospholipid synthesis pathways play a significant role to regulate the DAG required in Golgi-dependent membrane trafficking.     

Metabolic Activation of the HOG MAP Kinase Pathway by Snf1/AMPK Regulates Lipid Signaling at the Golgi

Phosphatidylinositol‐4‐phosphate (PI(4)P) is an important regulator of Golgi function. Metabolic regulation of Golgi PI(4)P requires the lipid phosphatase Sac1 that translocates between endoplasmic reticulum (ER) and Golgi membranes. Localization of Sac1 responds to changes in glucose levels, yet the upstream signaling pathways that regulate Sac1 traffic are unknown. Here, we report that mitogen‐activated protein kinase (MAPK) Hog1 transmits glucose signals to the Golgi and regulates localization of Sac1. We find that Hog1 is rapidly activated by both glucose starvation and glucose stimulation, which is independent of the well‐characterized response to osmotic stress but requires the upstream element Ssk1 and is controlled by Snf1, the yeast homolog of AMP‐activated kinase (AMPK). Elimination of either Hog1 or Snf1 slows glucose‐induced translocation of Sac1 lipid phosphatase from the Golgi to the ER and thus delays PI(4)P accumulation at the Golgi. We conclude that a novel cross‐talk between the HOG pathway and Snf1/AMPK is required for the metabolic control of lipid signaling at the Golgi.     

Lipid raft association of carboxypeptidase E is necessary for its function as a regulated secretory pathway sorting receptor

Membrane carboxypeptidase E (CPE) is a sorting receptor for targeting prohormones, such as pro-opiomelanocortin, to the regulated secretory pathway in endocrine cells. Its membrane association is necessary for it to bind a prohormone sorting signal at the trans-Golgi network (TGN) to facilitate targeting. In this study, we examined the lipid interaction of CPE in bovine pituitary secretory granule membranes, which are derived from the TGN. We show that CPE is associated with detergent-resistant lipid domains, or rafts, within secretory granule membranes. Lipid analysis revealed that these rafts are enriched in glycosphingolipids and cholesterol. Pulse-chase and subcellular fractionation experiments in AtT-20 cells show that the association of CPE with membrane rafts occurred only after it reached the Golgi. Cholesterol depletion resulted in dissociation of CPE from secretory granule membranes and decreased the binding of prohormones to membranes. In vivo cholesterol depletion using lovastatin resulted in the lack of sorting of CPE and its cargo to the regulated secretory pathway. We propose that the sorting receptor function of CPE necessitates its interaction with glycosphingolipid-cholesterol rafts at the TGN, thereby anchoring it in position to bind to its prohormone cargo.     

Cholesterol is required for the formation of regulated and constitutive secretory vesicles from the trans-Golgi network

We studied the role of cholesterol in regulated protein secretion in neuroendocrine cells by manipulating the cholesterol content of AtT-20 cells. Depletion of cellular cholesterol levels caused a reversible block of immature secretory granule biogenesis at the level of the trans -Golgi-network, whereas increased cholesterol levels promoted immature secretory granule formation. Cholesterol depletion also blocked the formation of constitutive secretory vesicles, but did not inhibit the transport between the endoplasmic reticulum and the Golgi complex. Our results indicate that the assembly of cholesterol-based lipid microdomains is required for the biogenesis of both regulated and constitutive secretory vesicles from the trans -Golgi-network in neuroendocrine cells.     

Connecting vesicular transport with lipid synthesis: FAPP2

Next to the protein-based machineries composed of small G-proteins, coat complexes, SNAREs and tethering factors, the lipid-based machineries are emerging as important players in membrane trafficking. As a component of these machineries, lipid transfer proteins have recently attracted the attention of cell biologists for their involvement in trafficking along different segments of the secretory pathway. Among these, the four-phosphate adaptor protein 2 (FAPP2) was discovered as a protein that localizes dynamically with the trans-Golgi network and regulates the transport of proteins from the Golgi complex to the cell surface. Later studies have highlighted a role for FAPP2 as lipid transfer protein involved in glycosphingolipid metabolism at the Golgi complex. Here we discuss the available evidence on the function of FAPP2 in both membrane trafficking and lipid metabolism and propose a mechanism of action of FAPP2 that integrates its activities in membrane trafficking and in lipid transfer. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

Inositol phosphorylceramide synthase is located in the Golgi apparatus of Saccharomyces cerevisiae

The plasma membrane of eukaryotic cells differs in lipid composition from most of the internal organelles, presumably reflecting differences in many of its functions. In particular, the plasma membrane is rich in sphingolipids and sterols, one property of which is to decrease the permeability and increase the thickness of lipid bilayers. In this paper, we examine the length of transmembrane domains throughout the yeast secretory pathway. Although the transmembrane domains of cis and medial Golgi residents are similar to those of endoplasmic reticulum proteins, these domains lengthen substantially beyond the medial Golgi, suggesting a thickening of the bilayer. Yeast sphingolipids have particularly long acyl chains, and Aur1p, the inositol phosphorylceramide synthase that initiates yeast sphingolipid synthesis, was found to be located in the Golgi apparatus by both immunofluorescence and membrane fractionation, with its active site apparently in the Golgi lumen. Thus, it appears that sphingolipid synthesis in yeast takes place in the Golgi, separated from glycerophospholipid synthesis in the endoplasmic reticulum. A similar separation has been found in mammalian cells, and this conservation suggests that such an arrangement of enzymes within the secretory pathway could be important for the creation of bilayers of different thickness within the cell.