Regulation of the Golgi complex by phospholipid remodeling enzymes

The mammalian Golgi complex is a highly dynamic organelle consisting of stacks of flattened cisternae with associated coated vesicles and membrane tubules that contribute to cargo import and export, intra-cisternal trafficking, and overall Golgi architecture. At the morphological level, all of these structures are continuously remodeled to carry out these trafficking functions. Recent advances have shown that continual phospholipid remodeling by phospholipase A (PLA) and lysophospholipid acyltransferase (LPAT) enzymes, which deacylate and reacylate Golgi phospholipids, respectively, contributes to this morphological remodeling. Here we review the identification and characterization of four cytoplasmic PLA enzymes and one integral membrane LPAT that participate in the dynamic functional organization of the Golgi complex, and how some of these enzymes are integrated to determine the relative abundance of COPI vesicle and membrane tubule formation. This article is part of a Special Issue entitled Lipids and Vesicular Transport.     

The Cirque du Soleil of Golgi membrane dynamics

The role of lipid metabolic enzymes in Golgi membrane remodeling is a subject of intense interest. Now, in this issue, Schmidt and Brown (2009. J. Cell Biol. doi:10.1083/jcb.200904147) report that lysophosphatidic acid–specific acyltransferase, LPAAT3, contributes to Golgi membrane dynamics by suppressing tubule formation.The idea that active remodeling of glycerolipid acyl chains contributes to the membrane transformations required for membrane trafficking is not new (Kozlov et al., 1989; Chernomordik et al., 1995). However, identification of specific enzymes that execute such functions in living cells has proven elusive. Now, an interesting study by Schmidt and Brown (see p. 211 of this issue) demonstrates that a lysophosphatidic acid acyltransferase (LPAAT) is directly involved in regulating mammalian Golgi trafficking functions. A variety of experimental approaches converge on a coherent model where LPAAT3 quenches the formation of Golgi-derived tubules. In doing so, LPAAT3 opposes what is most likely a phospholipase A₂–mediated tubulation pathway. This balance of PLA₂ and LPAAT3 activities has functional consequences for membrane trafficking from the mammalian Golgi complex.Glycerolipids, such as phosphatidic acid (PtdOH), consist of a glycerol backbone to which three additional constituents are esterified. Fatty acyl chains are attached at the sn-1 and sn-2 positions, and these lend glycerolipids their hydrophobic character. The headgroup at the sn-3 position can be very simple (an −OH group to generate diacylglycerol; DAG) or complex (i.e., another glycerolipid molecule). In the case of phospholipids, the headgroup is linked to the backbone by a phosphoester bond (PtdOH representing the simplest case). The three-dimensional shape of a phospholipid molecule (cone, inverted cone, cylinder) is governed by the ratio of the axial area of the headgroup to that of the acyl chain region. Because the sn-2 acyl chain is often unsaturated, and therefore kinked, a suitably bulky headgroup is required to match the axial area of the acyl chain region and generate a cylindrical molecule that packs into orderly membrane bilayers. The basic principle is lipid shape can be regulated at the level of either the headgroup or the acyl chains, and enrichment of non-cylindrical lipid molecules will physically deform membranes in predictable ways (Burger, 2000; Kooijman et al., 2005).Phospholipase A₂ (PLA₂) hydrolyzes the acyl chain from the sn-2 position of a glycerolipid molecule and, in doing so, generates a molecule with a glycerol backbone esterified to a fatty acid at sn-1 and to the headgroup at sn-3. This lyso-lipid exhibits a small axial area for the acyl chain region (and is shaped as an inverted cone that promotes positive membrane curvature). What LPAATs do is re-acylate the sn-2 position with a second fatty acid (or more accurately, a fatty acyl-CoA with release of CoA as product), often an unsaturated one in higher eukaryotes, so that the axial area of the acyl chain region is much increased. When the headgroup of the glycerolipid is small, as is the case with PtdOH and DAG, the renovated glycerolipid molecule now assumes a cone shape that promotes negative membrane curvature. The general deacylation/reacylation cycle driven by sequential PLA₂/LPAAT actions of this sort is termed the Lands cycle (Fig. 1; Lands and Hart, 1965). Although originally discovered as a metabolic pathway for phospholipid acyl chain remodeling in liver, the Lands cycle now resurfaces as a mechanism for controlling mammalian Golgi membrane dynamics.Open in a separate windowFigure 1.The Lands cycle. PLA₂ hydrolyzes the acyl-chain from a glycerophospholipid to generate a free fatty acid and a lysophospholipid product. Reacylation of lysophospholipid back to a glycerophospholipid (often with a different acyl chain at sn-2) is catalyzed by an LPAAT and involves consumption of a fatty acyl-CoA. This figure was adapted from Figure 5 in Shimizu (2009).LPAATs have been studied previously from the perspective of the enzymology of lipid metabolism, but their functions from the cell biological point of view remain poorly understood. The human genome sequence database identifies nine potential LPAATs (Leung, 2001; Shindou and Shimizu, 2009). A functional involvement of the Lands cycle (and LPAATs) with the Golgi complex was initially forecast by pharmacological studies with PLA₂ and LPAAT inhibitors—the former insults interfering with various membrane trafficking pathways and the latter promoting others (de Figueiredo et al., 1998, 2000; Drecktrah et al., 2003; Chambers et al., 2005). Unfortunately, inhibitor studies of this sort are difficult to interpret. For instance, do the pleiotropic effects of the drugs report inhibition of multiple enzyme isoforms with various execution points, or are these reflections of “off-target” effects?Schmidt and Brown (2009) now report the integral membrane protein LPAAT3 localizes to ER/Golgi membranes and exhibits lyso-PtdOH acyltransferase activity. Modulation of LPAAT3 expression has significant consequences for Golgi organization and function. siRNA-mediated silencing of LPAAT3 expression resulted in Golgi fragmentation into mini-stacks, an exquisite sensitivity of Golgi integrity to brefeldin A (BFA), and elevated mis-localization of Golgi resident proteins to the ER. Reciprocally, elevated LPAAT3 expression retards Golgi collapse into the ER upon BFA challenge. These various effects correlate with enhanced formation of Golgi-derived tubules in the face of LPAAT3 inhibitors (lyso-PtdOH formation favored) and depressed tubule biogenesis when LPAAT3 activity is increased (conversion of lyso-PtdOH to PtdOH favored). Tubulation is clearly relevant to membrane transport, as enhancement can (in specific cases) accelerate rates of cargo trafficking. Interference with tubule biogenesis, or maintenance, retards trafficking from the Golgi complex, and both anterograde and retrograde trafficking pathways are affected (Schmidt and Brown, 2009).The simple physical principle that connects the Lands cycle to tubulation is that production of inverted cone lyso-PtdOH by a PLA₂ strongly promotes positive membrane curvature and tubulation, whereas LPAAT3-mediated reacylation of lyso-PtdOH to PtdOH has the opposite effect (Fig. 2). Curvature parameters have been measured for lyso-PtdOH and PtdOH at physiological salt and pH concentrations, and the respective spontaneous radii of curvatures are +20Å and −46Å, respectively (for oleoyl molecular species; Kooijman et al., 2005). Interestingly, the measurements for lyso-PtdOH yield among the highest positive curvature values recorded to date. One testable question for future investigation is whether the PtdOH molecular species generated in Golgi membranes by LPAAT3 differ from those of bulk Golgi membrane PtdOH; that is, whether reacylation generates PtdOH molecular species with distinct properties such as unsaturated acyl chains at sn-2. Such a result would forecast an acyl-chain preference for LPAAT3 and the resultant molecular species would assume more extreme cone shapes that may contribute to the membrane transformations that accompany fission processes (Burger, 2000; Kooijman et al., 2005). It is also possible that newly remodeled PtdOH is a precursor for DAG, which may be the operative fission-ogenic glycerolipid. DAG assumes even more extreme negative curvatures than does PtdOH, and it is not subject to electrostatic penalties associated with packing the highly negatively charged PtdOH headgroup. Critical roles for DAG in Golgi membrane trafficking are well established (Kearns et al., 1997; Baron and Malhotra, 2002; Fernandez-Ulibarri et al., 2007; Asp et al., 2009).Open in a separate windowFigure 2.Protein domains consolidate the positive membrane curvature generated by lyso-PtdOH. PLA₂ hydrolyzes the acyl-chain from a phosphatidylcholine (PtdCho: cylindrical lipid) to generate a free fatty acid (FFA) and a lyso-PtdCho product (positive curvature). That lyso-PtdCho (LPC) species is further metabolized to lyso-PtdOH (LPA; greater positive curvature) by phospholipases with concomitant release of the choline (Cho) headgroup. The LPA is bound by proteins that “sense” curvature or bend membranes (e.g., BAR domain), leading to further sorting of LPA to the site of deformation (in this case a budding profile). LPAAT3 antagonizes this pathway by consuming lyso-PtdOH into PtdOH synthesis. The figure was adapted from one generously provided to V.A. Bankaitis by Wonhwa Cho (University of Illinois-Chicago, Chicago, IL).How may proteins interface with the Lands cycle in the Golgi system? Do proteins provide the primary driving force for membrane deformation, or is lipid metabolism the major factor? It is unlikely to be solely the latter—at least in this case. Simple activity of PLA₂ in generating lyso-PtdOH (or other lyso-lipids) is insufficient to impose significant positive curvature to membranes. The liberated fatty acid product will promote negative curvature—thereby countering the effects of the lyso-lipid. Enrichment of lyso-lipid into domains is a prerequisite for membrane deformation, and such enrichment can be reinforced by proteins in several ways. First, protein domains that bend membranes (e.g., BAR-domains; Frost et al., 2009) could bind lyso-lipids by virtue of their shape characteristics and thereby consolidate them into positively curved domains (Fig. 2). Second, coat or motor proteins that mechanically generate tubules could drive a physical rearrangement of membrane lipids to structures that best fit their shape (Roux et al., 2005; Krauss et al., 2008; Sorre et al., 2009). In this scenario, lyso-lipids re-sort preferentially to tubules and generate a positive feedback loop for membrane deformations with positive curvature.Reconstituted systems for membrane deformation use metabolically inert membranes, and are deprived of the active interface between lipid metabolism, proteins, and membrane dynamics. What blind spots are inherent in such protein-driven membrane deformation assays remains to be seen, but it will prove increasingly true that diverse pathways of lipid metabolism, including the Lands cycle, lubricate the actions of proteins in productive membrane deformation pathways. Which is more important—proteins or lipids—in this arena? Let’s call it an equal-opportunity collaboration.Given the intense interest concerning interfaces of lipid metabolism and Golgi function, it is difficult to believe that the concept of lipid metabolism as an active participant in membrane trafficking was ignored during the halcyon days when proteins involved in vesicle biogenesis were being discovered. Since the first demonstrations that specific lipid metabolic pathways are central to these processes (Bankaitis et al, 1990; Cleves et al., 1991), work from numerous laboratories has greatly expanded the lipid–Golgi interface. The report of Schmidt and Brown (2009) adds new sets of activities to the ever-growing roster of lipid metabolic enzymes whose actions contribute to the remarkable Cirque du Soleil of Golgi membrane dynamics. Indeed, we may soon wonder whether there is any such thing as a simple “housekeeping” lipid metabolic pathway in eukaryotic cells.     

Lipid metabolism in Chinese hamster V79-R membranes composed of unusual phospholipid molecular species

The relationship between the inhibition of cell growth and the changes in phospholipid metabolism in the presence of erucic acid was studied in Chinese hamster V79-R cells. 1. The addition of erucic acid to the medium inhibited cell growth. The degree of inhibition by erucic acid at a given concentration was dependent on cell density. 2. Exogenous erucic acid was incorporated into cellular phospholipids to form new phospholipid molecular species, which were identified to be the erucoyl/oleoyl, erucoyl/gondoyl and erucoyl/erucoyl species. 3. Synthesis of phosphatidylcholine and phosphatidylethanolamine in endoplasmic reticulum was reduced by erucic acid. Erucic acid had no effect on membrane flow of phospholipids from endoplasmic reticulum to plasma membrane. 4. The specific activity of sn-glycerol-3-phosphate acyltransferase in the membrane fraction from the cells supplemented with erucic acid was lower than that from the control cells. The reduction of phospholipid synthesis was attributed to the decrease in sn-glycerol-3-phosphate acyltransferase activity.     

A synthetic biology approach to the construction of membrane proteins in semi-synthetic minimal cells

Synthetic biology is an emerging field that aims at constructing artificial biological systems by combining engineering and molecular biology approaches. One of the most ambitious research line concerns the so-called semi-synthetic minimal cells, which are liposome-based system capable of synthesizing the lipids within the liposome surface. This goal can be reached by reconstituting membrane proteins within liposomes and allow them to synthesize lipids. This approach, that can be defined as biochemical, was already reported by us (Schmidli et al. J. Am. Chem. Soc. 113, 8127-8130, 1991). In more advanced models, however, a full reconstruction of the biochemical pathway requires (1) the synthesis of functional membrane enzymes inside liposomes, and (2) the local synthesis of lipids as catalyzed by the in situ synthesized enzymes. Here we show the synthesis and the activity - inside liposomes - of two membrane proteins involved in phospholipids biosynthesis pathway. The proteins, sn-glycerol-3-phosphate acyltransferase (GPAT) and lysophosphatidic acid acyltransferase (LPAAT), have been synthesized by using a totally reconstructed cell-free system (PURE system) encapsulated in liposomes. The activities of internally synthesized GPAT and LPAAT were confirmed by detecting the produced lysophosphatidic acid and phosphatidic acid, respectively. Through this procedure, we have implemented the first phase of a design aimed at synthesizing phospholipid membrane from liposome within from within — which corresponds to the autopoietic growth mechanism.     

Phospholipase D and the Maintenance of Phosphatidic Acid Levels for Regulation of Mammalian Target of Rapamycin (mTOR)

Phosphatidic acid (PA) is a critical metabolite at the heart of membrane phospholipid biosynthesis. However, PA also serves as a critical lipid second messenger that regulates several proteins implicated in the control of cell cycle progression and cell growth. Three major metabolic pathways generate PA: phospholipase D (PLD), diacylglycerol kinase (DGK), and lysophosphatidic acid acyltransferase (LPAAT). The LPAAT pathway is integral to de novo membrane phospholipid biosynthesis, whereas the PLD and DGK pathways are activated in response to growth factors and stress. The PLD pathway is also responsive to nutrients. A key target for the lipid second messenger function of PA is mTOR, the mammalian/mechanistic target of rapamycin, which integrates both nutrient and growth factor signals to control cell growth and proliferation. Although PLD has been widely implicated in the generation of PA needed for mTOR activation, it is becoming clear that PA generated via the LPAAT and DGK pathways is also involved in the regulation of mTOR. In this minireview, we highlight the coordinated maintenance of intracellular PA levels that regulate mTOR signals stimulated by growth factors and nutrients, including amino acids, lipids, glucose, and Gln. Emerging evidence indicates compensatory increases in one source of PA when another source is compromised, highlighting the importance of being able to adapt to stressful conditions that interfere with PA production. The regulation of PA levels has important implications for cancer cells that depend on PA and mTOR activity for survival.     

Comparative gene identification 58/α/β hydrolase domain 5 lacks lysophosphatidic acid acyltransferase activity

Mutations in the gene encoding comparative gene identification 58 (CGI-58)/α/β hydrolase domain 5 (ABHD5) cause Chanarin-Dorfman syndrome, characterized by excessive triacylglycerol storage in cells and tissues. CGI-58 has been identified as a coactivator of adipose TG lipase (ATGL) and a lysophosphatidic acid acyltransferase (LPAAT). We developed a molecular model of CGI-58 structure and then mutated predicted active site residues and performed LPAAT activity assays of recombinant WT and mutated CGI-58. When mutations of predicted catalytic residues failed to reduce LPAAT activity, we determined that LPAAT activity was due to a bacterial contaminant of affinity purification procedures, plsC, the sole LPAAT in Escherichia coli. Purification protocols were optimized to reduce plsC contamination, in turn reducing LPAAT activity. When CGI-58 was expressed in SM2-1(DE3) cells that lack plsC, lysates lacked LPAAT activity. Additionally, mouse CGI-58 expressed in bacteria as a glutathione-S-transferase fusion protein and human CGI-58 expressed in yeast lacked LPAAT activity. Previously reported lipid binding activity of CGI-58 was revisited using protein-lipid overlays. Recombinant CGI-58 failed to bind lysophosphatidic acid, but interestingly, bound phosphatidylinositol 3-phosphate [PI(3)P] and phosphatidylinositol 5-phosphate [PI(5)P]. Prebinding CGI-58 with PI(3)P or PI(5)P did not alter its coactivation of ATGL in vitro. In summary, purified recombinant CGI-58 that is functional as an ATGL coactivator lacks LPAAT activity.     

Membrane topology of human AGPAT3 (LPAAT3)

Integral membrane lysophospholipid acyltransferases (AT) are involved in many reactions that produce phospholipids and triglycerides. Enzymes that utilize lysophosphatidic acid (LPA) as an acceptor substrate have been termed LPAATs, and several are members of the 1-acylglycerol-3-phosphate O-acyltransferase (AGPAT) gene family. Amino acid sequence comparisons with other acyltransferases reveal that AGPATs contain four conserved motifs (I-IV), whose invariant residues appear to be important for catalysis and/or substrate recognition. Although the enzymatic activities of many AGPATs are known, for many members their structural organization within membranes and their exact biological functions are unclear. Recently, a new function for AGPATs was discovered when it was determined that human AGPAT3/LPAAT3 is involved in the structure and function of the Golgi complex. Here we have determined the topological orientation of human AGPAT3/LPAAT3. AGPAT3/LPAAT3 possesses two transmembrane domains, one of which separates motifs I and II, which are thought to form a functional unit that is critical for enzymatic activity. This is a surprising result but similar to a recent study on the topology of human LPAAT 1. The data is consistent with a structural arrangement in which motif I is located in the cytoplasm and motif II is in the endoplasmic reticulum and Golgi lumen, suggesting a different model for AGPAT3/LPAAT3’s enzymatic mechanism.     

ULTRASTRUCTURAL LOCALIZATION OF PHOSPHOLIPID SYNTHESIS IN THE RAT TRIGEMINAL NERVE DURING MYELINATION

A method for the ultrastructural localization of acyltransferase enzymes involved in phospholipid metabolism has been applied to the developing rat trigeminal nerve. Determination of acyltransferase levels in the nerve indicated that a peak of activity occurs at the 8th day after birth with gradual declines of activity up to 15 days. Morphological surveys and determinations of cholesterol levels suggested that heavy myelin formation occurs in the nerve during this latter period. Fixed nerves incubated in a medium for localization of acyltransferases indicated deposition of reaction product associated with Golgi cisternae, intracellular smooth vesicles, and the plasma membrane of the Schwann cell in the incipient stages of myelin formation. Golgi-derived vesicles appeared to move toward the Schwann cell surface and fuse with the plasma membrane. Activity continued to be detectable in the plasma membrane of the internal mesaxon as long as cytoplasm was evident and mature myelin membrane was not yet formed. Cells in which myelin formation appeared advanced showed little or no enzyme marker. Consistent with cytochemical observations were biochemical determinations of acyltransferases which showed high levels of the enzymes in microsomes, while no activity could be detected in the myelin fraction. Acyltransferase reaction product was also observed in the Golgi apparatus of ganglion cell bodies, axoplasmic smooth vesicles, and the axolemma. Localization of acyltransferase enzymes in Schwann cells, ganglion cell bodies, and axons during development of the nerve is discussed in relation to membrane biogenesis in the nervous system.     

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.     

Inhibition of a Golgi complex lysophospholipid acyltransferase induces membrane tubule formation and retrograde trafficking

Recent studies have suggested that formation of Golgi membrane tubules involves the generation of membrane-associated lysophospholipids by a cytoplasmic Ca2+-independent phospholipase A2 (PLA2). Herein, we provide additional support for this idea by showing that inhibition of lysophospholipid reacylation by a novel Golgi-associated lysophosphatidylcholine acyltransferase (LPAT) induces the rapid tubulation of Golgi membranes, leading in their retrograde movement to the endoplasmic reticulum. Inhibition of the Golgi LPAT was achieved by 2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide (CI-976), a previously characterized antagonist of acyl-CoA cholesterol acyltransferase. The effect of CI-976 was similar to that of brefeldin A, except that the coatomer subunit beta-COP remained on Golgi-derived membrane tubules. CI-976 also enhanced the cytosol-dependent formation of tubules from Golgi complexes in vitro and increased the levels of lysophosphatidylcholine in Golgi membranes. Moreover, preincubation of cells with PLA2 antagonists inhibited the ability of CI-976 to induce tubules. These results suggest that Golgi membrane tubule formation can result from increasing the content of lysophospholipids in membranes, either by stimulation of a PLA2 or by inhibition of an LPAT. These two opposing enzyme activities may help to coordinately regulate Golgi membrane shape and tubule formation.     

Arachidonoyl-CoA:lysophosphatidylcholine acyltransferase activity in ras-transformed NIH 3T3 fibroblasts depends on the membrane composition.

Investigations were performed on the influence of membrane lipids on arachidonoyl-CoA:lysophosphatidylcholine acyltransferase in microsomal membranes from control and ras-transformed NIH 3T3 fibroblasts. Of all the tested phospholipids only sphingomyelin induced activation of acyltransferase in membranes from ras-transformed cells. No specific phospholipid effect on the acyltransferase was observed in microsomal membranes from control fibroblasts. Diacylglycerol was found to inhibit acyltransferase in both cell lines, whereas ceramide accumulation induced inhibition only in membranes from the transformed cells. The effects of diacylglycerol, ceramide, sphingomyelin and sphingomyelinase are discussed with respect to their putative roles in the signal transduction pathways in oncogene-expressing cells.     

Turnover of fatty acids in the 1-position of phosphatidylethanolamine in Escherichia coli

Phosphatidylethanolamine is the major membrane phospholipid of Escherichia coli, and two experimental approaches were used to investigate the metabolic activity of the fatty acids occupying the 1-position of this phospholipid. [3H]Acetate pulse-chase experiments with logarithmically growing cells indicated that 3-5% of the acyl groups were removed from the phosphatidylethanolamine pool/generation. The reacylation aspect of the turnover cycle was demonstrated by the incorporation of fatty acids into the 1-position of pre-existing phosphatidylethanolamine when de novo phospholipid biosynthesis was inhibited using the plsB acyltransferase mutant. 2- Acylglycerophosphoethanolamine would be the intermediate in a 1-position turnover cycle, and this lysophospholipid was identified as a membrane component that could re-esterified by a membrane-bound acyltransferase. The acyltransferase either utilized acyl-acyl carrier protein directly as an acyl donor or activated fatty acids for acyl transfer in the presence of ATP and Mg2+. Acyl-acyl carrier protein was also indicated as an intermediate in the latter reacylation reaction by the complete inhibition of phosphatidylethanolamine formation from fatty acids by acyl carrier protein-specific antibodies and by the observation that the inhibition of the acyltransferase by LiCl was reversed by the addition of acyl carrier protein. Coenzyme A thioesters were not substrates for this acyltransferase. These results suggest the existence of a metabolic cycle for the utilization of 1-position acyl moieties of phosphatidylethanolamine followed by the resynthesis of this membrane phospholipid from 2- acylglycerophosphoethanolamine by an acyl carrier protein-dependent 1-position acyltransferase.     

Cell-free analysis of Golgi apparatus membrane traffic in rat liver

 Cell-free systems for the analysis of Golgi apparatus membrane traffic rely either on highly purified cell fractions or analysis by specific trafficking markers or both. Our work has employed a cell-free transfer system from rat liver based on purified fractions. Transfer of any constituent present in the donor fraction that can be labeled (protein, phospholipid, neutral lipid, sterol, or glycoconjugate) may be investigated in a manner not requiring a processing assay. Transition vesicles were purified and Golgi apparatus cisternae were subfractionated by means of preparative free-flow electrophoresis. Using these transition vesicles and Golgi apparatus subfractions, transfer between transitional endoplasmic reticulum and cis Golgi apparatus was investigated and the process subdivided into vesicle formation and vesicle fusion steps. In liver, vesicle formation exhibited both ATP-independent and ATP-dependent components whereas vesicle fusion was ATP-independent. The ATP-dependent component of transfer was donor and acceptor specific and appeared to be largely unidirectional, i.e., ATP-dependent retrograde (cis Golgi apparatus to transitional endoplasmic reticulum) traffic was not observed. ATP-dependent transfer in the liver system and coatomer-driven ATP-independent transfer in more refined yeast and cultured cell systems are compared and discussed in regard to the liver system. A model mechanism developed for ATP-dependent budding is proposed where a retinol-stimulated and brefeldin A-inhibited NADH protein disulfide oxidoreductase (NADH oxidase) with protein disulfide-thiol interchange activity and an ATP-requiring protein capable of driving physical membrane displacement are involved. It has been suggested that this mechanism drives both the cell enlargement and the vesicle budding that may be associated with the dynamic flow of membranes along the endoplasmic reticulum-vesicle-Golgi apparatus-plasma membrane pathway. Accepted: 26 January 1998     

A missense mutation accounts for the defect in the glycerol-3-phosphate acyltransferase expressed in the plsB26 mutant

The sn-glycerol-3-phosphate acyltransferase (plsB) catalyzes the first step in membrane phospholipid formation. A conditional Escherichia coli mutant (plsB26) has a single missense mutation (G1045A) predicting the expression of an acyltransferase with an Ala349Thr substitution. The PlsB26 protein had a significantly reduced glycerol-3-phosphate acyltransferase specific activity coupled with an elevated Km for glycerol-3-phosphate.     

Cloning of a coconut endosperm cDNA encoding a 1-acyl-sn-glycerol-3-phosphate acyltransferase that accepts medium-chain-length substrates.

Immature coconut (Cocos nucifera) endosperm contains a 1-acyl-sn-glycerol-3-phosphate acyltransferase (LPAAT) activity that shows a preference for medium-chain-length fatty acyl-coenzyme A substrates (H.M. Davies, D.J. Hawkins, J.S. Nelsen [1995] Phytochemistry 39:989-996). Beginning with solubilized membrane preparations, we have used chromatographic separations to identify a polypeptide with an apparent molecular mass of 29 kD, whose presence in various column fractions correlates with the acyltransferase activity detected in those same fractions. Amino acid sequence data obtained from several peptides generated from this protein were used to isolate a full-length clone from a coconut endosperm cDNA library. Clone pCGN5503 contains a 1325-bp cDNA insert with an open reading frame encoding a 308-amino acid protein with a calculated molecular mass of 34.8 kD. Comparison of the deduced amino acid sequence of pCGN5503 to sequences in the data banks revealed significant homology to other putative LPAAT sequences. Expression of the coconut cDNA in Escherichia coli conferred upon those cells a novel LPAAT activity whose substrate activity profile matched that of the coconut enzyme.     

A PLA1-2 punch regulates the Golgi complex

The mammalian Golgi complex, trans Golgi network (TGN) and ER-Golgi intermediate compartment (ERGIC) are comprised of membrane cisternae, coated vesicles and membrane tubules, all of which contribute to membrane trafficking and maintenance of their unique architectures. Recently, a new cast of players was discovered to regulate the Golgi and ERGIC: four unrelated cytoplasmic phospholipase A (PLA) enzymes, cPLA(2)α (GIVA cPLA(2)), PAFAH Ib (GVIII PLA(2)), iPLA(2)-β (GVIA-2 iPLA(2)) and iPLA(1)γ. These ubiquitously expressed enzymes regulate membrane trafficking from specific Golgi subcompartments, although there is evidence for some functional redundancy between PAFAH Ib and cPLA(2)α. Three of these enzymes, PAFAH Ib, cPLA(2)α and iPLA(2)-β, exert effects on Golgi structure and function by inducing the formation of membrane tubules. We review our current understanding of how PLA enzymes regulate Golgi and ERGIC morphology and function.     

Acylation of lysophosphatidylcholine plays a key role in the response of monocytes to lipopolysaccharide.

Mononuclear phagocytes play a pivotal role in the progression of septic shock by producing tumor necrosis factor-alpha (TNF-alpha) and other inflammatory mediators in response to lipopolysaccharide (LPS) from Gram-negative bacteria. Our previous studies have shown monocyte and macrophage activation correlate with changes in membrane phospholipid composition, mediated by acyltransferases. Interferon-gamma (IFN-gamma), which activates and primes these cells for enhanced inflammatory responses to LPS, was found to selectively activate lysophosphatidylcholine acyltransferase (LPCAT) (P < 0.05) but not lysophosphatidic acid acyltransferase (LPAAT) activity. When used to prime the human monocytic cell line MonoMac 6, the production of TNF-alpha and interleukin-6 (IL-6) was approximately five times greater in cells primed with IFN-gamma than unprimed cells. Two LPCAT inhibitors SK&F 98625 (diethyl 7-(3,4,5-triphenyl-2-oxo2,3-dihydro-imidazole-1-yl)heptane phosphonate) and YM 50201 (3-hydroxyethyl 5,3'-thiophenyl pyridine) strongly inhibited (up to 90%) TNF-alpha and IL-6 production in response to LPS in both unprimed MonoMac-6 cells and in cells primed with IFN-gamma. In similar experiments, these inhibitors also substantially decreased the response of both primed and unprimed peripheral blood mononuclear cells to LPS. Sequence-based amplification methods showed that SK&F 98625 inhibited TNF-alpha production by decreasing TNF-alpha mRNA levels in MonoMac-6 cells. Taken together, the data from these studies suggest that LPCAT is a key enzyme in both the pathways of activation (priming) and the inflammatory response to LPS in monocytes.     

sn-glycerol-3-phosphate acyltransferase tubule formation is dependent upon heat shock proteins (htpR)

Overexpression of the Escherichia coli sn-glycerol-3-phosphate (glycerol-P) acyltransferase, an integral membrane protein, causes formation of ordered arrays of the enzyme in vitro. The formation of these tubular structures did not occur in an E. coli strain bearing a mutation in the htpR gene, the regulatory gene for the heat shock response. The htpR165 mutation was shown by genetic analysis to be the lesion responsible for blockage of tubule formation. Similar amounts of glycerol-P acyltransferase were produced in isogenic htpR+ and htpR165 strains, ruling out an effect of htpR165 on expression of glycerol-P acyltransferase. Further, phospholipid metabolism was not altered in either strain after induction of glycerol-P acyltransferase synthesis. Increased glycerol-P acyltransferase synthesis caused a partial induction of the heat shock response which was dependent upon a wild type htpR gene. The heat shock proteins induced were identified as the groEL and dnaK gene products on two-dimensional gels. These two proteins have been implicated in the assembly of bacteriophage coats. These heat shock proteins appear essential for tubule formation.