Sphingolipid Transport: Rafts and Translocators*
- Gerrit van Meer‡ and
- Quirine Lisman
- From the Department of Cell Biology and Histology, Academic Medical Center, University of Amsterdam, P. O. Box 22700, 1100 DE Amsterdam, and Department of Membrane Enzymology, Centre for Biomembranes and Lipid Enzymology, Institute of Biomembranes, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands
Until some 15 years ago, sphingolipids were generally believed to protect the cell surface against harmful factors in the environment by forming a mechanically stable and chemically resistant outer leaflet of the plasma membrane lipid bilayer. Furthermore, complex glycosphingolipids were found to be involved in specific functions like recognition and signaling (1). Whereas the first feature would depend on physical properties of the sphingolipids, the signaling functions involve specific interactions of the complex glycan structures on the glycosphingolipids with similar lipids on neighboring cells or with proteins. Since then, two findings have revolutionized the field. (i) Simple sphingolipid metabolites, like ceramide and sphingosine 1-phosphate, have been found to be important mediators in signaling cascades of apoptosis, proliferation, and stress responses (reviews by Hannun and Obeid (66) and Spiegel and Milstien (67)). (ii) It has been realized that ceramide-based lipids self-aggregate in cellular membranes to form a separate phase that is less fluid (liquid-ordered) than the bulk liquid-disordered phospholipids based on diacylglycerol. Sphingolipid-based microdomains or “rafts” were originally proposed to sort membrane proteins along the cellular pathways of membrane transport (2). Presently, most excitement focuses on their organizing functions in signal transduction (3).
Sphingolipids are synthesized in the ER1 and the Golgi but are enriched in plasma membrane and endosomes where they perform many of their functions. Thus, sphingolipids travel between organelles. Transport occurs via transport vesicles and via monomeric transport through the cytosol. Furthermore, some sphingolipids efficiently translocate across cellular membranes. That transport is not random is clear from the heterogeneous distribution of sphingolipids over the cell; sphingolipids are virtually absent from mitochondria and the ER but constitute 20–35 mol % of the plasma membrane lipids (TableI). Furthermore, signaling pools of sphingolipids do not freely mix with pools of biosynthesis and degradation (reviews by Hannun and Obeid (66), Merrill (68), and Spiegel and Milstien (67)). The specificity in sphingolipid transport is the topic of the present review.
Biosynthetic Traffic and Lipid Translocators
The first steps in sphingolipid synthesis are the condensation of l-serine and palmitoyl-CoA to ketosphinganine and its reduction to sphinganine in the ER membrane. In yeast, these lipids do not feed into signaling pools (4), and exogenous sphingoid bases need to go through a cycle of phosphorylation and dephosphorylation before they can be utilized for ceramide synthesis (5). This suggests that sphingoid bases synthesized de novoare channeled through the pathway into ceramide without being able to escape. In yeast, ceramide is then converted to inositolphosphoceramide and the mannosyl derivatives mannosylinositolphosphoceramide and mannosyldiinositolphosphoceramide on the lumenal surface of the Golgi (6). In mammals, ceramide is utilized for the synthesis of glucosylceramide (GlcCer) on the cytosolic side of the Golgi, sphingomyelin (SM) on the lumenal surface of the Golgi, and in specialized cells, e.g. many epithelial cells, of galactosylceramide (GalCer) in the lumen of the ER (Fig.1) (7). Because ceramide synthesis occurs on the cytosolic side of the ER, the rate of ceramide translocation toward the lumena of ER and Golgi affects the relative synthesis of the various products. If the t of spontaneous ceramide translocation would be tens of minutes (8), this is slow compared with the vesicular transport between ER and Golgi (minutes). However, translocation may be faster in the unsaturated lipid environment of the ER. In addition, ER and Golgi may possess proteins that stimulate ceramide translocation. Ceramide transport to the site of SM synthesis can be inhibited under conditions where transport to the site of GlcCer synthesis and ER-Golgi vesicle transport are normal (9), and besides the vesicular pathway, a non-vesicular mechanism delivers ceramide to the Golgi in mammalian cells and yeast (10, 11). In yeast, this alternative pathway depends on ER-Golgi membrane contact and on a cytosolic factor and is energy-independent (11). Interestingly, close apposition of the ER to cisternae of the trans-Golgi has been observed in mammalian cells (12). In the model of Fig. 1, GlcCer synthase in the cis-Golgi receives ceramide via the vesicular pathway whereas GlcCer synthase and SM synthase in the trans-Golgi (13) receive ceramide from the ER via membrane contacts. Similar contacts have often been observed between ER and mitochondria (12). They may be responsible for the transfer of signaling ceramide to mitochondria. A mitochondrial ceramidase has been identified (Hannun and Obeid (66)).
Sphingolipid synthesis and translocation in the Golgi. Ceramide (Cer) from the cytosolic surface of the ER is converted to GalCer in the ER lumen or transported to the Golgi (G). The GlcCer synthase is found at two locations in the hepatocyte Golgi by sucrose gradient centrifugation (65). One peak colocalized with SM synthase, which was not relocated to the ER by brefeldin A in these cells (7), and thus probably situated in the trans-Golgi/TGN (13). Ceramide reaches the cis-Golgi by vesicular transport whereas ER-TGN contacts allow ceramide transport by exchange. These contacts have been suggested to be sites of general lipid exchange (12), which also holds for similar contacts between ER and mitochondria (M). GlcCer translocates toward the lumen of the Golgi, where it is galactosylated to LacCer. LacCer is the precursor for the various complex glycosphingolipid series. For simplicity, the seven cisternae of the Golgi have been reduced to just two.
GlcCer synthesized on the cytosolic surface of the Golgi is partially converted to complex glycosphingolipids in the Golgi lumen (review by Kolter et al. (69)). Experiments with brefeldin A, which fuses the cis-medial Golgi with the ER, have suggested that the enzymes synthesizing lactosylceramide (LacCer; Fig.1) and the first complex glycosphingolipids are in the early Golgi. However, the bulk of these events is thought to occur in the trans-Golgi or trans-Golgi network (TGN) in vivo (14, 15). GlcCer is probably translocated across the Golgi membrane by an energy-independent translocator (14, 16). Alternatively, GlcCer may be translocated toward the lumen by MDR1 P-glycoprotein, an ATP-binding cassette transporter that causes multidrug resistance (17). However, so far, translocation of GlcCer by MDR1 has only been proven for short chain analogs (18, 19). MDR1 is mostly found at the plasma membrane, where it may clear the cytosolic surface of GlcCer by translocation toward the exoplasmic leaflet. GlcCer has access to this cytosolic surface via the cytosolic side of transport vesicles or, alternatively, via monomeric transport throughout the cytosol (20), possibly mediated by the glycolipid transfer protein (21). An apical GlcCer translocator could thus enrich GlcCer on the apical as compared with the basolateral surface of epithelial cells, after which the difference in lipid composition between the two domains would be maintained by tight junctions acting as a barrier to lipid diffusion in the outer leaflet of the lipid bilayer (22). It is probably by a similar translocator that sphingosine 1-phosphate after synthesis in the cytosol reaches the outside of the plasma membrane and is secreted.
Complex Sphingolipids and Sphingomyelin
GalCer synthesized in the ER lumen may flip toward the cytosolic surface (16), from where it has access to the same sites as GlcCer. In contrast, complex glycosphingolipids and SM synthesized in the lumen of the Golgi appear unable to translocate from the lumenal toward the cytosolic surface (14, 16). As a consequence, they can only leave the Golgi via the lumenal surface of transport vesicles (Fig. 1). This has been confirmed for the complex glycosphingolipid GM3 (sialyl-LacCer (23)), SM (7), and for the yeast inositol sphingolipids (24). The enrichment of complex glycosphingolipids and SM in the exoplasmic leaflet of the apical plasma membrane of epithelial cells (7) as compared with the basolateral surface (Table I) has led to the proposal that these sphingolipids self-aggregate at the site of budding of apical transport vesicles in the TGN (25). Basolateral vesicles would have lower sphingolipid levels but the same high concentration of cholesterol. Sphingolipid and cholesterol concentrations are low in the ER, implying that retrograde transport vesicles are devoid of sphingolipids and cholesterol (22). This has been experimentally confirmed (26). These data led to the simple model for sphingolipid sorting of Fig.2A. Sphingolipid rafts are thought to occur in the early Golgi (27), possibly even in the ER (28).
Lateral segregation of lipids into microdomains.A, the Golgi complex of epithelial cells buds vesicles with at least three different lipid compositions: an apical composition, characterized by high levels of complex glycosphingolipids, SM, and cholesterol (a), a basolateral composition, having a high content of cholesterol (b), and an ER composition, with a low concentration of sphingolipids and cholesterol and a high concentration of unsaturated glycerophospholipids (34) (c). The three phases, displaying different thicknesses, must be recognized by the respective budding machineries in the cytosol, probably via membrane-spanning proteins. The segregation into three phases may occur in one single Golgi cisterna. B, caveolae. In an environment of glycerophospholipids (green), sphingolipid/cholesterol domains enriched in GPI proteins (blue) may contain subdomains enriched in GM1 (red; Ref. 39), with lipid domains enriched in caveolin and dually acylated kinases (brown) oriented toward the cytosol.
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A large body of evidence supports the notion that the lipids of eukaryotic plasma membranes display a heterogeneous lateral distribution. Biophysical studies on model membranes have firmly established the principles by which mixtures of sphingolipids, unsaturated glycerophospholipids, and cholesterol can segregate into two fluid phases, where the sphingolipids and part of the cholesterol segregate into a “liquid-ordered” domain from the unsaturated lipids in a “liquid-disordered” phase. At the same time, these studies have delimited the applicability of detergents in the cold to isolate the domains as detergent-insoluble remnants that float in sucrose gradients (3). A number of questions concerning the structural characteristics of the liquid-ordered domains remain to be solved.
(a) What percentage of the cell surface is occupied by rafts? The diameter of sphingolipid/cholesterol rafts on the outer surface of the plasma membrane has been estimated by a number of approaches to be small (tens to hundreds of nm) compared with that of cells (tens of μm) and to occupy some 10% of the cell surface (29,30). In contrast, sphingolipids constitute 20–50% of the polar lipids of the plasma membrane (Table I) where they are concentrated in the outer bilayer leaflet. Thus, they completely cover the apical surface of epithelial cells, whereas the relative occupancy will be close to 40% in non-epithelial cells. In support of the latter, roughly one-half of the plasma membrane resisted extraction by cold detergent (31, 32). In a monolayer consisting of apical membrane lipids from kidney, only 50% was covered by liquid-ordered rafts whereas the outer leaflet of the apical membrane would consist exclusively of sphingolipids (33).
(b) However, in the experimental monolayer the lipids of the outer and inner leaflets of the plasma membrane mixed, and the domain properties of the lipids of the cytosolic leaflet are unknown. From the fact that dually acylated proteins colocalize with the sphingolipid/cholesterol domains as measured by various techniques, it is assumed that liquid-ordered rafts exist in the cytosolic leaflet of the plasma membrane as well. Of the phosphatidylserine, confined to the cytosolic leaflet by the aminophospholipid translocase, 70% may be disaturated (34), whereas in yeast PI contained some disaturated species (35). These lipids could thus form the basis for a liquid-ordered phase. In pure lipid membranes, rafts on one side of the membrane perfectly match rafts in the opposite leaflet (36). However, from the low concentration of raft-lipids in the cytosolic leaflet it is unlikely that cytosolic rafts fully complement rafts in the outer leaflet of the plasma membrane.
(c) If the rafts measured by biophysical techniques are different from the rafts as defined by detergent insolubility (see question a), does this imply that different types of raft exist within a single membrane? Indeed, studies locating the gangliosides GM1, GM3, and GD3, various proteins with a glycosylphosphatidylinositol (GPI) anchor, and caveolin have clearly established that different liquid-ordered domains co-exist on the cell surface (see Refs. 37 and 38). Small ganglioside-rich microdomains can exist within larger ordered domains in both natural and model membranes (39, 40). Caveolae are examples of such “super” rafts being coupled to cytosolic rafts as defined by the acylated kinases (Fig.2B). Cytosolic rafts may colocalize with each type of domain in the outer leaflet or with only one of the various types of domains. Coupling may involve caveolin or membrane-spanning proteins or may depend on phase-coupling between the opposed lipid domains.
(d) By what mechanisms do membrane proteins locate to domains? One determinant may be a long transmembrane domain that would fit the thicker raft (41, 42). Membranes in cells occur in at least three thicknesses (Fig. 2A). The ER has the thickness of a pure phospholipid bilayer (hydrophobic thickness of some 3.5 nm), the liquid-disordered phase of the plasma membrane displays the thickness of phospholipid plus cholesterol (4 nm), and the sphingolipid rafts may be 4.5–5.5 nm thick (42). Because the thickness of a raft depends on whether it is matched by a cytosolic raft and on the length of the amide-linked fatty acids, the various types of raft may display a distinct thickness and recruit unique sets of proteins. The mechanism of raft association of proteins with multiple transmembrane domains and of protein-protein complexes is more difficult to understand. A GPI anchor targets proteins to specific rafts. The mechanism is not clear. They can be displaced from rafts by gangliosides (see Ref. 33). A reduction of mobility by for example binding of a multivalent ligand also stimulates raft association. Acylation is a signal for raft localization on the cytosolic side (43).
(e) What are the physical properties that determine the affinity of a certain lipid for rafts? Although this question has been answered for model membranes of simple lipid compositions (3), biomembranes contain mixtures of 50–100 lipid species and various types of rafts. This means that many aspects of lipid-lipid immiscibility in these membranes remain to be resolved. Fluorescent reporter molecules have been helpful in spotting lipid sorting events. However, mostly it is not clear to what extent such a molecule mimics a natural lipid.
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Uptake into the Cell
Plasma membrane sphingolipids are continuously taken up into the cell via the membrane flux of endocytosis. In addition, lipids on the cytosolic surface may transfer to other membranes as monomers.
Non-vesicular Uptake from the Plasma Membrane
Most sphingolipids in the exoplasmic leaflet of the plasma membrane bilayer have no access to the cytosolic side under resting conditions. One exception is sphingosine. When added exogenously to cells or when produced in lysosomes, it spontaneously translocates to the cytosolic surface and equilibrates with intracellular membranes. It has been suggested that during cell stimulation, SM may translocate via a scramblase protein to the cytosolic surface where it is then hydrolyzed to ceramide by a neutral sphingomyelinase (see Refs. 44 and45). It is not fully clear how this ceramide reaches sites where it is reutilized for synthesis of SM and GlcCer. Surprisingly, a Golgi protein with lipid transfer specificity for SM strongly stimulated SM resynthesis (46). Ceramide appears unable to leave the lumen of the lysosome (47), possibly due to its inability to leave the internal membranes where it is produced. Exogenous sphingosine 1-phosphate, which binds to specific cell surface receptors, apparently translocates to the cytosolic surface via the ABC transporter CFTR, the cystic fibrosis transmembrane conductance regulator (48). In addition, galactosylsphingosine and glucosylsphingosine, when added to cells, are acylated probably after translocation toward the cytosolic surface. After translocation, lysosphingolipids can freely move through the cell due to their high off-rate from membranes, whereas the resulting GalCer and GlcCer may fulfill functions on the cytosolic surface (49).
Only in one study of many, an exogenous GlcCer (analog) was reported to flip toward the cytosolic surface of the plasma membrane (see Refs. 7and 50). It is not clear whether complex glycosphingolipids ever reach the cytosolic surface of the plasma membrane, nor is it clear what would be their fate. Interestingly, specific interactions of glycosphingolipids have been reported with cytosolic proteins like calmodulin (51). In addition, if gangliosides reach mitochondria during signaling events (52), they must first have reached a cytosolic surface (Fig. 1).
Like other lipids, sphingolipids follow the bulk membrane flow through the exocytotic and endocytotic vesicular transport pathways. From studies on the transport of (mainly) membrane proteins a complex pattern of pathways and compartments has been identified (Fig. 3). Sphingolipids have been shown to pass through each of these compartments. High concentrations of complex glycosphingolipids have been observed in the internal membranes of late endosomes (53, 54), most likely the site of their degradation. On the other hand, studies on the transport and Golgi glycosylation of exogenous glycosphingolipid (analogs) have established that most sphingolipids recycle from the early (sorting) endosomes, the late endosomes, and the recycling endosomes to the plasma membrane. At the same time, a fraction of the complex sphingolipids, but particularly GlcCer, reaches the Golgi complex (7). The latter is also true for the glycolipid-binding toxins like cholera and Shiga toxin and Escherichia coli verotoxin. From the Golgi, the toxin-glycolipid complexes follow the retrograde pathway all the way to the ER, where the active subunit is translocated across the membrane into the cytosol (e.g. Ref. 55). Also in the absence of toxin, a small fraction of the complex glycosphingolipids reaches the ER (53).
Endocytotic recycling of sphingolipids.Sphingolipids can be endocytosed via clathrin-dependent and -independent pathways. From early endosomes (EE) they are recycled to the plasma membrane or shuttled to the recycling endosome (RE), the late endosome (LE), or the TGN. Endosomes and Golgi are connected via a bidirectional vesicular route. In epithelial cells, one leg of the system is connected to the apical and one to the basolateral surface.
Although ample evidence supports lipid sorting by domains in the various endocytotic organelles, most of this evidence is derived from using lipid analogs, toxins, antibodies and virus bound to glycosphingolipids, and GPI-proteins as raft markers. The quantitative behavior of the natural lipids and the size of the various pathways remain to be established. Analogs of LacCer and globoside were endocytosed by a clathrin-independent subclass of the vesicles that took up SM, indicating sorting at the plasma membrane (56). The two pathways led to different classes of early endosomes, both of which had a connection to the Golgi. Both clathrin-dependent and -independent pathways are followed by glycolipid-bound toxins (e.g. Refs. 55 and 56). The clathrin-independent pathway of toxin transport has been suggested to provide very efficient access to the Golgi for GPI-proteins (55) and might be a raft pathway. Interestingly, a rise in the cellular cholesterol concentration misrouted LacCer to the lysosomes. The latter situation was also encountered in a number of sphingolipid storage diseases (see Ref. 56). Either high cholesterol levels abolished the clathrin-independent pathway to the Golgi, or alternatively, high cholesterol affected the partitioning of the LacCer analog into the proper membrane domain. The biophysical basis for domain-mediated sorting in the endosomes, notably the interaction between the various domains and coat proteins, remains to be established.
Endocytotic lipid sorting is particularly interesting in epithelial cells, as these display a transcellular vesicular pathway but at the same time need to maintain the difference in apical and basolateral lipid composition. In initial studies in Madin-Darby canine kidney cells no specificity was observed in the transcytosis of SM and GlcCer analogs, which allowed for their use as bulk membrane markers (7). However, sorting between these analogs was observed in hepatocyte-derived HepG2 cells. It was concluded that the lipids are sorted by lateral segregation in an apical endosome, termed the “subapical compartment” (see Ref. 57) or “apical recycling compartment” (58), which functionally resembles the recycling endosome in non-epithelial cells. Under normal conditions in fully polarized hepatocytes, a GlcCer analog was recycled to the bile canalicular surface and SM to the basolateral surface (see Ref. 57), and evidence was provided for transient activation during polarity development of a pathway for SM to the bile canalicular surface by protein kinase A (59). Transcytosis of GalCer from the apical to the basolateral membrane of enterocytes has been held responsible for allowing the passage of human immunodeficiency virus across the intestinal epithelium (see Ref. 60).
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The exciting developments in the fields of sphingolipid-mediated signal transduction and sphingolipid-mediated protein sorting have led to a tremendous activity in the studies of sphingolipid organization, especially the structural role of sphingolipids in membrane rafts. It is now being realized that such rafts exist in most cellular membranes. To fully grasp raft function, it will be necessary to identify and characterize the different types of raft, to follow their fate in time, and to understand the role of the various sphingolipids in their structure. Although one important challenge will be to unravel the biophysical complexity of lipid mixtures, it will be most important to define the interactions between sphingolipids and proteins. These are proteins involved in signaling but also proteins involved in vesicular transport. Except for structural functions, sphingolipids serve regulatory functions in their own right. Because sphingolipid functions are governed by the enzymes that make, break, and transport the sphingolipids, another major challenge will be to identify these enzymes and establish how their activity is regulated in the living cell.
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↵* This minireview will be reprinted in the 2002 Minireview Compendium, which will be available in December, 2002. This work was supported by the Dr. Anton Meelmeijer Program (AMC) (to Q. L.), European Community Research Training Network on Sphingolipids Grant HPRN-CT-2000-00077, Zorgonderzoek Nederland Medical Sciences (ZonMW), and the Ministry of Economic Affairs (Senter) (to G. v. M.). This is the fourth article of five in the “Sphingolipid Metabolism and Signaling Minireview Series.”
↵‡ To whom correspondence should be addressed. Tel.: 31-30-2533427; Fax: 31-30-2522478; E-mail: email@example.com.
Published, JBC Papers in Press, May 13, 2002, DOI 10.1074/jbc.R200010200
- endoplasmic reticulum
- trans-Golgi network
- The American Society for Biochemistry and Molecular Biology, Inc.
Sphingolipid content of plasma membranes
Biosynthesis (also called anabolism) is a multi-step, enzyme-catalyzed process where substrates are converted into more complex products in living organisms. In biosynthesis, simple compounds are modified, converted into other compounds, or joined together to form macromolecules. This process often consists of metabolic pathways. Some of these biosynthetic pathways are located within a single cellular organelle, while others involve enzymes that are located within multiple cellular organelles. Examples of these biosynthetic pathways include the production of lipid membrane components and nucleotides.
The prerequisite elements for biosynthesis include: precursor compounds, chemical energy (e.g. ATP), and catalytic enzymes which may require coenzymes (e.g.NADH, NADPH). These elements create monomers, the building blocks for macromolecules. Some important biological macromolecules include: proteins, which are composed of amino acid monomers joined via peptide bonds, and DNA molecules, which are composed of nucleotides joined via phosphodiester bonds.
Properties of chemical reactions
Biosynthesis occurs due to a series of chemical reactions. For these reactions to take place, the following elements are necessary:
- Precursor compounds: these compounds are the starting molecules or substrates in a reaction. These may also be viewed as the reactants in a given chemical process.
- Chemical energy: chemical energy can be found in the form of high energy molecules. These molecules are required for energetically unfavorable reactions. Furthermore, the hydrolysis of these compounds drives a reaction forward. High energy molecules, such as ATP, have three phosphates. Often, the terminal phosphate is split off during hydrolysis and transferred to another molecule.
- Catalytic enzymes: these molecules are special proteins that catalyze a reaction by increasing the rate of the reaction and lowering the activation energy.
- Coenzymes or cofactors: cofactors are molecules that assist in chemical reactions. These may be metal ions, vitamin derivatives such as NADH and acetyl CoA, or non-vitamin derivatives such as ATP. In the case of NADH, the molecule transfers a hydrogen, whereas acetyl CoA transfers an acetyl group, and ATP transfers a phosphate.
In the simplest sense, the reactions that occur in biosynthesis have the following format:
Some variations of this basic equation which will be discussed later in more detail are:
- Simple compounds which are converted into other compounds, usually as part of a multiple step reaction pathway. Two examples of this type of reaction occur during the formation of nucleic acids and the charging of tRNA prior to translation. For some of these steps, chemical energy is required:
- Simple compounds that are converted into other compounds with the assistance of cofactors. For example, the synthesis of phospholipids requires acetyl CoA, while the synthesis of another membrane component, sphingolipids, requires NADH and FADH for the formation the sphingosine backbone. The general equation for these examples is:
- Simple compounds that join together to create a macromolecule. For example, fatty acids join together to form phospholipids. In turn, phospholipids and cholesterol interact noncovalently in order to form the lipid bilayer. This reaction may be depicted as follows:
Many intricate macromolecules are synthesized in a pattern of simple, repeated structures. For example, the simplest structures of lipids are fatty acids. Fatty acids are hydrocarbon derivatives; they contain a carboxyl group "head" and a hydrocarbon chain "tail". These fatty acids create larger components, which in turn incorporate noncovalent interactions to form the lipid bilayer. Fatty acid chains are found in two major components of membrane lipids: phospholipids and sphingolipids. A third major membrane component, cholesterol, does not contain these fatty acid units.
The foundation of all biomembranes consists of a bilayer structure of phospholipids. The phospholipid molecule is amphipathic; it contains a hydrophilic polar head and a hydrophobic nonpolar tail. The phospholipid heads interact with each other and aqueous media, while the hydrocarbon tails orient themselves in the center, away from water. These latter interactions drive the bilayer structure that acts as a barrier for ions and molecules.
There are various types of phospholipids; consequently, their synthesis pathways differ. However, the first step in phospholipid synthesis involves the formation of phosphatidate or diacylglycerol 3-phosphate at the endoplasmic reticulum and outer mitochondrial membrane. The synthesis pathway is found below:
The pathway starts with glycerol 3-phosphate, which gets converted to lysophosphatidate via the addition of a fatty acid chain provided by acyl coenzyme A. Then, lysophosphatidate is converted to phosphatidate via the addition of another fatty acid chain contributed by a second acyl CoA; all of these steps are catalyzed by the glycerol phosphate acyltransferase enzyme. Phospholipid synthesis continues in the endoplasmic reticulum, and the biosynthesis pathway diverges depending on the components of the particular phospholipid.
Like phospholipids, these fatty acid derivatives have a polar head and nonpolar tails. Unlike phospholipids, sphingolipids have a sphingosine backbone. Sphingolipids exist in eukaryotic cells and are particularly abundant in the central nervous system. For example, sphingomyelin is part of the myelin sheath of nerve fibers.
Sphingolipids are formed from ceramides that consist of a fatty acid chain attached to the amino group of a sphingosine backbone. These ceramides are synthesized from the acylation of sphingosine. The biosynthetic pathway for sphingosine is found below:
As the image denotes, during sphingosine synthesis, palmitoyl CoA and serine undergo a condensation reaction which results in the formation of dehydrosphingosine. This product is then reduced to form dihydrospingosine, which is converted to sphingosine via the oxidation reaction by FAD.
This lipid belongs to a class of molecules called sterols. Sterols have four fused rings and a hydroxyl group. Cholesterol is a particularly important molecule. Not only does it serve as a component of lipid membranes, it is also a precursor to several steroid hormones, including cortisol, testosterone, and estrogen.
Cholesterol is synthesized from acetyl CoA. The pathway is shown below:
More generally, this synthesis occurs in three stages, with the first stage taking place in the cytoplasm and the second and third stages occurring in the endoplasmic reticulum. The stages are as follows:
- 1. The synthesis of isopentenyl pyrophosphate, the "building block" of cholesterol
- 2. The formation of squalene via the condensation of six molecules of isopentenyl phosphate
- 3. The conversion of squalene into cholesterol via several enzymatic reactions
The biosynthesis of nucleotides involves enzyme-catalyzed reactions that convert substrates into more complex products. Nucleotides are the building blocks of DNA and RNA. Nucleotides are composed of a five-membered ring formed from ribose sugar in RNA, and deoxyribose sugar in DNA; these sugars are linked to a purine or pyrimidine base with a glycosidic bond and a phosphate group at the 5' location of the sugar.
The DNA nucleosides adenosine and guanosine consist of a purine base attached to a ribose sugar with a glycosidic bond. In the case of RNA nucleotides deoxyadenosine and deoxyguanosine, the purine bases are attached to a deoxyribose sugar with a glycosidic bond. The purine bases on DNA and RNA nucleotides are synthesized in a twelve-step reaction mechanism present in most single-celled organisms. Higher eukaryotes employ a similar reaction mechanism in ten reaction steps. Purine bases are synthesized by converting phosphoribosyl pyrophosphate (PRPP) to inosine monophosphate (IMP), which is the first key intermediate in purine base biosynthesis. Further enzymatic modification of IMP produces the adenosine and guanosine bases of nucleotides.
- The first step in purine biosynthesis is a condensation reaction, performed by glutamine-PRPP amidotransferase. This enzyme transfers the amino group from glutamine to PRPP, forming 5-phosphoribosylamine. The following step requires the activation of glycine by the addition of a phosphate group from ATP.
- GAR synthetase performs the condensation of activated glycine onto PRPP, forming glycineamide ribonucleotide (GAR).
- GAR transformylase adds a formyl group onto the amino group of GAR, forming formylglycinamide ribonucleotide (FGAR).
- FGAR amidotransferase catalyzes the addition of a nitrogen group to FGAR, forming formylglycinamidine ribonucleotide (FGAM).
- FGAM cyclase catalyzes ring closure, which involves removal of a water molecule, forming the 5-membered imidazole ring 5-aminoimidazole ribonucleotide (AIR).
- N5-CAIR synthetase transfers a carboxyl group, forming the intermediate N5-carboxyaminoimidazole ribonucleotide (N5-CAIR).
- N5-CAIR mutase rearranges the carboxyl functional group and transfers it onto the imidazole ring, forming carboxyamino- imidazole ribonucleotide (CAIR). The two step mechanism of CAIR formation from AIR is mostly found in single celled organisms. Higher eukaryotes contain the enzyme AIR carboxylase, which transfers a carboxyl group directly to AIR imidazole ring, forming CAIR.
- SAICAR synthetase forms a peptide bond between aspartate and the added carboxyl group of the imidazole ring, forming N-succinyl-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR).
- SAICAR lyase removes the carbon skeleton of the added aspartate, leaving the amino group and forming 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR).
- AICAR transformylase transfers a carbonyl group to AICAR, forming N-formylaminoimidazole- 4-carboxamide ribonucleotide (FAICAR).
- The final step involves the enzyme IMP synthase, which performs the purine ring closure and forms the inosine monophosphate intermediate.
Other DNA and RNA nucleotide bases that are linked to the ribose sugar via a glycosidic bond are thymine, cytosine and uracil (which is only found in RNA). Uridine monophosphate biosynthesis involves an enzyme that is located in the mitochondrial inner membrane and multifunctional enzymes that are located in the cytosol.
- The first step involves the enzyme carbamoyl phosphate synthase combining glutamine with CO2 in an ATP dependent reaction to form carbamoyl phosphate.
- Aspartate carbamoyltransferasecondenses carbamoyl phosphate with aspartate to form uridosuccinate.
- Dihydroorotase performs ring closure, a reaction that loses water, to form dihydroorotate.
- Dihydroorotate dehydrogenase, located within the mitochondrial inner membrane, oxidizes dihydroorotate to orotate.
- Orotate phosphoribosyl hydrolase (OMP pyrophosphorylase) condenses orotate with PRPP to form orotidine-5'-phosphate.
- OMP decarboxylase catalyzes the conversion of orotidine-5'-phosphate to UMP.
After the uridine nucleotide base is synthesized, the other bases, cytosine and thymine are synthesized. Cytosine biosynthesis is a two-step reaction which involves the conversion of UMP to UTP. Phosphate addition to UMP is catalyzed by a kinase enzyme. The enzyme CTP synthase catalyzes the next reaction step: the conversion of UTP to CTP by transferring an amino group from glutamine to uridine; this forms the cytosine base of CTP. The mechanism, which depicts the reaction UTP + ATP + glutamine ⇔ CTP + ADP + glutamate, is below:
Cytosine is a nucleotide that is present in both DNA and RNA. However, uracil is only found in RNA. Therefore, after UTP is synthesized, it is must be converted into a deoxy form to be incorporated into DNA. This conversion involves the enzyme ribonucleoside triphosphate reductase. This reaction that removes the 2'-OH of the ribose sugar to generate deoxyribose is not affected by the bases attached to the sugar. This non-specificity allows ribonucleoside triphosphate reductase to convert all nucleotide triphosphates to deoxyribonucleotide by a similar mechanism.
In contrast to uracil, thymine bases are found mostly in DNA, not RNA. Cells do not normally contain thymine bases that are linked to ribose sugars in RNA, thus indicating that cells only synthesize deoxyribose-linked thymine. The enzyme thymidylate synthetase is responsible for synthesizing thymine residues from dUMP to dTMP. This reaction transfers a methyl group onto the uracil base of dUMP to generate dTMP. The thymidylate synthase reaction, dUMP + 5,10-methylenetetrahydrofolate ⇔ dTMP + dihydrofolate, is shown to the right.
Although there are differences between eukaryotic and prokaryotic DNA synthesis, the following section denotes key characteristics of DNA replication shared by both organisms.
DNA is composed of nucleotides that are joined by phosphodiester bonds.DNA synthesis, which takes place in the nucleus, is a semiconservative process, which means that the resulting DNA molecule contains an original strand from the parent structure and a new strand. DNA synthesis is catalyzed by a family of DNA polymerases that require four deoxynucleoside triphosphates, a template strand, and a primer with a free 3'OH in which to incorporate nucleotides.
In order for DNA replication to occur, a replication fork is created by enzymes called helicases which unwind the DNA helix.Topoisomerases at the replication fork remove supercoils caused by DNA unwinding, and single-stranded DNA binding proteins maintain the two single-stranded DNA templates stabilized prior to replication.
DNA synthesis is initiated by the RNA polymeraseprimase, which makes an RNA primer with a free 3'OH. This primer is attached to the single-stranded DNA template, and DNA polymerase elongates the chain by incorporating nucleotides; DNA polymerase also proofreads the newly synthesized DNA strand.
During the polymerization reaction catalyzed by DNA polymerase, a nucleophilic attack occurs by the 3'OH of the growing chain on the innermost phosphorus atom of a deoxynucleoside triphosphate; this yields the formation of a phosphodiester bridge that attaches a new nucleotide and releases pyrophosphate.
Two types of strands are created simultaneously during replication: the leading strand, which is synthesized continuously and grows towards the replication fork, and the lagging strand, which is made discontinuously in Okazaki fragments and grows away from the replication fork. Okazaki fragments are covalently joined by DNA ligase to form a continuous strand. Then, to complete DNA replication, RNA primers are removed, and the resulting gaps are replaced with DNA and joined via DNA ligase.
A protein is a polymer that is composed from amino acids that are linked by peptide bonds. There are more than 300 amino acids found in nature of which only twenty, known as the standard amino acids, are the building blocks for protein. Only green plants and most microbes are able to synthesize all of the 20 standard amino acids that are needed by all living species. Mammals can only synthesize ten of the twenty standard amino acids. The other amino acids, valine, methionine, leucine, isoleucine, phenylalanine, lysine, threonine and tryptophan for adults and histidine, and arginine for babies are obtained through diet.
Amino acid basic structure
The general structure of the standard amino acids includes a primary amino group, a carboxyl group and the functional group attached to the α-carbon. The different amino acids are identified by the functional group. As a result of the three different groups attached to the α-carbon, amino acids are asymmetrical molecules. For all standard amino acids, except glycine, the α-carbon is a chiral center. In the case of glycine, the α-carbon has two hydrogen atoms, thus adding symmetry to this molecule. With the exception of proline, all of the amino acids found in life have the L-isoform conformation. Proline has a functional group on the α-carbon that forms a ring with the amino group.
One major step in amino acid biosynthesis involves incorporating a nitrogen group onto the α-carbon. In cells, there are two major pathways of incorporating nitrogen groups. One pathway involves the enzyme glutamine oxoglutarate aminotransferase (GOGAT) which removes the amide amino group of glutamine and transfers it onto 2-oxoglutarate, producing two glutamate molecules. In this catalysis reaction, glutamine serves as the nitrogen source. An image illustrating this reaction is found to the right.
The other pathway for incorporating nitrogen onto the α-carbon of amino acids involves the enzyme glutamate dehydrogenase (GDH). GDH is able to transfer ammonia onto 2-oxoglutarate and form glutamate. Furthermore, the enzyme glutamine synthetase (GS) is able to transfer ammonia onto glutamate and synthesize glutamine, replenishing glutamine.
The glutamate family of amino acids
The glutamate family of amino acids includes the amino acids that derive from the amino acid glutamate. This family includes: glutamate, glutamine, proline, and arginine. This family also includes the amino acid lysine, which is derived from α-ketoglutarate.
The biosynthesis of glutamate and glutamine is a key step in the nitrogen assimilation discussed above. The enzymes GOGAT and GDH catalyze the nitrogen assimilation reactions.
In bacteria, the enzyme glutamate 5-kinase initiates the biosynthesis of proline by transferring a phosphate group from ATP onto glutamate. The next reaction is catalyzed by the enzyme pyrroline-5-carboxylate synthase (P5CS), which catalyzes the reduction of the ϒ-carboxyl group of L-glutamate 5-phosphate. This results in the formation of glutamate semialdehyde, which spontaneously cyclizes to pyrroline-5-carboxylate. Pyrroline-5-carboxylate is further reduced by the enzyme pyrroline-5-carboxylate reductase (P5CR) to yield a proline amino acid.
In the first step of arginine biosynthesis in bacteria, glutamate is acetylated by transferring the acetyl group from acetyl-CoA at the N-α position; this prevents spontaneous cyclization. The enzyme N-acetylglutamate synthase (glutamate N-acetyltransferase) is responsible for catalyzing the acetylation step. Subsequent steps are catalyzed by the enzymes N-acetylglutamate kinase, N-acetyl-gamma-glutamyl-phosphate reductase, and acetylornithine/succinyldiamino pimelate aminotransferase and yield the N-acetyl-L-ornithine. The acetyl group of acetylornithine is removed by the enzyme acetylornithinase (AO) or ornithine acetyltransferase (OAT), and this yields ornithine. Then, the enzymes citrulline and argininosuccinate convert ornithine to arginine.
There are two distinct lysine biosynthetic pathways: the diaminopimelic acid pathway and the α-aminoadipate pathway. The most common of the two synthetic pathways is the diaminopimelic acid pathway; it consists of several enzymatic reactions that add carbon groups to aspartate to yield lysine:
- Aspartate kinase initiates the diaminopimelic acid pathway by phosphorylating aspartate and producing aspartyl phosphate.
- Aspartate semialdehyde dehydrogenase catalyzes the NADPH-dependent reduction of aspartyl phosphate to yield aspartate semialdehyde.
- 4-hydroxy-tetrahydrodipicolinate synthase adds a pyruvate group to the β-aspartyl-4-semialdehyde, and a water molecule is removed. This causes cyclization and gives rise to (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate.
- 4-hydroxy-tetrahydrodipicolinate reductase catalyzes the reduction of (2S,4S)-4-hydroxy-2,3,4,5-tetrahydrodipicolinate by NADPH to yield Δ'-piperideine-2,6-dicarboxylate (2,3,4,5-tetrahydrodipicolinate) and H2O.
- Tetrahydrodipicolinate acyltransferase catalyzes the acetylation reaction that results in ring opening and yields N-acetyl α-amino-ε-ketopimelate.
- N-succinyl-α-amino-ε-ketopimelate-glutamate aminotransaminase catalyzes the transamination reaction that removes the keto group of N-acetyl α-amino-ε-ketopimelate and replaces it with an amino group to yield N-succinyl-L-diaminopimelate.
- N-acyldiaminopimelate deacylase catalyzes the deacylation of N-succinyl-L-diaminopimelate to yield L,L-diaminopimelate.
- DAP epimerase catalyzes the conversion of L,L-diaminopimelate to the meso form of L,L-diaminopimelate.
- DAP decarboxylase catalyzes the removal of the carboxyl group, yielding L-lysine.
The serine family of amino acids
The serine family of amino acid includes: serine, cysteine, and glycine. Most microorganisms and plants obtain the sulfur for synthesizing methionine from the amino acid cysteine. Furthermore, the conversion of serine to glycine provides the carbons needed for the biosynthesis of the methionine and histidine.
During serine biosynthesis, the enzyme phosphoglycerate dehydrogenase catalyzes the initial reaction that oxidizes3-phospho-D-glycerate to yield 3-phosphonooxypyruvate. The following reaction is catalyzed by the enzyme phosphoserine aminotransferase, which transfers an amino group from glutamate onto 3-phosphonooxypyruvate to yield L-phosphoserine. The final step is catalyzed by the enzyme phosphoserine phosphatase, which dephosphorylates L-phosphoserine to yield L-serine.
There are two known pathways for the biosynthesis of glycine. Organisms that use ethanol and acetate as the major carbon source utilize the glyconeogenic pathway to synthesize glycine. The other pathway of glycine biosynthesis is known as the glycolytic pathway. This pathway converts serine synthesized from the intermediates of glycolysis to glycine. In the glycolytic pathway, the enzyme serine hydroxymethyltransferase catalyzes the cleavage of serine to yield glycine and transfers the cleaved carbon group of serine onto tetrahydrofolate, forming 5,10-methylene-tetrahydrofolate.
Cysteine biosynthesis is a two-step reaction that involves the incorporation of inorganic sulfur. In microorganisms and plants, the enzyme serine acetyltransferase catalyzes the transfer of acetyl group from acetyl-CoA onto L-serine to yield O-acetyl-L-serine. The following reaction step, catalyzed by the enzyme O-acetyl serine (thiol) lyase, replaces the acetyl group of O-acetyl-L-serine with sulfide to yield cysteine.
The aspartate family of amino acids
The aspartate family of amino acids includes: threonine, lysine, methionine, isoleucine, and aspartate. Lysine and isoleucine are considered part of the aspartate family even though part of their carbon skeleton is derived from pyruvate. In the case of methionine, the methyl carbon is derived from serine and the sulfur group, but in most organisms, it is derived from cysteine.
The biosynthesis of aspartate is a one step reaction that is catalyzed by a single enzyme. The enzyme aspartate aminotransferase catalyzes the transfer of an amino group from aspartate onto α-ketoglutarate to yield glutamate and oxaloacetate. Asparagine is synthesized by an ATP-dependent addition of an amino group onto aspartate; asparagine synthetase catalyzes the addition of nitrogen from glutamine or soluble ammonia to aspartate to yield asparagine.
The diaminopimelic acid biosynthetic pathway of lysine belongs to the aspartate family of amino acids. This pathway involves nine enzyme-catalyzed reactions that convert aspartate to lysine.
- Aspartate kinase catalyzes the initial step in the diaminopimelic acid pathway by transferring a phosphoryl from ATP onto the carboxylate group of aspartate, which yields aspartyl-β-phosphate.
- Aspartate-semialdehyde dehydrogenase catalyzes the reduction reaction by dephosphorylation of aspartyl-β-phosphate to yield aspartate-β-semialdehyde.
- Dihydrodipicolinate synthase catalyzes the condensation reaction of aspartate-β-semialdehyde with pyruvate to yield dihydrodipicolinic acid.
- 4-hydroxy-tetrahydrodipicolinate reductase catalyzes the reduction of dihydrodipicolinic acid to yield tetrahydrodipicolinic acid.
- Tetrahydrodipicolinate N-succinyltransferase catalyzes the transfer of a succinyl group from succinyl-CoA on to tetrahydrodipicolinic acid to yield N-succinyl-L-2,6-diaminoheptanedioate.
- N-succinyldiaminopimelate aminotransferase catalyzes the transfer of an amino group from glutamate onto N-succinyl-L-2,6-diaminoheptanedioate to yield N-succinyl-L,L-diaminopimelic acid.
- Succinyl-diaminopimelate desuccinylase catalyzes the removal of acyl group from N-succinyl-L,L-diaminopimelic acid to yield L,L-diaminopimelic acid.
- Diaminopimelate epimerase catalyzes the inversion of the α-carbon of L,L-diaminopimelic acid to yield meso-diaminopimelic acid.
- Siaminopimelate decarboxylase catalyzes the final step in lysine biosynthesis that removes the carbon dioxide group from meso-diaminopimelic acid to yield L-lysine.
Protein synthesis occurs via a process called translation. During translation, genetic material called mRNA is read by ribosomes to generate a protein polypeptide chain. This process requires transfer RNA (tRNA) which serves as an adaptor by binding amino acids on one end and interacting with mRNA at the other end; the latter pairing between the tRNA and mRNA ensures that the correct amino acid is added to the chain. Protein synthesis occurs in three phases: initiation, elongation, and termination.Prokaryotic translation differs from eukaryotic translation; however, this section will mostly focus on the commonalities between the two organisms.
Before translation can begin, the process of binding a specific amino acid to its corresponding tRNA must occur. This reaction, called tRNA charging, is catalyzed by aminoacyl tRNA synthetase. A specific tRNA synthetase is responsible for recognizing and charging a particular amino acid. Furthermore, this enzyme has special discriminator regions to ensure the correct binding between tRNA and its cognate amino acid. The first step for joining an amino acid to its corresponding tRNA is the formation of aminoacyl-AMP:
This is followed by the transfer of the aminoacyl group from aminoacyl-AMP to a tRNA molecule. The resulting molecule is aminoacyl-tRNA:
The combination of these two steps, both of which are catalyzed by aminoacyl tRNA synthetase, produces a charged tRNA that is ready to add amino acids to the growing polypeptide chain.
In addition to binding an amino acid, tRNA has a three nucleotide unit called an anticodon that base pairs with specific nucleotide triplets on the mRNA called codons; codons encode a specific amino acid. This interaction is possible thanks to the ribosome, which serves as the site for protein synthesis. The ribosome possesses three tRNA binding sites: the aminoacyl site (A site), the peptidyl site (P site), and the exit site (E site).
There are numerous codons within an mRNA transcript, and it is very common for an amino acid to be specified by more than one codon; this phenomenon is called degeneracy. In all, there are 64 codons, 61 of each code for one of the 20 amino acids, while the remaining codons specify chain termination.
Translation in steps
As previously mentioned, translation occurs in three phases: initiation, elongation, and termination.
Step 1: Initiation
The completion of the initiation phase is dependent on the following three events:
1. The recruitment of the ribosome to mRNA
2. The binding of a charged initiator tRNA into the P site of the ribosome
3. The proper alignment of the ribosome with mRNA's start codon
Step 2: Elongation
Following initiation, the polypeptide chain is extended via anticodon:codon interactions, with the ribosome adding amino acids to the polypeptide chain one at a time. The following steps must occur to ensure the correct addition of amino acids:
1. The binding of the correct tRNA into the A site of the ribosome
2. The formation of a peptide bond between the tRNA in the A site and the polypeptide chain attached to the tRNA in the P site
3. Translocation or advancement of the tRNA-mRNA complex by three nucleotides
Translocation "kicks off" the tRNA at the E site and shifts the tRNA from the A site into the P site, leaving the A site free for an incoming tRNA to add another amino acid.
Step 3: Termination
The last stage of translation occurs when a stop codon enters the A site. Then, the following steps occur:
1. The recognition of codons by release factors, which causes the hydrolysis of the polypeptide chain from the tRNA located in the P site
2. The release of the polypeptide chain
3. The dissociation and "recycling" of the ribosome for future translation processes
A summary table of the key players in translation is found below:
|Key players in Translation||Translation Stage||Purpose|
|tRNA synthetase||before initiation||Responsible for tRNA charging|
|mRNA||initiation, elongation, termination||Template for protein synthesis; contains regions named codons which encode amino acids|
|tRNA||initiation, elongation, termination||Binds ribosomes sites A, P, E; anticodon base pairs with mRNA codon to ensure that the correct amino acid is incorporated into the growing polypeptide chain|
|ribosome||initiation, elongation, termination||Directs protein synthesis and catalyzes the formation of the peptide bond|
Diseases associated with macromolecule deficiency
Errors in biosynthetic pathways can have deleterious consequences including the malformation of macromolecules or the underproduction of functional molecules. Below are examples that illustrate the disruptions that occur due to these inefficiencies.
Uridine monophosphate (UMP) biosynthesis
As DNA polymerase moves in a 3' to 5' direction along the template strand, it synthesizes a new strand in the 5' to 3' direction
The tRNA anticodon interacts with the mRNA codon in order to bind an amino acid to growing polypeptide chain.
The process of tRNA charging