All of the references to this I can find refer to enzymes like Flippase making it 'easier' or 'more likely' that the translocation will occur, rather than actually make it possible.
The following is from Alberts
In the ER, however, phospholipids equilibrate across the membrane within minutes, which is almost 100,000 times faster than can be accounted for by spontaneous “flip-flop.”
Does this mean that left unattended, any phospholipid membrane will 'flip-flop' spontaneously, just very slowly, and can this be observed?
Bilayer components will 'flip-flop' at measurable rates, but these are very different for different lipid classes. Here are the results of an experiment using fluorescently-labelled analogues.
Bai, JN and Pagano, RE (1997) Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles. Biochemistry 36:8840-8848 DOI: 10.1021/bi970145r
The authors followed the transbilayer (flip-flop) and interbilayer movement of fluorescently-labelled analogues of various membrane lipids: sphingomyelin (C-5-DMB-SM), ceramide (C-5-DMB-Cer), phosphatidylcholine (C-5-DMB-PC) and diacylglycerol (C-5-DMB-DAG) in a system of unilamellar vesicles consisting of 1-palmitoyl-2-oleoyl phosphatidylcholine.
The results were analysed in terms of a model in which the labelled molecules could move between vesicles and between the two monolayers of the bilayer.
half times: interbilayer transbilayer (flip-flop) C-5-DMB-SM >21 s 3.3 h C-5-DMB-Cer 350 s 22 min C-5-DMB-PC 400 s 7.5 h C-5-DMB-DAG 100 h 70 ms
So, for the phospholipid tested the half-time for flip-flop was 7.5 hours.
Model studies of lipid flip-flop in membranes
Biomembranes, which are made of a lipid bilayer matrix where proteins are embedded or attached, constitute a physical barrier for cell and its internal organelles. With regard to the distribution of their molecular components, biomembranes are both laterally heterogeneous and transversally asymmetric, and because of this they are sites of vital biochemical activities. Lipids may translocate from one leaflet of the bilayer to the opposite either spontaneously or facilitated by proteins, hence they contribute to the regulation of membrane asymmetry on which cell functioning, differentiation, and growth heavily depend. Such transverse motion—commonly called flip-flop—has been studied both experimentally and computationally. Experimental investigations face difficulties related to time-scales and probe-induced membrane perturbation issues. Molecular dynamics simulations play an important role for the molecular-level understanding of flip-flop. In this review we present a summary of the state of the art of computational studies of spontaneous flip-flop of phospholipids, sterols and fatty acids. Also, we highlight critical issues and strategies that have been developed to solve them, and what remains to be solved.
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First step: across the IM. Model systems
Once the cytoplasmic steps of biosynthesis are complete, newly synthesized phospholipids and Ra-lipid A are transported across the IM bilayer. The mechanisms used to shuttle lipids across biological membranes (flip-flop) remain poorly understood. Transport of amphipathic lipids across a hydrophobic lipid bilayer is predicted to be thermodynamically unfavourable, requiring input of energy ( Singer and Nicolson, 1972 ). Transport of lipids across bilayers has been studied in many excellent in vitro models. In this discussion, I will use the general term flip-flop to describe the outward movement of lipids across a bilayer. While this term does not discern outward from inward movement of lipids, only the former will be considered. Lipid flip-flop in model lipid bilayers is extremely slow, with half-lives on the order of hours to days, but is very fast in biological membranes, occurring in seconds to tens of seconds ( Rothman and Kennedy, 1977 ). This has led to the conclusion that lipid flip-flop in biological membranes is protein catalysed. Several candidate proteins have been identified in eukaryotes that catalyse transbilayer movement of different classes of lipids ( Daleke, 2003 Holthuis and Levine, 2005 Pohl et al., 2005 ). However, in prokaryotes, the only protein thus far identified with a proven role in lipid transport is MsbA (see below).
Differing conclusions have been reached with regard to energy requirements for IM lipid transport. In some systems, lipid flip-flop is ATP dependent but not in others. Work by deKruijff and colleagues suggests that lipid flip-flop in reconstituted vesicles is energy independent and requires only hydrophobic membrane-spanning α-helical peptides ( Kol et al., 2004 ). In their studies, the authors tested the ability of membrane-spanning peptides to induce flip-flop of short chained, fluorescently labelled phospholipids (C6-NBD-PE and C6-NBD-PG) in artificial bilayers ( Kol et al., 2001 ). They found that vesicles containing the model transmembrane α-helical peptide GKKL(AL)12KKA supported flip-flop of the short-chain lipids, whereas flip-flop was negligible in lipid vesicles alone. This work represents an interesting alternative to the concept that a dedicated protein machinery catalyses transbilayer lipid movement and that suggests this activity may be a general property of membrane proteins containing α-helical membrane-spanning domains.
Menon and colleagues have presented evidence for an ATP-independent flippase activity in membranes of Gram-positive bacteria ( Hrafnsdottir and Menon, 2000 ) and this activity has also been detected in membranes from Gram-negative bacteria ( Kubelt et al., 2002 ). Using di-C4-phosphatidylcholine as a substrate for the flippase assay, it was possible to solubilize, reconstitute and partially purify the activity. However, to date, a protein(s) has not been identified nor has a mechanism been established.
On the other hand, work with reconstituted ATP binding cassette (ABC) transporters from bacteria ( Margolles et al., 1999 ) or humans ( Romsicki and Sharom, 2001 ) has shown that they possess ATP-dependent phospholipid flippase activity in vitro. ABC transporter proteins use the energy of ATP hydrolysis to transport a variety of lipid and non-lipid substrates across membranes, and are present in all three domains of living organisms. Konings and colleagues have demonstrated (again using short-chain fluorescent phospholipid analogues) that purified, reconstituted LmrA, an ABC transporter from Lactococcus lactis, possesses phospholipid flippase activity in vitro that is dependent on exogenous ATP ( Margolles et al., 1999 ). In a similar manner, reconstituted human p-glycoprotein/MDR1 (ABCB1) has been shown to possess ATP-dependent flippase activity with a wide variety of long- and short-chain phospholipid analogues ( Romsicki and Sharom, 2001 ). While in vitro studies of phospholipid flip-flop activity have proven informative, caution should be used when extrapolating from these results to the in vivo situation, as they typically employ short-chain, water-soluble lipid analogues or contain other modifications that create structurally unique molecules that may differ in important ways from their naturally occurring counterparts.
One enigma in lipid biology is the dynamic organization of cholesterol. Although it can spontaneously move across and between membranes, many proteins have been found to stimulate its movement. Its high affinity for sphingolipids contrasts with the experimental results regarding its transbilayer organization. It also remains unclear how cells move domains of sphingolipids and cholesterol, which have an increased resistance against bending, into highly curved budding Golgi vesicles. Moreover, biophysicists do not see membrane proteins move into such ‘ordered domains’ in artificial reconstituted membranes, whereas they do in the cell. The molecular mechanism of flippases and their lipid specificity are not yet understood nor is intermembrane lipid transport through membrane contact sites. The biogenesis of lipid droplets and their relationship with the ER constitutes another unresolved issue. Finally, we lack a basic understanding of how and why our cells synthesize the multitude of lipid species that we now observe with our sharpened analytical tools. We don't understand their impact on membrane structure and function, and we probably miss half of the functions that are exerted by lipids in signal transduction and homeostasis because they occur in minor amounts.
Many enzymes are involved in the synthesis, remodeling and conversion of cellular lipids, and their intermembrane and intramembrane transport. It is a challenge to unravel lipid homeostasis at the systems level stable isotope labeling and mass spectrometry might allow us to do just that. A bigger challenge is to find out how lipid homeostasis ties in with all other protein-based systems in the cell to regulate cell physiology at large. Mapping the lipids is only a start!
Disproven: Rafts on the cell membrane
Tiny structures made of lipid molecules and proteins have been believed to wander within the membrane of a cell, much like rafts on the water. This "raft hypothesis" has been widely accepted, but now scientists at TU Wien (Vienna) have shown that in living cells these lipid rafts do not exist. This result has now been published in the journal Nature Communications.
"We should not think of the cell membrane as a static, solid surface," says Eva Sevcsik from the biophysics group at TU Wien. "The membrane, the outermost layer of the cell, is fluid. Its molecules -- lipids and proteins -- are constantly in motion."
The proteins can serve different purposes. They can act as docking stations for substances from outside, or as channels transporting molecules into the cell. As many proteins influence each other, it appeared likely that such proteins may move within the membrane together as a "nano-raft."
This hypothesis became more and more popular among cell biologists, and "rafts" have been associated with many cellular processes. But the evidence for this hypothesis has only been derived from studies with model systems or dead cells. "Rafts" have never been directly observed in a living cell.
Many researchers used to think that "rafts" are just too small and short-lived to be detected with conventional microscopic methods. In the biophysics labs at TU Wien, a combination of several cutting-edge techniques has now been used to tackle this problem. "On the one hand, we use super-resolution microscopy, which allows us to study single molecules, on the other hand, we can influence the cell membrane using micro- and nanostructured surfaces," says Eva Sevcsik. "That way we can analyse the structure of the cell membrane in completely new ways."
First, surfaces were structured on a micrometer scale, so that cells which were grown on this surface could interact with the structure. "It is like molecular Lego," says Eva Sevcsik. "We place molecular building blocks on the micro structured surface, which bind to specific proteins in the cell membrane." The proteins attach to the structured surface and cannot travel across the cell membrane any more.
Therefore, a protein can be selected, which is considered to be an important building block of the nano-raft, it can be fixed at particular positions on the surface and then one can study how the other proteins and lipids react.
The molecules become visible using a special microscopic technique. Tiny amounts of fluorescent markers are attached to proteins or lipids, and then molecules can be filmed as they travel within the membrane. "When we study the motion of single proteins, we can see whether we are dealing with membrane rafts or not," says Eva Sevcsik. "Such a raft, anchored at the artificial nanostructures on the surface below, would offer more resistance to the wandering proteins than the surrounding regions. Therefore, the motion of the proteins would be slower. In our measurements, however, the diffusive motion of the molecules is the same everywhere."
For Eva Sevcsik, the fact that the raft hypothesis has remained popular for such a long time, even though there has never been any direct evidence for it, is not that surprising: "It is always tempting to interpret one's results in the context of an established hypotheses, this is a common problem in science. Our goal was to test the raft hypothesis without any bias or prejudice."
The raft hypothesis as it has been taught up until now has taken a blow. But if raft-like structures travelling across the membrane do not exist, are there other mechanisms providing order among proteins and lipids? "Perhaps the actin cytoskeleton plays a more important role than we had thought," says Eva Sevcsik. The cytoskeleton lies directly below the cell membrane and provides stability. Now, Sevcsik wants to study its function using biophysical research methods.
Will lipid molecules 'flip-flop' over a membrane without the use of an enzyme? - Biology
a Optogenetics & Synthetic Biology Interdisciplinary Research Center, State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130# Meilong Road, Shanghai 200237, China
E-mail: [email protected], [email protected]
b Key Laboratory for Advanced Materials and Joint International Research Laboratory of Precision Chemistry and Molecular Engineering, Feringa Nobel Prize Scientist Joint Research Center, School of Chemistry and Molecular Engineering, East China University of Science & Technology, 130# Meilong Road, Shanghai, China
Nature or synthetic systems that can self-assemble into biomimetic membranes and form compartments in aqueous solution have received extensive attention. However, these systems often have the problems of requiring complex processes or lacking of control in simulating lipid synthesis and membrane formation of cells. This paper demonstrates a conceptually new strategy that uses a photoligation chemistry to convert nonmembrane molecules to yield liposomes. Lysosphingomyelin (Lyso) and 2-nitrobenzyl alcohol derivatives (NBs) are used as precursors and the amphiphilic character of Lyso promotes the formation of mixed aggregates with NBs, bringing the lipid precursors into close proximity. Light irradiation triggers the conversion of NBs into reactive aldehyde intermediates, and the preassembly facilitates the efficient and specific ligation between aldehyde and Lyso amine over other biomolecules, thereby accelerating the synthesis of phospholipids and forming membrane compartments similar to natural lipids. The light-controllable transformation represents the use of an external energy stimulus to form a biomimetic phospholipid membrane, which has a wide range of applications in medicinal chemistry, synthetic biological and abiogenesis.
3. Compartmentalisation of Metabolic Pathways
All reactions occurring in cells take place in certain space – compartment, which is separated from other compartments by means of semipermeable membranes. They help to separate even chemically quite heterogeneous environments and so to optimise the course of chemical reactions.
Enzymes catalysing individual reactions often have different temperature and pH optimums and if there was only one cellular compartment a portion of enzymes would probably not function or them-catalysed reactions would not be sufficiently efficient. By dividing the cellular space, optimal conditions for individual enzymatically catalysed reactions are created.
At the same time, cell also protects itself against the activity of lytic enzymes. For example, sealing the cellular digestion in lysosomes prevents an unwanted auto-digestion of other organelles within cell. A common processes that accompany the disruption of some of the compartments (like spilling the content of lysosomes or mitochondria) are necrosis or activation of apoptosis (the process of programmed cell death).
Compartmentalization affects the regulation of metabolic pathways as well, making them more accurate and targeted and less interfering with each other. It is sometimes possible to regulate the course of the reaction at the point of entry of particular substrate into the compartment (transport across the membrane, often mediated by transport mechanisms).
Despite its advantages, compartmentalization at the same time puts greater demand on the energy consumption. It arises from a frequent need to use ATP-dependent transporters, transporting substances across membranes against the concentration gradient and thus creating different environments in different compartments.
One of the necessary conditions for the emergence of live was the separation of cellular environment from the outside (external) environment. Resulting from this, there was a need for selective transport mechanisms between both spaces and later a need for intercellular communication.
Cytoplasmic membrane creates the border with the extracellular compartment and membranes of a similar composition separate other compartments inside the cell.
The width of a cell membrane is approximately 6-10 nm. The core of its architecture is made of phospholipid bilayer with embedded proteins and cholesterol molecules. The latter two can bind various saccharides and so form glycolipids and glycoproteins. This basic structure is, in the case of membranes of different organelles, modified to a certain degree, thus affecting the physico-chemical properties of the membrane (especially its permeability), which are in close connection to the function and course of the biochemical processes in the organelle.
A good example is the myelin sheath of a neuron, which has a ratio of proteins to lipids 19 % to 81 % and thus has excellent insulating properties. On the other hand, the inner mitochondrial membrane has the ration reversed in a favor of proteins (76 % to 24 %) and that is the reason for its relative impermeability (even for substances that pass through standard membranes).
Molecules of phospholipids consist of two physically distinct parts:
1) Polar (hydrophilic) part
The polar part is made up of a phosphate group with other optionally attached groups – this part of the molecule faces the aqueous medium (or other polar solvent).
2) Non-polar (hydrophobic) part
The non-polar part consists of fatty acid chains turned against each other and thus forming the hydrophobic core of the membrane. The hydrophobic interactions of lipid molecules are responsible for their tendency to aggregate and form membranes.
Phospholipid molecule, containing polar as well as non-polar part, belongs to the group of amphipathic molecules.
The currently accepted model of the structure of biological membranes was created in year 1972 by S.J. Singer and G.L. Nicols. According to this, so-called fluid-mosaic model, we can consider the biological membrane as a 2-dimensional liquid in which lipid and protein molecules diffuse more or less easily.
The diffusion rate of phospholipids is much faster than that of other membrane components. Places of membrane with higher content of proteins and cholesterol therefore have lower lateral diffusion rate and stabilise membrane (this applies especially to cholesterol). On the other hand, parts of membrane containing mostly lipids have the ability to flip to the opposite side (through so-called flip-flop mechanism).
The fluidity of membrane depends mostly on:
1) The temperature: the higher the temperature the more mobile the membrane is (sol phase), at lower temperatures it is stiffer (gel phase).
2) The proportion of unsaturated FA: the higher the content of unsaturated FA, the more mobile the phase is.
Proteins form a basic component of cellular membranes. According to their localisation within the membrane, they can be divided into peripheral and integral:
1) Peripheral proteins
Peripheral proteins do not penetrate to the hydrophobic core of the membrane they only bind to its surface area (on extra- or intracellular side) and thereby can be separated from the membrane without damaging it. The main binding interactions include electrostatic forces and hydrogen bonds.
2) Integral proteins
Integral proteins permeate the membrane, either through its full thickness (transmembrane proteins) or only up to a certain depth. Separation of these proteins is associated with the disruption of membrane integrity.
Membrane proteins perform various functions, for example receptor, enzymatic or transport.
Cholesterol makes up approximately ¼ of all membrane lipids. Its molecule, similarly to the molecule of phospholipids, has amphipathic character (due to the presence of OH- group attached to the 3 rd carbon atom). The main function of cholesterol is to stabilise the membrane and lower its fluidity.
Permeability, expressing the rate of passive diffusion of molecules through the membrane, follows the Fick’s law of diffusion and depends on:
1) The size and the polarity of diffusing molecules
2) The concentration gradient
3) The thickness of a membrane
4) The surface area of a membrane
1) The size and polarity of diffusing molecules
In general, small and non-polar molecules pass through the membrane quite easily while larger and polar ones usually need transporters or channels.
2) The concentration gradient
The higher the concentration of a substance on one side of the membrane, the higher its tendency to pass to the other side. This rule applies to other gradients as well – electrochemical (given by the difference of charges on both sides) or osmotic (given by the difference of osmotically active particles on both sides of the membrane).
3) The thickness of a membrane
Substances pass more slowly through a membrane that is thicker.
4) The surface area of a membrane
Larger quantity of substance diffuses through the membrane per unit of time if the membrane has larger surface area.
Other properties of membranes are: the degree of thermal and electrical insulation, electric charge (the total charge of a cell membrane is negative – primarily due to the presence of negative sialic acids residues attached to the glycoproteins and glycolipids) and the ability of selective transport.
Selective transport can be divided to:
3) Transport of macromolecules
This diagram shows an example of transport processes through the blood-brain barrier (barrier between the blood and nervous tissue):
1) Passive transport
Passive transport takes place without energy consumption, as it is based on the physical principle of diffusion (driven by the concentration gradient of a substance on both sides of the membrane). Without the existence of such gradient, the passive transport halts. There are two basic types of passive transport:
b) Facilitated diffusion
A) Simple diffusion
Simple diffusion is a transfer of substances through a membrane without the help of transport proteins. Substances must pass through the hydrophobic core of the membrane, which explains why is this type of transport mechanism typical for:
1. Small non-polar molecule: like gases (CO2, O2, …)
2. Small polar molecule: water, urea
3. Larger non-polar molecules: FA, cholesterol or fat-soluble vitamins
Hydrophilic and larger molecules (mostly with Mr over 200) pass through the membrane by simple diffusion only very slowly or not at all. Transport of ions, whose molecules are relatively small, is difficult due to the presence of a large hydration shell consisting of water molecules.
B) Facilitated diffusion
Facilitated diffusion is a type of passive transport assisted by transport proteins that non-covalently bind the molecule and transport it to the other side of the membrane. It is faster than the simple diffusion and can be accompanied by a transport of other substance in the opposite direction (so-called antiport, for example ATP with ADP or Cl – with HCO3 – ). Another possibility is the transport with the help of channel protein that penetrates the whole width of the membrane. The transfer of a molecule is associated with the change of the channel’s conformation. Some channels are controlled through the changes in membrane electric potential (voltage-gated channels).
There is a difference between the kinetics of the simple and facilitated diffusion. In the case of simple diffusion, increasing the concentration of transferred substance leads to the linear increase in the rate of diffusion. Transport proteins involved in the facilitated diffusion, on the other hand, have limited capacity (given by their total number in the membrane) and when the concentration reaches high values, the rate of the diffusion slows down until it reaches its maximum speed (where the transport capacity is fully saturated).
Among the most important examples of facilitated diffusion are glucose GLUT transporters. Intracellular transformation of glucose to glucose-6-phosphate and its subsequent usage ensures the existence of continuous concentration gradient. Overall, there exist seven types of GLUT transporters of which the most important are:
1. GLUT 1 and 3, serving to maintain the basal glucose uptake by tissues, whose metabolism is dependent on glucose (brain, erythrocytes, kidneys or placenta).
2. GLUT 2, localised on the membrane of pancreatic β-cells and hepatocytes. It also enables the transfer of glucose form absorptive epithelia (e.g. the epithelia of proximal convoluted tubule of kidney, intestinal epithelia or enterocytes) to the blood.
3. GLUT 4 is the glucose transporter of so-called insulin-dependent tissues (skeletal muscle, myocardium and adipose tissue). Its presence on the membrane of these tissues is subject to the effect of insulin. This mainly occurs after the meal when are the above-mentioned tissues responsible of up to 80% of glucose metabolism. Between the meals, on the contrary, they do not absorb glucose and save it for the tissues that are dependent upon it.
2) Active transport
Active transport can take place against the concentration and electrochemical gradient as well. It is possible because the transport is coupled with the ATP hydrolysis (ATP → ADP a Pi) and the energy released is used in the process of transport. We recognize two basic types of this transport:
A) Primary active transport
Energy of ATP is used directly in the process of transportation of a particular substance. Examples are Na + /K + -ATPase, H + /K + -ATPase or Ca 2+ -ATPase.
Na + /K + -ATPase is a tetramer made up of two alpha and two beta subunits. Alpha subunits are transmembrane, their intracellular domains have a binding site for Na + and extracellular domains for K + . Beta subunits are glycolysed (as opposed to alpha) and do not penetrate through the whole membrane. Their oligosaccharide chains are facing the extracellular space. There exist two distinct conformational states of the enzyme, depending on whether it is phosphorylated or not. Na + /K + -ATPase serves as an antiport and in return for the energy released from the ATP it transports 3 Na + cations out of the cell in exchange for 2 K + cations. The result is an uneven distribution of ions on the membrane that forms the basis for the resting membrane potential. Na + /K + -ATPase is ubiquitous – it probably present in all human cells.
Animation shows function of this transporter (located in the center) during the action potential:
H + /K + -ATPase is an antiport operating similar to Na + /K + -ATPase. It can be found in the parietal cells of the stomach (where it produces the gastric juices) and in the cells of the proximal convoluted tubule of kidney. It transports one H + out of the cell in exchange for one K + ion.
Ca 2+ -ATPase, a calcium pump, is mostly localized in muscle and nerve cells. It actively pumps calcium ions out of the cytoplasm, either into the sarcoplasmic reticulum or extracellularly, thus lowering the Ca 2+ concentration back to the previous level (for example before the contraction of a muscle cell).
B) Secondary active transport (or cotransport)
In this case of active transport, the energy released from ATP is not directly used to transport the particular molecule (like glucose). Instead, it is used to transport other substances (e.g. sodium cation), which causes the formation of the concentration or chemical gradient across the membrane. It is this gradient that drives the transport of the (relevant) substance with the help of other transporters (e.g. SGLT – Sodium Glucose Transporter).
A transporter carrying out the secondary active transport (SGLT) thus displaces at least two particles – firstly the one, that is supposed to be transported (glucose) and secondly the one, that drives (through the existence of its gradient across the membrane) the transport (Na + ). To maintain this gradient, a second transporter is necessary (e.g. Na + /K + -ATPase), which can be located in other portion of the membrane. It is this second transporter that consumes the energy (ATP) – that is why this kind of transport mechanism belongs to the group of active transport. In parentheses, we’ve provided an example of secondary active transport of glucose through SGLT transporter, which uses the gradient of Na + created by Na + /K + -ATPase. According to the direction of the transport we recognise symport (both particles are transported in the same direction, from or into the cell) and antiport (particles are transported in the opposite direction – one is carried into the cell and the other out of it). SGLT provides the symport of glucose and Na + .
Tertiary active transport works on the similar principle.
3) Transport of macromolecules across the membrane
According to the direction:
a) Exocytosis is a process by which macromolecules leave the cell. Cytoplasmic membrane fuses with the membrane of the transport vesicle and the macromolecule is either released in the extracellular space or remains a part of the cell membrane.
b) Endocytosis is the process of uptake of macromolecules into the cells. Cytoplasmic membrane invaginates inwards and creates a transport vesicle. According to the nature of the transported particles, the process is carried out differently:
1. Pinocytosis: the transport of macromolecules in solution. The process can be selective (only occurring through specific membrane receptors) or non-selective (the place of invagination is random).
2. Phagocytosis: ingestion of large particles. The cell initially surrounds the particle with protrusions of the cytoplasmic membrane (pseudopodia) and then encloses it to the vesicle.
Transport inside the cell usually occurs through:
1) Diffusion: particles dissolved in aqueous medium of cytosol
2) Transport in secretory vesicles: proteins are most commonly formed at rough endoplasmic reticulum, followed by their transport to Golgi apparatus. Secretory vesicles or lysosomes break free from GA and are further transported within the cell. This kind of transport is carried out by motor proteins (dyneins and kinesins) that use the ATP in order to move across the surface of microtubules (dynein moves towards their – end and kinesin towards their + end) carrying the vesicle attached to their second end.
Compartmentalization of metabolic pathways
Cytosol (cytoplasm without organelles):
1) Metabolism of saccharides: glycolysis, part of gluconeogenesis, glycogenolysis and synthesis of glycogen, pentose cycle
2) Metabolism of fatty acids: FA synthesis
3) Metabolism of amino acids: synthesis of nonessential AA, some of the transamination reactions
4) Other pathways: parts of heme and urea synthesis pathways, metabolism of purines and pyrimidines
1) Metabolism of saccharides: PDH, part of gluconeogenesis (conversion of pyruvate to OAA)
2) Metabolism of fatty acids: beta-oxidation of FA (Linen’s spiral), synthesis (hepatocytes only) and degradation (extrahepatic tissues) of ketone bodies
3) Metabolism of amino acids: oxidative deamination, some of the transamination reactions
4) Other pathways: Krebs cycle, respiratory chain and oxidative phosphorylation, parts of heme and urea synthesis pathways
1) Proteosynthesis (translation of mRNA)
2) Posttranslational modifications (oxidations, cleavage, methylations, phosphorylations, glycosylations)
1) TAG and phospholipid synthesis
2) FA elongation (to a maximal length of 24 carbon atoms – in nerve tissue) and desaturation (maximally at 9 th carbon atom – counted from carboxyl group)
3) Parts of steroid synthesis pathway
4) Biotransformation of xenobiotics
5) Conversion of glucose-6-phosphate to glucose (only in tissues with glucose-6-phosphatase)
New understanding of how bacteria build their protective cell wall
Using a series of chemical and genetic tricks to interrogate a dizzying cast of characters involved in the process of building a cell wall, researchers believe they have discovered the hidden identity of a key enzyme involved in flipping precious cargo from the inside to the outside of a bacterial cell.
It sounds like a hardboiled mystery, but it's the results of research published this month in Science from a team led by microbiologists at Harvard Medical School and Ohio State University.
The bacterial membrane is like an overinflated balloon that would burst without the cell wall, a molecular cage that surrounds the membrane and gives the membrane integrity in the face of the great osmotic pressure exerted on free-living, single-cell organisms. The building blocks of the wall are made inside the cell and need to be secreted through the membrane to the exterior to construct the wall where it's needed. The keys to the hidden passageways that export these building blocks through the membrane have remained mysterious, despite repeated efforts to bring them to light.
Cell wall construction is an important target for antibiotics such as penicillin and bacitracin. When these drugs interfere with cell wall production, bacteria burst and die. Growing concern about the increase in antibiotic resistance has fueled the search for alternative weapons in the fight against resistant bacteria. Because of its prior success as a target, researchers have been trying to learn more about cell wall assembly to discover new ways of blocking it for therapeutic development.
"The more you know about a process, the easier it is to break it," said Thomas Bernhardt, associate professor of microbiology and immunobiology at HMS.
For many years, scientists have known how the basic building blocks of the cell wall are assembled inside the cell cytoplasm, and how the blocks are stitched together on the cell surface to construct the wall. What has remained puzzling is how the bricks needed to build the cell wall are transported across the membrane to the outside, where the wall is assembled.
The wall building blocks consist of sugar molecules linked to a lipid carrier that anchors them to the cell membrane. It has long been thought that bacterial cells possess a transport protein that promotes a flip-flop reaction to move the lipid-linked building blocks from one side of the membrane to the other. However, the identity of this transporter, known as a flippase, has remained mysterious. But now a team of scientists from HMS and OSU have found evidence that the flippase is a protein called MurJ. The researchers are hopeful that this discovery could eventually lead to a new category of antibiotics that block the flipping reaction.
A few candidates for the flippase have been considered, but researchers have been in disagreement over which candidate truly catalyzes the flip-flop. To resolve the issue, a method of detecting the reaction in living cells was needed. However, this was easier said than done.
"It's a subtle change, shifting molecules from one side of the membrane to the other," Bernhardt said. "We needed an exquisitely specific and sensitive assay."
In addition to being small in scale, the cell wall building blocks are rare. In the experiments that the researchers ran, only a few thousand of the millions of lipid molecules in the cell membrane are cell-wall related. Bernhardt's team developed a method using colicins, protein toxins that work like a molecular razor blade, slicing the sugar blocks off their lipid anchors. This releases free sugar blocks not normally produced by cells into the medium that the bacterial cells are floating in. Because the toxins can't penetrate the membrane, they can only snip sugar blocks on the outside. If the sugar blocks are outside, that shows that flipping is underway.
"The first time we were able to see the colicin product I was incredibly excited because I knew we were detecting the flipping reaction," said Lok To (Chris) Sham, a postdoctoral fellow in Bernhardt's lab.
The next step was to use a combination of genetic and chemical techniques to test what happened when the candidate flippases were switched off.
Co-author Natividad Ruiz and her laboratory at OSU had developed strains of bacteria with mutated versions of MurJ that were uniquely susceptible to a chemical that reacts with certain amino acids in proteins. When the chemical was introduced to populations of the bacteria with the mutated MurJ proteins, none of the colicin- generated sugar blocks were found in the medium. This finding indicated that flipping stopped in the absence of MurJ, revealing that MurJ was the hidden flippase.
The team performed similar experiments to test whether turning off the other proteins suspected of being the missing flippase would stop flipping, but in those tests, the bacteria continued to flip sugar blocks.
Each experiment needed to happen quickly. Sham recalled adding the reagent at his bench and running across the hall to the lab's centrifuge to harvest the cells before they could burst and thus destroy the evidence that he needed to collect.
The researchers added that finding a way to make MurJ work as a flippase in test tube models will be important in the next phase of research, as they work to purify MurJ and to monitor the mechanism of the flipping process more closely.
The experiments were done in E. coli, but the researchers suspect this process is common to all bacteria with cell walls.
"We now know what protein is involved in the process," Bernhardt said. "Next we need to delve into the mechanics of the operation, the structure of MurJ and how it promotes transport so that we can plug it with small drug molecules to interfere with the flipping process."
As is often true in the complex world of microbiology, solving one case only leads to new mysteries.