28 Biomembranes -III

Prof. M. N. Gupta

 

  1. Objectives
  •  To learn about biomembranes of the cells of extremophiles
  •  To learn how freeze-fracture and deep etching techniques have helped us in understanding membrane structure.
  •  To learn how fluroscence technique have helped us in gaining quantitative insight into movements /motions of lipids and proteins in biomembranes.
  1. Concept Map
  1. Description

Most of the times discussions on biomembranes tend to focus on outer plasma membrane of mammalian system. These membranes in the case of microbial world, especially that of extremophiles have to deal with different challenges. Cellular organelles also have biomembranes, some one, others two. We will look at this and some other aspects before we conclude our discussion on structure of biomembranes.

 

Membranes allow selective transport across and movements/ motions within their bilayer structures. These multiple objectives are met by these assemblies of lipids and proteins. Microbes that live under harsh conditions of temperature and pH or Cl- presence at high concentration have tweaked the biomembrane design to cope with conditions of their habitat. Integral proteins are not limited to membrane spanning proteins, about which we have mentioned and will further discuss while talking of membrane transport. There are also lipid anchored proteins which are essentially lipoproteins but the lipids are of different kinds from those that form bilayers.

 

Fluoroscence microscopy with its different approaches have proved valuable in gathering some quantitative aspects of what freedom of movement protein and lipids leave within biomembranes.

 

Membranes are outer boundaries of both cells (plasma membrane) as well as sub cellular compartments. All membranes contain lipids and proteins. Some membranes also contain carbohydrates. As we have seen these are part of the proteins as oligosaccharide chains.

 

The composition of these three components varies from one membrane to another. A good example of this is the inner mitochondrial membrane which hosts the proteins of the respiratory chain.

In both bilayers, lipids are generally free to move around. The rotational movement and lateral movements are not disallowed. However, the transverse movements generally described as flip-flop movement is highly restricted.

 

These allowed movements ofcourse depend upon the overall membrane fluidity. It can also be said that in turn, the extent of these movements, along with those of proteins define the overall membrane fluidity.

 

To further emphasize the role cholesterol at physiological temperature, if the cholesterol content is increased in the bilayers, the overall membrane fluidity decreases.

 

The condensed ring systems of the steroid is rigid and interferes with lateral movement of the fatty acid chains. As this is a property of steroid rings and not just of cholesterol, it is not surprising that in some biomembranes, cholesterol is actually replaced with other steroids.

 

Maize leaves have 7% by weight sitosterol. Yeast has 4% of ergosterol. Paramecium (a ciliated protist) has 4% of stigmasterol. Sterols also regulate the fluidity at extreme conditions as well as under different growth conditions.

 

Below the membrane transition temperature, presence of sterols prevent highly ordered packing of lipids in bilayers. Bilayers, otherwise, can pack rather tightly. Self assembly feature is based upon these being able to order themselves.

 

Above the transition temperature, the bilayer core is on its own and has considerable freedom of motion. Such unrestricted movements would have destroyed any semblance of biomembrane structure. Steroid (cholesterol being the most prevent and important one) prevent this structural collapse by restricting this motion.

 

In case of different growth temperatures, the organisms, as we mentioned earlier, alter the membrane lipid composition itself. When cultured at higher temperature, membrane lipids have higher saturated fatty acid.

Thus, both fatty acids of lipids and cholesterol are involved in connecting the membrane fluidity in cells under different conditions. Organisms live and survive under variety of conditions. The cell membrane design is tailored as a part of the survival kit.

 

Hyperthermophiles can growth at very high temperatures. Their optimal temperature of growth is >80 oC. Karl Stetter’group in Germany isolated Pyrolobus fumarii that can grow at even 113 oC. Their enzymes, called extremozymes remain catalytically active at even 140 oC. What happens to the bilayers at very high temperature so that the cellular integrity can be preserved?

 

It turns out that cells of hyperthemophiles do not have same membrane structure. In case of their membranes, the hydrophobic tail are bonded to form just a monolayer. The presence of monolayer rather than bilayer prevents the collapse of the membrane structure.

 

At the other end of the thermal spectrum are psychrophiles. Psychrotolerant organism can grow at low temperature (~ 4-8 °C), though they grow better at 25 – 35 °. True psychrophiles grow best at ≤ 15 °C. Their natural habitat is cold environments like Antarctic.

 

For their membranes, these cold psychrophiles use the strategy of having appropriate fatty acids with regard to length of the hydrocarbon chain and unsaturation.

 

To visualize how effective this strategy is, think of margarine. It remains softer than butter at cold temperature, because of the higher content of unsaturated fatty acids.

 

Alkalophiles and halophiles are other example of extremophiles. Most of the alkalophiles are also halophiles. As outer surface their membrane is exposed to high OH-, it is moot question: how does proton motive force exists and leads to formation of ATP for their growth and survival.

 

It so happens that these organisms donot use proton gradient to form ATP. Instead, an ion gradient of Na+ rather than H+ is used to get the required energy.

 

In the next modules, we will talk of pumps and channels which allow transport of polar and charged molecules across the membranes. Right now, it suffices to say that many halophiles have chloride pumps that push Cl-1 inside the cells.

 

Halophiles also in some cases accumulate K+ inside the cells. These strategies are used to maintain osmotic balance with their external environments.

 

Barophiles are microorganisms that grow better when pressure is > 1 atm. A bacterial strain MT41 can only grow at least 500 atm. Optimal pressure required is 700 atm.

 

It was isolated from marine sediments near the Philippines from a depth of 10,000 meters. We donot know much about their membrane design. Some moderately basophilic bacteria which can also grow without pressure have provided some clues. Some key nutrient transport proteins in cytoplasmic membranes seem to be structurally modified.

 

Thus, it is misleading to think that structure of fatty acids and presence of sterols can take care of all contigencies under which cell membranes help organisms retain their shape and integrity.

 

The bacterium Deinococcus radiodurans is registant to radiation. While a human cannot survive at 5 Grays level of radiation, this extremophile can survive even 30,000 Grays of radiation. Radiations are ionising and thereby destroy living organisms. What kind of membrane design the organism has? So, it is important to note that fluid mosaic model is not universal.

 

It may be interesting to recall how the initial clues about membrane structure were obtained.

 

In 1925, E. Gorter and F. Grendel extracted membrane lipids from red blood cells.

 

The only membrane in these cells is the plasma membrane as there are no cellular organelles. So, it was an intelligent choice. The extraction was done by acetone and lipids obtained on water.

 

Careful estimates showed that this continuous macromolecular layer had an area of two times the size of the surface area of red blood cells. As the lipid molecules self assemble, this floating layer was continuous. The conclusion was that these lipids in the plasma membrane were present as bilayers.

 

As we mentioned earlier, both X-ray diffraction and election microscopy confirmed the bilayer nature of the membranes. It was freeze fracture electron microscopy which enabled isolation of separate monolayers.

 

Freeze-fracture electron microscopy especially turned out to be valuable for finding that proteins are located in the membrane interior as well. This was not initially believed in view of the hydrophobic interior of the membranes.

 

In this technique, cells or membrane fragments are rapidly frozen in the liquid nitrogen. The frozen sample is then fractured by a microtome knife. Microtomes are used to precisely and uniformly cut thin sections of tissue. It is the tissue which slides to the steel knife and is forced through the knife resulting in the section.

 

The plane of fracture is generally in the middle of the bilayer, separating the two leaflets. These exposed regions are shadowed by carbon and platinum to create a replica of the middle of the bilayer.

 

A combination of freeze fracture and deep etching techniques helps in looking at the exposed surfaces. The ice covering the adjacent membrane is sublimed in the process called deep etching.

 

The combined technique is called freeze –etching electron microscopy. This technique provided the first proof that proteins are present in the membrane interior. The integral membrane proteins were seen as dense globular particles.

 

Such particles were seen in the case of erythrocyte membranes and sarcoplasmic reticulum membranes. In any new technique or its new application, validation is important to rule out artifacts.

 

Synthetic bilayers of phosphatidyl choline were prepared and examined by this technique. The fractured faces were smooth. Myelin membranes are relatively inert insulators. When examined, these also showed large smooth areas free of the dense globular particles.

 

Lipid-Anchored Proteins

 

Some authors distinguish between membrane spanning proteins and integral proteins. In such case, the latter term refers to lipid anchored proteins. These proteins are covalently bonded. A significant fraction of the non-peripheral proteins in eukaryotes are lipid anchored. These proteins are linked to the lipids of either of the two leaflets.

Prion protein is responsible for what is popularly called mad cow disease. It is covalently linked via the C-terminal amino acid to the polar head of the phosphotidyl inositol. Many proteins have similar linkage.

 

The linkage is called ethanolamine-phosphate-trimannose bridge and proteins are called glycosyl phosphotidyl inositol (GPI) anchored proteins. Sequential addition of sugar residues and ethanolamine phosphate to the phosphotidyl ethanolamine takes place.

 

The protein to be anchored has a signal peptide at the C-terminal which is removed in the lumen of the rough endoplasmic reticulum and this preformed GPI-anchor attached to the C-terminal.

 

Some proteins are transiently attached to the cytosolic face of the membrane by its N-terminal glycine amino group forming an amide bond with myristate (C14:0) molecule. Such proteins are called Myristoylated proteins.

 

Alternatively, instead of an amide linkage, a thioether linkage is formed between C- terminal cys residue and 15-C farnesyl or 20-C geranylgeranyl polyolefinic chain. Such proteins are called Prenylated proteins.

 

Some other proteins form the thioether linkage with palmitate. These are called Palmitoylated Proteins.

 

The lipid anchored proteins are involved in some key biological processes. For example, cell signaling G-proteins and Ras family of protein are lipid modified or lipid anchored protein.

 

G-protein-coupled receptors (GPCRs) are membrane spanning proteins which contain 7 α-helical regions. Examples of these receptors include receptor for several hormones and neuroreceptors, light activated rhodopsins of the eye, odorant receptors in the nose.

 

On binding its ligand, a GPCR activates the trimeric G-proteins with α,β,γ subunits. The Gα and Gγ subunits are the lipid anchored proteins on the cytosolic face of the plasma membrane.

 

Ras proteins, on the other hand, are examples of monomeric G-proteins. These activate a Ser/Thr phosphorylation cascade. Like Gα subunit, Ras proteins are also anchored to the cytosolic surface of the membrane.

 

Along with other membrane proteins, lipid anchored proteins constitute the functional part of the biological membrane, which is, other than controlling transport.

 

Let us also look at some important examples of peripheral proteins. These proteins interact with the lipid and/or other membrane proteins. The interaction is limited to the surface but to both inner and outer surface of the membranes. Thus, in case of lipids, it is limited to the polar heads.

The cytosolic face of the erythrocyte plasma membrane has a network of peripheral proteins which constitute its cytoskeleton. Cytoskeleton is an internal scaffold and consists of microfilaments, intermediate filaments and microtubules.

 

In case of erythrocyte plasma membrane, the major component of the cytoskeleton is spectrin. This protein has triple-stranded α-helical coiled-coil which form long chains. These chains interact with two other peripheral proteins called ankyrin and protein band 4.1.

 

Ankyrin constitutes a crosslink between spectrin and the cytosolic domain of integral anion exchange band 3 proteins. Band 4.1 facilitates the binding of actin filaments to the spectrin chains which links them to the cytosolic domain of glycophorin.

 

Like with other cells, cytoskeleton of erythrocyte cells provides both strength and flexibility to the plasma membrane. In other cells, though, cytoskeleton cris-crosses the cytoplasm.

 

Membranes of the cellular organelles

 

-Nucleus has inner and outer membranes which fuse together at nuclear pores. These pores allow transport between nucleus and cytoplasm. The outer nuclear membrane is continuous with rough endoplasmic reticulum.

 

-ER is a network interconnected membrane vesicles. Golgi consists of flat membrane bound sacs. Mitochondria has both inner and outer membrane. In addition, these have interconnected flattened vesicles which assume the shape of discs called thylakoids.

 

  •  Lysosomes and Peroxisomes, both have a single membrane. Peroxisomes have enzymes which

degrades fatty acids and amino acids. Their metabolism produces H2O2 (hence the name), so these also have catalase to decompose H2O2.

 

Diffusion within the bilayers

Figure 6: Fusion of a mouse cell and a human cell

 

Fluorescence microscopy provided vivid proof of how proteins can move rapidly in the membranes. A hybrid cell, fusion of a human and mouse cell was prepared. The fluorescent labeled antibodies specific to membrane proteins of each component cells were used to track these marker proteins. Red and green fluorescent labels were used for the two component cells.

 

As expected, the freshly prepared hybrid cells showed segregated red and green patches. However, within 1 hr and at 37 oC, the mixing of green and fluorescent molecules was observed. The estimate was that in about one minute, a membrane protein can move by several microns.

Fluorescence photobleaching recovery technique allow quantitative measurement of the lateral movement of the membrane molecules. In this, a cell surface component is labeled and viewed through a microscope which looks at ~ 3 μm2 region.

 

A light pulse from laser is used to destroy this region. This is called bleaching. It is found that fluorescence in this region is again recovered with time. The bleached molecules move away and fluorescent molecules move in. The rate of recovery can be related to the diffusion coefficient D. (cm-2 sec-1).

 

S = 4 (Dt)1/2

 

This powerful technique allowed measurement of the diffusion coefficients of both lipids as well as proteins. Any of the two classes of molecules can be specially labeled with fluorescent tags. The conjugation chemistry with many fluorescent tags is available.

 

Phospholipid molecules were found to move at the rate of ~ 2 μm/ sec. The diffusion coefficient of lipids were found to be of the order of 10-8 cm2 sec-1 by measurements with a large variety of membranes.

 

Proteins, on the other hand, have a wide range of lateral mobility. Rhodopsin was found to be among the highly mobile proteins with diffusion coefficient of 4 ×10-9 cm2 sec-1. Fibronectin, a peripheral protein has diffusion coefficient of < 10-12 cm2 sec–1. This protein is anchored to actin filaments through integrin. The mobility is thus related to the function of the membrane protein.

Figure 8: Lateral diffusion of lipids is much more rapid than transverse diffusion (flip-flop)

-Transverse diffusion, sometime referred to as flip-flop is the movement of a molecule from one face of the membrane to another. The ESR shows that a phospholipid molecule can flip-flop once in several hours. These measurements were made by preparing phosphotidyl choline vesicles with some molecules labeled with nitroxide spin label.

 

-Proteins donot show flip flop. It may be obvious now that the biological processes involving membranes are largely mediated by proteins and not lipids. One “structural role” of the protein is to shape membrane asymmetry. Their own polar nature and selective glycosytation ensures this flip flop is not possible.

 

A major role of membranes is to ensure necessary, selective transport. This is one aspect which we have not discussed yet. We do that in the next module.

 

What we have focused on so far are the structural aspects of diverse biomembranes. Again, it is not a comprehensive view but enough for us to get an overall idea about how the biomembrane design- simple and elegant – discharges diverse multiple functions. We will see more of that in the context of transport of molecules across these structures.

 

Summary

 

  •  Biomembranes of extremophiles and cellular organelles.
  •  Freeze fracture and deep etching techniques
  •  Lipid anchored proteins
  •  Lateral and transverse motion within bilayers.