Question

1. Cholesterol is a membrane plasticizer or a membrane buffer. Explain what are the effects of cholesterol at high and low temperatures.

2. What is the difference between integral and peripheral proteins?

3. Membrane lipids are distributed asymmetrically. Explain the concept and the reason behind this asymmetrical distribution

Answer 1

1. Cholesterol enhances membrane fluidity: Lipid bilayers undergo a highly cooperative phase transition with a defined Tm:
Below Tm the the acyl chains are tightly packed and the lipids exist as a solid-like gel;
. Above Tm the acyl chains are disordered and the lipids are in a liquid-like liquid crystal phase
. Rapid lateral diffusion of lipids and proteins occurs in the plane of the membranes above but not below Tm.
Lipid bilayers can be made more fluid (higher Tm) either by decreasing fatty acyl chain length and/or increasing the degree of unsaturation. (Chain length and the presence of cis-double bonds can greatly affect Tm and fluidity through affecting the extent of van der Waals packing).
Membrane fluidity is essential for the biological function of membranes. Therefore organisms go to great lengths to maintain a fluid membrane bilayer (eg, bacteria regulate acyl chain length).
But simply increasing the degree of unsaturation can compromise the integrity of the cell membrane. Animals cells have another way of increasing membrane fluidity:
Cholesterol: - Is a natural steroid,
- About the same length as a C16 fatty acid; therefore it reaches across half of the bilayer.
- Essential component of most mammalian membranes (~20% of cell membrane lipid)
- Destroys the phase transition of pure lipid membranes, thereby keeping the membranes fluid below the phase transition and more rigid above the phase transition. Often referred to as a membrane plasticizer.
Cholesterol, another type of lipid that is embedded among the phospholipids of the membrane, helps to minimize the effects of temperature on fluidity. At low temperatures, cholesterol increases fluidity by keeping phospholipids from packing tightly together, while at high temperatures, it actually reduces fluidity. In this way, cholesterol expands the range of temperatures at which a membrane maintains a functional, healthy fluidity.

2. Membrane Proteins
Peripheral Membrane Proteins: Loosely attached to membranes via electrostatic interactions – released with high salt. Unlike integral membrane proteins, peripheral membrane proteins do not stick into the hydrophobic core of the membrane, and they tend to be more loosely attached.Peripheral proteins localized to the cytosolic face of the plasma membrane include the cytoskeletal proteins spectrin and actin in erythrocytes and the enzyme protein kinase C. This enzyme shuttles between the cytosol and the cytosolic face of the plasma membrane and plays a role in signal transduction. Other peripheral proteins, including certain proteins of the extracellular matrix, are localized to the outer (exoplasmic) surface of the plasma membrane.

Integral Membrane Proteins: Largely contained within the membrane (solubilization requires disruption of the membrane by detergents). Often span the entire membrane. Stability energetics are similar to water soluble proteins, except that non-polar groups interact with acyl chains in the membrane. The rule here is: "hydrophobic outside - hydrophilic or hydrophobic inside", thereby matching the location. Asymmetry across the bilayer is required for most functions (both lipid and protein). No flip-flop! Integral membrane proteins are, as their name suggests, integrated into the membrane: they have at least one hydrophobic region that anchors them to the hydrophobic core of the phospholipid bilayer. Some stick only partway into the membrane, while others stretch from one side of the membrane to the other and are exposed on either side. Proteins that extend all the way across the membrane are called transmembrane proteins.In all the transmembrane proteins examined to date, the membrane-spanning domains are α helices or multiple β strands. In contrast, some integral proteins are anchored to one of the membrane leaflets by covalently bound fatty acids. In these proteins, the bound fatty acid is embedded in the membrane, but the polypeptide chain does not enter the phospholipid bilayer.
The portions of an integral membrane protein found inside the membrane are hydrophobic, while those that are exposed to the cytoplasm or extracellular fluid tend to be hydrophilic. Transmembrane proteins may cross the membrane just once, or may have as many as twelve different membrane-spanning sections. A typical membrane-spanning segment consists of 20-25 hydrophobic amino acids arranged in an alpha helix, although not all transmembrane proteins fit this model. Some integral membrane proteins form a channel that allows ions or other small molecules to pass.

3.
Major lipid components of the eukaryotic plasma membrane include glycerophospholipids, sphingolipids, and cholesterol. Lipids are irregularly distributed between the two leaflets, thus causing lipid asymmetry, or within the same leaflet, forming a lipid microdomain. Glycerophospholipids and sphingolipids both contribute to the lipid asymmetry, whereas cholesterol and sphingolipids form lipid microdomains. Maintenance of proper lipid asymmetry is required for the mechanical stability of the membrane and for vesicular transport. On the other hand, local or global changes in lipid asymmetry are important for cell cycle progression, apoptosis, and platelet coagulation. Three classes of lipid translocases, P-type ATPases, ABC transporters, and scramblases, are known to be involved in the regulation of lipid asymmetry. In this review, we describe the physiological and pathological functions of lipid asymmetry and the current knowledge of lipid translocases.
Of the glycerophospholipids, phosphatidylcholine (PC) is located mainly in the outer (extracytosolic) leaflet, and the aminophospholipids phosphatidylserine (PS) and phosphatidylethanolamine (PE), as well as minor lipids like phosphatidylinositol (PI) and phosphatidic acid (PA), are abundant in the inner (cytosolic) leaflet. The sphingolipids, sphingomyelin (SM) and glycosphingolipids, are confined to the outer leaflet. Translocation of lipids from one leaflet to the other is called ‘flip-flop’, with ‘flip’ being the movement from the extracytosolic leaflet to cytosolic leaflet, and ‘flop’ being the reverse. Since the polar head groups of glycerophospholipids and sphingolipids make it difficult for them to traverse the hydrophobic interior membrane, spontaneous transbilayer movement is very slow in protein-free model membranes.1) Therefore, the existence of enzymes that catalyze transbilayer movement, called lipid translocases or flippases, has been assumed. Recent studies have identified at least three protein families as lipid translocases:
(1)   a subfamily of P-type ATPases, also known as aminophospholipid translocases, which flip PS and PE specifically; (2) ATP-binding cassette (ABC) transporters that flop glycerophospholipids with little selectivity; and (3) lipid scramblases, which randomize the transbilayer distribution of all glycerophospholipid classes

Maintenance of lipid asymmetry is important for certain cellular processes. For example, interactions between PhosphatidylSerine(PS) located in the inner leaflet and skeletal proteins like spectrin improve the mechanical stability of the membranes of red blood cells. In yeast, mutational analyses demonstrated that members of the aminophospholipid translocase family function in intracellular trafficking, maintenance of organelle structure, and cell polarity.Local or global changes in lipid asymmetry also cause a variety of cellular responses. For instance, transient Phosphotidylethanol(PE) exposure and a complete loss of cell surface SM have been observed at the cleavage furrow during cytokinesis in cultured cells. Moreover, when cell surface PE was trapped by either a PE-binding peptide conjugated to streptavidin, or a mutation in PE biosynthesis, cell division stopped at the late stage of cytokinesis due to inhibited disassembly of the contractile ring.These results suggest that locally and temporally regulated exposure of PE is essential for cell cycle progression. Likewise, in budding yeast, PE is predominantly located on the extracytosolic side of the plasma membrane at the bud neck. When cell surface PE was trapped, actin filaments accumulated at the bud neck and small bud, suggesting that redistribution of PE at specific regions is involved in cell polarity. PS exposure on phagocytic cells is also important. The ABC transporter ABCA1 promotes phagocytosis by redistributing aminophospholipids on the macrophage membrane surface.Likewise, CED-7, an ortholog of ABCA1 in Caenorhabditis elegans, is required for optimal engulfment during apoptosis.