1. Molecular Biology of the Cell
Alberts • Johnson • Lewis • Raff • Roberts • Walter
Molecular Biology of the Cell
Fifth Edition
Chapter 10
Membrane Structure
Copyright © Garland Science 2008

2. The lipid bilayer is composed of amphiphilic lipids
Cell membranes are composed of a lipid bilayer with proteins held by non-covalent interactions
The lipid bilayer is composed of amphiphilic lipids
Figure Molecular Biology of the Cell (© Garland Science 2008)

3. Lipid molecules constitute about 50% of the mass of most animal cell membranes (remainder being proteins)
5x106 lipid molecules in 1mm x 1mm area
Most abundant lipids are the phospholipids
They have a polar head group and a hydrophobic hydrocarbon tail
If the tail has double bonds = not saturated – makes kinks
If the tail has only single bonds = saturated
Differences in length and saturation of the fatty acid tails influence how phospholipid molecules pack against one another, thereby affecting fluidity of the membrane.

4. PHOSPHOGLYCERIDES, THE MAIN PHOSPHOLIPIDS IN ANIMAL CELLS
Figure Molecular Biology of the Cell (© Garland Science 2008)

5. PHOSPHOGLYCERIDES, and SPHINGOSINE
Have three carbon glycerol as a backbone with two long chains of fatty acids, 3rd Carbon is attached to a phosphate group that is linked to one of several different types of head group.
There are different types of phosphoglycerides, main ones are:
Phosphatidyl ethanolamine
Phosphatidyl serine
Phosphatidyl choline.
Sphingomyelin has sphingosine as a backbone – because of the amino and hydroxyl groups on it it contributes to polarity and to forming H-bonds with other proteins or water or lipid molecules.

6. PHOSPHOGLYCERIDES, and SPHINGOSINE STRUCTURES
Figure Molecular Biology of the Cell (© Garland Science 2008)

7. Cholesterol structure: A sterol ring structure and polar OH head
Figure Molecular Biology of the Cell (© Garland Science 2008)

8. ORIENTATION OF CHOLESTEROL IN THE LIPID BILAYER
Figure Molecular Biology of the Cell (© Garland Science 2008)

9. EFFECTS OF CHOLESTEROL ON THE LIPID BILAYER
Enhances the permeability barrier; makes the membrane less permeable to small water soluble molecules, because of the cholesterol stiffened region of the phospholipids.
Tightens the packing of the lipids but does not make the membrane less fluid.
Prevents the hydrocarbon chains from coming together and crystallizing.

10. Hydrophobic and hydrophilic molecules interact differently with water
Insoluble molecule in water: Ice-like packing of water
Higher Energy
Figure Molecular Biology of the Cell (© Garland Science 2008)

11. Lipids organize together in the lowest Energy structure
Figure Molecular Biology of the Cell (© Garland Science 2008)

12. Membranes have a self-healing property; free edges are not energetically favorable
Figure Molecular Biology of the Cell (© Garland Science 2008)

13. THE LIPID BILAYER IS A FLUID
In 1970 researchers recognized that individual lipid molecules are able to diffuse freely within lipid bilayers:
This came from studies on synthetic lipid bilayers:
Bilayers made in the form of spherical liposomes (size 25nm-1mm)
Black membranes: planar bilayers formed across a hole in partition between two aqueous compartments

14. Liposomes can be made artificially in the lab to mimic membranes and study their properties
Figure 10-9a Molecular Biology of the Cell (© Garland Science 2008)

15. “Black membranes” are made to study diffusion and transport of material across a membrane
Figure Molecular Biology of the Cell (© Garland Science 2008)

16. Computer simulation of lipid molecules to show that they are disordered, with irregular surface
Figure Molecular Biology of the Cell (© Garland Science 2008)

17. Studies that showed the mobility of lipid molecules
Constructing a lipid molecule with a fluorescent dye attached to the polar head group and follow the diffusion of individual molecules in the membrane.
Lipid molecules rarely move from one leaflet to the one on the other side; flip-flop – phospholipid translocators catalyse the rapid flip-flop.
Lipid molecules have high lateral diffusion 2mm/sec
Modify a lipid head group to carry a “spin label” such as nitroxyl group with unpaired electrons; if it spins it generates a signal detected by electron spin resonance ESR spectroscopy.
Lipid molecules do rotate about their long axis.
Cholesterol does flip-flop easily

18. The fluidity of the membrane depends on its composition and on the temperature
Figure Molecular Biology of the Cell (© Garland Science 2008)

19. The fluidity of the membrane depends on its composition and on the temperature
If hydrocarbon chains are short or with double bonds : this decreases the freezing temperature (becomes more difficult to freeze).
In addition, lipid molecules with double bonds are more spread and so the lipid bilayer becomes thinner than bilayers of saturated lipids. (idea of lipid domains, dicussed in the next slides)
Cholesterol affects the freezing temperature of lipid bilayers; so it lowers the freezing point. ( do not forget the other roles cholesterol play in the lipid bilayer- discussed earlier)

20. Despite their fluidity lipid bilayers can form domains of different compositions
With certain lipid mixtures, different lipids can come together transiently creating a dynamic patchwork of different domains
1:1:1 phosphatidylcholine:sphingomelin +/- : cholesterol
Without Cholesterol
With Cholesterol
Figure Molecular Biology of the Cell (© Garland Science 2008)

21. Domains called lipid rafts – a specialized region of the plasma membrane
Enriched in sphingolipids and cholesterol
Figure 10-14a Molecular Biology of the Cell (© Garland Science 2008)

22. Domains called lipid rafts – a specialized region of the plasma membrane –thicker due to lipid composition
Specific proteins that assemble there help to stabilize these rafts and also rafts help concentrate specific proteins in them to do a specific function.
Figure 10-14b Molecular Biology of the Cell (© Garland Science 2008)

23. Lipid droplets are surrounded by a phospholipid monolayer
Lipid droplets function : to store fat (Adipocyte = fat cells) :
Food source
To liberate fatty acids on demand
Used to synthesize triacylglycerides and cholesterol esters, from enzymes in ER membrane
Used to make lipids.
Figure Molecular Biology of the Cell (© Garland Science 2008)

24. ASYMMETRY IS FUNCTIONALLY IMPORTANT
Lipid bilayers are asymmetrical
Example: Red Blood cell
Phosphatidylcholine and Sphingomyelin are in outer monolayer
Phosphatidylserine in inner leaflet is – charged, and so there this results in a charge difference
ASYMMETRY IS FUNCTIONALLY IMPORTANT
Figure Molecular Biology of the Cell (© Garland Science 2008)

25. Example 1: a lipid kinase (PI-3K) can add phosphate onto Phosphatidylinositol creating a binding site for docking of specific proteins.
Figure 10-17a Molecular Biology of the Cell (© Garland Science 2008)

26. Example 2: PLC is activated by an extracellular signal to cleave specific phospholipids molecules generating an intracellular mediator
Figure 10-17b Molecular Biology of the Cell (© Garland Science 2008)

27. Phospholipids asymmetry is exploited by animal cells
When animal cell undergoes apoptosis phosphatidylserine, normally present in cytosolic leaflet only rapidly moves to extracellular monolayer
This on the cell surface signals other cells, like macrophages o come to phagocytose (eat) the dead cell and digest it.
How this occurs?
Translocator that moves this lipid from noncytosolic to cytosolic layer gets inactivated
A scramblase that transfers phospholipids randomly gets activated

28. Glycolipids (Sugar containing lipid molecules) – found only in non-cytosolic monolayer
Made from
Sphingosine
Most complex
Figure Molecular Biology of the Cell (© Garland Science 2008)

29. GLYCOLIPIDS Sugars get added in the lumen of the ER and the golgi
Gangliosides are the most complex : contain oligosaccharides with one or more sialic acid residue – net negative charge – most abundant in nerve cells
FUNCTION:
May help protect the membrane from harsh conditions (pH and enzymes) (on Apical surface of intestinal epithelium).
Charged glycolipids may be important to change electric field across the membrane (ex. Ca++ at membrane surface)
Cell-cell recognition processes (sperm and egg cell-cell adhesion)
Provide entry point for certain bacteria toxins (cholera toxin binds to GM1 ganglioside) [causes too much Na+ and water to enter intestine]

30. MEMBRANE PROTEINS Perform most of the membrane’s specific task
Amount and types of proteins in a membrane are highly variable
If a membrane is 50% protein by mass it will have more lipid molecules than protein molecules (as lipids are smaller than proteins) so about 50 lipids for one protein

31. Different ways in which a membrane protein can associate with the membrane
Associated with outer monolayer (GPI anchor)
Transmembrane proteins (integral membrane proteins)
amphiphilic
Peripheral membrane proteins
Associated with inner cyosolic monolayer
Figure Molecular Biology of the Cell (© Garland Science 2008)

32. Different ways proteins attach to membranes
Transmembrane proteins / integral membrane proteins – amphiphilic
Some have covalent attachment of fatty acid chains – more hydrophobic (1)
Some associate only with inner leaflet (by alpha helix or lipid chain) (4)(5)
Figure Molecular Biology of the Cell (© Garland Science 2008)

33. Different ways proteins attach to membranes
Some are exposed only to the outside attached my specific oligosaccharides (covalent) attached to a lipid PI. Such as GPI (glycophosphatidylinositol) (in the ER protein is cleaved and GPI added) PI specific PLC cuts these to be released. (6)
Some attach by non-covalent interactions with other membrane proteins – can be removed with gentle extraction with solution of high or low ionic strength or extreme pH. = peripheral membrane proteins.
Figure Molecular Biology of the Cell (© Garland Science 2008)

34. Different ways proteins attach to membranes:
Attachment by fatty acid chains
These function on one side of the membrane (ex. Intracellular signaling proteins)
Figure Molecular Biology of the Cell (© Garland Science 2008)

35. In transmembrane proteins, they have hydrophobic domains (alpha helices that span the membrane)
Can be Single pass (as example shown here) or multipass (transmembrane protein crosses multiple times).
Domains that are inside the bilayer are hydrophobic, they are composed of amino acids that are hydrophobic.
Figure Molecular Biology of the Cell (© Garland Science 2008)

36. THE AMINO ACIDS AND THEIR PROPERTIES

40. REQUIREMENTS FOR A TRANSMEMBRANE PROTEIN
Segments of amino acids with high degree of hydrophobicity are long enough to span a lipid bilayer as an alpha-helix (an alpha helix has maximum hydrogen-bonds between amino acids).

41. QUESTION
Monomeric single pass transmembrane proteins span the membrane with a single a-helix that has specific characteristic chemical properties in the region of the bilayer. Which of the three 20-amino acid sequences below is the most likely candidate for such transmembrane segment? A I T L Y F G V M S B P C E R D

42. Hydropathy plot used to predict from the amino acid sequence the stretch that is hydrophobic (TM domain)
20% of an organisms total protein are TM proteins
Figure Molecular Biology of the Cell (© Garland Science 2008)

43. Multi-spanning proteins Structure determined by X-RAY crystalography
Figure Molecular Biology of the Cell (© Garland Science 2008)

44. TM (transmembrane) domains of single pass proteins do not contribute to the folding of cytoplasmic or extracellular parts; so they can be produced each independently in the cells to see there function as soluble proteins.
Sometimes TM domains of different proteins or within the same protein interact together. This is a property of the TM domain alone, as if it is cut it can still associate with the other TM domain.

45. TM domains associate together even if they are separated
Done by engineering genes encoding separate pieces of a multipass protein in living cells.
Figure Molecular Biology of the Cell (© Garland Science 2008)

46. TM domains enter the membrane with the help of protein “translocators” to insert in bilayer first, then they associate with other TM domains
Figure Molecular Biology of the Cell (© Garland Science 2008)

47. Beta barrel proteins tend to be more rigid:
Some are pore forming (water channels) polar amino acids located on the inside, nonpolar outside
Some b-barrel proteins are receptors or enzymes
Figure Molecular Biology of the Cell (© Garland Science 2008)

48. Beta barrel proteins
Restricted to the outer membrane of bacteria, mitochondria and chloroplast.
In Eukaryotes and bacteria; most multipass TM proteins have alpha helices.
Alpha helices can slide past each other to open/close channels
Beta barrels are more rigid

49. In animal cells most TM proteins are glycosylated
Oligosaccharides are present on non-cytosolic side (remember sugars added in lumen of ER/golgi)
the cytosol has a reducing environment, so S-S (disulfide) bonds form only in the extracellular space.
Long polysaccharides chains linked to a protein core in the extracellular matrix make up the proteoglycans (they can be attached to the PM as well).
Figure Molecular Biology of the Cell (© Garland Science 2008)

50. Glycocalyx = sugar coat
Glycocalyx is seen by specific stain ruthenium red, or with fluorescent lectins (a carbohydrate-binding protein).
Figure 10-28a Molecular Biology of the Cell (© Garland Science 2008)

51. Different types of sugar attachment
Proteoglycans also are found in the extracellular matrix
Figure 10-28b Molecular Biology of the Cell (© Garland Science 2008)

52. Table 2-1 Molecular Biology of the Cell (© Garland Science 2008)

53. Membrane proteins can be solubilized and purified by detergents
Agents that disrupt the hydrophobic interactions and destroy the lipid bilayer can solubilize TM proteins:
Detergents are amphiphilic much more soluble in water than lipids
Ionic charged (SDS)
Uncharged nonionic (TX-100, and b-octy).
At [low] are monomer, at [high ] they form micelles
They depend on Temp, pH and [salt]
Figure 10-29a Molecular Biology of the Cell (© Garland Science 2008)

54. Detergent micelles have irregular shapes
Figure 10-29c Molecular Biology of the Cell (© Garland Science 2008)

55. When mixed with membranes – hydrophobic ends of detergent bind to hydrophobic membrane parts – displace lipid molecules  soluble detergent-protein complexes
Figure Molecular Biology of the Cell (© Garland Science 2008)

56. Figure 10-31 Molecular Biology of the Cell (© Garland Science 2008)

57. Bacteriorhodopsin: first membrane protein with known structure
Figure Molecular Biology of the Cell (© Garland Science 2008)

58. Bacteriorhodopsin: first membrane protein with known structure
On membrane of the archaean Halobacterium Salinarum
Lives in seawater, exposed to light
It is a light activated proton pump
Each contains a light absorbing chromophore called retinal (vitamin A)
Light causes a change in conformation of the protein and H+ goes from inside to outside of the cell
In bright light each molecule pumps several hundred protons per second
The H+ gradient drives ATP production by other protein in the plasma membrane.
other proteins in same family is rhodopsin, similar structure with different function in vertebrate retina (this is a signal transducer and not a transporter; functions to activate G-protein inside the cell).

59. Bacteriorhodopsin: 7 TM alpha helices
Figure Molecular Biology of the Cell (© Garland Science 2008)

60. Membrane proteins can be quite complex in structure: multicomponent
Figure Molecular Biology of the Cell (© Garland Science 2008)

61. Membrane proteins diffuse in plane of PM: heterokaryons of human and mouse cells
Figure Molecular Biology of the Cell (© Garland Science 2008)

62. Diffusion rate measure: fluorescent recovery after photobleaching
Figure 10-36a Molecular Biology of the Cell (© Garland Science 2008)

63. Diffusion rate measure: fluorescent loss in photobleaching
Figure 10-36b Molecular Biology of the Cell (© Garland Science 2008)

64. FRAP and FLIP Diffusion coefficient is measured.
Monitor movement of large proportion of molecules, in a relatively large area of the cell (single particle tracking techniques have overcome this problem).
Found that the viscosity of membrane similar to olive oil (without proteins in it).

65. Cells can confine proteins and lipids to specific domains within a membrane: tight junctions
Figure Molecular Biology of the Cell (© Garland Science 2008)

66. Sperm cells have specific membrane domains : not known how they are maintained
Figure Molecular Biology of the Cell (© Garland Science 2008)

67. Common ways to restrict mobility of proteins
Figure Molecular Biology of the Cell (© Garland Science 2008)

68. Membrane of RBCs maintained by a network of cytoskeletal protein complexes
Figure Molecular Biology of the Cell (© Garland Science 2008)

69. Membrane of RBCs maintained by a network of cortical cytoskeletal protein complexes
Figure 10-41a Molecular Biology of the Cell (© Garland Science 2008)

70. Figure 10-41b Molecular Biology of the Cell (© Garland Science 2008)

71. The cortical barriers act as a mechanical barriers for membrane proteins; movement measured by high speed single particle tracking
Figure Molecular Biology of the Cell (© Garland Science 2008)