Chapter 7
Membranes: Their Structure, Function and Chemistry
I. Why do cells have membrane?
A. maintain condition of low entropy
1. this is the MAJOR FUNCTION for all cells
2. membrane defines outermost part of living cell
a. separates cell from environment
b. must maintain appropriate
concentrations of molecules
c. w/o membranes, important stuff drifts away
3. compartmentalize the cell
a. isolate incompatible reactions
i) eg: lysosomes contain hydrolytic enzymes
not compatible with other macromolecules
ii) eg: peroxisomes contain enzymes that
produce H2O2 (H2O2 is toxic to rest of cell)
b. easier to maintain appropriate
concentrations in smaller volumes
B. locus for specific functions of cell
1. eg: reactions of oxidative e-transport and
phosphorylation found
only in mitochondrial membrane
a. the ATP synthetic reaction mechanism
of the mitochondrion REQUIRES
a semipermeable membrane.
2. eg: reactions of photosynthetic electron
transport and photophosphorylation
found only in the thylakoid membranes
of chloroplast (chlp)
a. the ATP synthetic reaction mechanism
of the thylakoid REQUIRES
a semipermeable membrane
C. control movement of substances in/out of cell or organelle
1. membrane is impermeable to most
hydrophilic molecules
2. cells use transport proteins to control
permeability of specific molecules
3. cells use "gated channels" to allow
rapid changes in permeability
D. role in cell communication and recognition
1. plasma membrane interacts with other cells
a. cells must recognize other cells
- immune response to foreign cells
b. cells must interact with other cells
- gap junctions, tight junctions, etc
- adhesions
c. cells receive communications from
other cells
i. chemical communications = hormones, self recognition
ii. electrical communications = nerve impulse
d. cells transmit communication
-info enters cell through plasma membrane
-info leaves cells through plasma membrane
E. help detect and transmit external signals
1. hormones bind receptors, elicit response
II. Models of Membrane Structure
A. two main components of membranes
1. amphoteric lipids
-eg: phospholipids such as
phosphatidylcholine chime
2. proteins
-often amphoteric
B. amphoteric lipids interact with aqueous media
1. form lipid bilayers
-model bilayer (gif)
-model bilayer (chime)
2. form micelles
-favored if head groups are large
relative to tails
-eg: detergents have only ONE
hydrocarbon tail
3. form liposomes
-favored if some head groups are
very small relative to large tails
-enclose an aqueous region
C. importance of liposomes
1. used in drug delivery
-eg: drugs such doxorubicin can be trapped
in the aqueous interior of liposomes
-liposomes can be made that expose
an antibody directed against the
Her-2 growth factor receptor
-about 30% of aggressive breast cancers
OVERexpress the Her-2 receptor
-antibody of the liposome attaches the
liposome specifically to the cancer cells
-cancer cells internalize the Her-2 with its
bound liposome into endosomes
-endosomes fuse with lysosomes and
are degraded
-drug leaks into cancer cell
-drug intercalates into DNA structure
-causes errors in transcription and replication
-target cells die
D. really know and understand Singer and Nicolson/1972
1. fluid mosaic model (see Fig 7-6)
a. key features of fluid mosaic model
(1) developed by Singer and Nicolson/1972
(2) retains lipid bilayer of other models,
but, proteins seen as discrete globular
bodies that penetrate through the lipid bilayer
(3) lipid tails are in a fluid state
(4) proteins form a "mosaic" pattern as
they "float" in a phospholipid "sea"
(5) most proteins and lipids free to
move laterally across face of membrane
(a) proteins are large (move relatively slow)
(b) lipids are much smaller (move
relatively fast)
-see animation of lipid diffusion
-NOTE: this illustrates lipid diffusion
in one monolayer (not a bilayer)
-imagine all lipids (not just the yellow one)
moving much faster!
(6) some membrane proteins anchored to cytoskeleton
(a) when anchored, protein mobility more limited
(b) eg: RBCs
i. spectrin linked to plasma membrane
proteins via ankyrin
and band 4.1 (see page 664)
(6) some lipids bound to protein
(a) limits mobility of the lipids that are bound
b. fluid mosaic model accepted by virtually all scientists today
E. Henderson and Unwin
2. used Electron Microscopy to determine
the first (1990) 3-D struct of a membrane protein (BR)
(bacteriorhodopsin) in lipid bilayer
3. found that BR was a single polypeptide
folded back and forth 7 times
with hydrophilic domains both
sides membrane
a. see a more recent (1996) crystal structure of BR
b. see BR with membrane lipids
4. seven transmembrane spans
a. each span in an alpha helical
configuration
b. alpha helix amino acids are mostly
hydrophobic in character
c. see BR structure (red = polar; blue = hydrophobic)
5. one bound retinal molecule
a. retinal functions in light absorption
b. functions in light induced H+ transport
6. relationship between BR and you
a. BR related to rhodopsin, the protein
involved in vision (Bovine Rhodopsin)
-Note: still 7 transmembrane spans,
still has retinal molecule
b. see cartoon of human rhodopsin
6. most integral proteins have related structure
a. one to several transmembrane spans
(may differ slightly in length)
(1) transmembrane spans are highly hydrophobic
-see glycophorin transmembrane span
(2) about 20 aa minimum
-count the amino acids in the
glycophorin transmembrane span
(3) longer spans due to angular
transection of membrane
(a) no spans lie parallel to the plane of
membrane between the two lipid bilayers
b. differ in number of transmembrane spans
(1) one to many spans
(a) glycophorin has 1 transmembrane span
(b) light harvesting complex has 3 spans
(c) bacteriorhodopsin has 7 spans
(d) other proteins have more than 12 spans
c. differ in extent of hydrophilic domains
(a) glycophorin has large hydrophilic domains
(b) the K+ channel has very small hydrophilic domains
7. membrane spanning regions can
be deduced directly from DNA sequence
with computer algorithms
a. can identify a membrane protein from
DNA w/o knowing anything else
about the protein
III. Molecular Organization and Membrane Function
A. much work done with red blood
cell (RBC) membrane
B. why so much work done on RBC membrane?
1. RBC easy to get
-blood banks
-slaughter houses
-student fingers
2. mammalian RBC, 2, 3, have no internal membranes
-see formation of mammalian RBCs
-therefore, no contamination of RBC plasma membrane
-is this true for all RBCs?
-birds?
-see chicken RBCs stained for DNA
-reptiles?
-other non-mammels
-nucleus free RBCs rare in nature
a. easy to get pure plasma membrane
(PM) preparations from RBCs of mammels
C. types of membrane proteins
1. integral membrane protein =
intrinsic membrane protein
a. see generic cartoon
-black proteins (X & Y) are integral proteins
-type Y is very common
-type X exceedingly rare
b. all type Y have at least one long (20 aa) sequence
hydrophobic aa residues span membrane
(1) eg: glycophorin
(b) one transmembrane span
(c) hydrophilic domains on
both sides of membrane
(d) only outer domain has
covalently attached carbohydrates
(2) eg: see the anion channel of RBC membrane
(3) most membrane proteins have
multiple transmembrane spans
b. integral membrane protein typically
more difficult to work with than
water soluble protein
(1) must use detergents to isolate
protein and to stabilize tertiary structure
-see octylglucoside ( detergent)
-see OG micelle
-see OG micelle (spacefilling)
-cartoon of PSII prep
(2) only a few x-ray crystal structures are
available because membrane
proteins very hard to crystalize
2. peripheral protein = extrinsic proteins
a. no transmembrane spans
-see generic cartoon
-red "Z" is a peripheral protein
-see peripheral protein associated with lipids
-may also bind to other memb proteins
b. located on surface of membrane
-eg: Band 4 protein
-eg: spectrin
c. usually bound electrostatically to membrane
-bind to lipid head groups
-bind to other proteins
d. the key feature is no hydrophobic interactions
with interior of membrane
d. peripheral proteins much easier to isolate
(1) usually come off membrane with
high salt treatment
(2) detergents not usually needed
e. usually easy to work with
(like water soluble protein)
D. proteins in RBC membrane
1. see Fig 7-10
2. integral proteins
a. glycophorin
b. anion channel
c. MANY more are not shown
3. peripheral proteins
a. spectrin
b. ankyrin
c. actin
d. band 4.1
e. a few more are not shown
E. membrane components
1. separation and analysis of components
a. chromatography
(1) thin layer chromatography
(a) analytical separation membrane
lipids
(b) small scale preparitive separation
of lipids
(2) column chromatography
(a) preparative separation of lipids
(b) preparative separation proteins
(3) HPLC = high performance liquid
chromatography
(a) very high resolution
(b) separate lipids
(c) separate peptides
(d) separate proteins
b. electrophoresis (Fig 7-23)
(1) review PAGE technique
-use detergent (SDS) to solubilize memb protein
(2) PAGE = polyacrylamide gel electrophoresis
(a) very widely used to separate protein
(b) see resolution possible with
SDS-PAGE = sodium dodecyl sulfate-PAGE
i) separate protein on basis of
molecular weight (MW)
ii) proteins are completely denatured
with strong detergent (SDS)
iii) proteins have no biological activity
iv) molecular weight can be estimated
with good accuracy by comparing
migrations with proteins of known MW
c. microscopy
-light
-electron
-transmission EM
-freeze-fracture analysis (Fig 7-16, 7-17, 7-18)
-read pp 175-176
2. membrane lipids
a. see Table 7-2, Figure 7-7
b. cartoon of any membrane lipid
c. classes membrane lipids
(1) major phospholipids
(a) phosphatidylcholine (PC), parts labeled
i) found in most membranes
ii) spontaneously forms bilayers or liposomes in water
iii) shape of PC doesn't favor micelles
iv) note the full + and full - charge on the head group
-highly hydrophilic!
v) note the highly hydrophobic tails!
(b) phosphatidylethanolamine (PE)
i) found in most membranes
ii) form bilayers
(c) phosphatidylserine (PS)
i) found in most membranes
ii) form bilayers
(2) minor phospholipids
(a) cardiolipin
i) mitochondrial inner membrane
ii) activates cytochromes
(b) phosphatidylinositol (PI)
i) found in most membrane
ii) source inositol triphosphates
(3) sphingolipids
i) eg: sphingomyelin
ii) sphingolipids found in most
mammalian cell membrane
iii) barrier function
iv) activates certain enzymes
(4) glycolipids (found in plant membrane)
(a) monogalactosyldiacylglycerol
i) major lipid of plant thylakoid membrane
ii) doesn't form bilayers by itself
- (head group too small)
iii) but, small head allows acute
curving of inner leaflet of
the thylakoid membrane
(b) digalactosyldiacylglycerol
i) major bilayer forming lipid in
thylakoid membrane
(5) steroids
i) eg: cholesterol
i) found in most animal membrane
ii) reduces bilayer permeability
iii) modulates membrane fluidity
(6) summary of membrane composition
(7) detailed model of a lipid bilayer
3. membrane proteins
a. classification by function
(1) structural proteins
(a) spectrin
i) see model
(b) actin
i) see model
(c) clathrin
i) see p 349
(d) BAR domain of integral proteins
i) just reported
ii) relate structure to function
-NOTE: the BAR domain gives the
membrane its tubular/ vesicular SHAPE
(2) transport proteins
(a) carrier proteins
i) eg: lactose permease view 2
-part of the lac operon
-lets lactose enter bacterial cells
-structure only recently determined
ii) eg: maltoporin
-facilitate the diffusion of molecules or ions
such as sugars & amino acids
across membrane
(b) channels
i) eg: K+ channel
ii) facilitate the diffusion of ions
iii) ions pass under certain conditions
(c) active transport proteins
i) use energy (ATP, ion gradients) to produce &
maintain gradients of ions & molecules
(3) light transduction
(a) eg: bovine rhodopsin chime
i) absorb photons of light
ii) triggers nervous impulse
(b) eg: bacterial bacteriorhodopsin
i) absorb light
ii) uses light energy to transport
H+ across membrane
iii) simplest example of an ion pump
(c) light harvesting proteins
i) absorb light
ii) transfer energy to reaction center protein
(d) reaction center proteins (chime)
i) structure (gif)
ii) receive light energy (from light or
from light harvesting proteins)
iii) transfer light energy to other protein
iv) ultimately, H+ transported
across membrane
(4) electron transport proteins
(a) transfer e- from one molecule to
another molecule
i) ET proteins may be integral or peripheral
-integral
-eg: succinate dehydrogenase gif
-eg: reaction center proteins
-eg: bc1 complex gif
-peripheral
-eg: cytochrome C
-eg: ferredoxin
-eg: plastocyanin
(5) receptor proteins
(a) bind other molecules
(b) elicit cell response
(c) eg: acetylcholine receptor
i) opens Na+ channels
ii) no crystal structure available for the
globular domain that binds acetylcholine
and sits on the surface of the membrane
(d) eg: insulin receptor
i) changes cell level of cyclic AMP in
presence/absence of insulin
4. membrane carbohydrates
a. external surface plasma membrane (PM) has
covalently bound carbohydrates
(1) see model or Fig 7-5
(2) see cartoon of membrane carbohydrates
(2) RBC plasma membrane composition (by weight)
(a) 52% protein
(b) 40% lipid
-Note: Most of the membrane mass
IS NOT due to lipids!!!
(c) 8% carbohydrate by weight
(d) most carbohydrate attached to protein
i) see glycophorin
(e) some carbohydrates attached to lipid
b. internal surface golgi vesicles, secretion vesicles
and lysosomes also have covalently bound
carbohydrates
(1) carbohydrates attached to protein
or lipid are never exposed to
cytoplasmic side of membrane
(2) how is this shown:
(a) lectins = plant proteins that bind
specific carbohydrates very tightly
i) eg: concanavalin A (a lectin) binds
internal mannose
-note Con A is a tetramer
-note all binding sites are peripheral
ii) eg: wheat germ agglutinin (a lectin) binds
terminal N-acetylglucosamine
(b) ferritin is Fe containing protein
isolated from liver
i) hollow sphere of many subunits
-full sphere
-part of sphere
ii) sphere forms a "cage" capable of holding many Fe atoms
-volume large enough to hold several
1000 Fe atoms
iii) Fe atoms make ferritin visible with EM
iv) protein sphere allows easy covalent linkage
v) ferritin is visible with electron microscope
due to many Fe
-NOTE: the image above shows that the
Fe of ferritin is visible, it doesn't specifically
illustrate the experiments below
(c) ferritin can be covalently
attached to a specific lectin
(d) Exp 1: add ferritin-lectin to a
suspension of cells
i) look at cells with EM
ii) see ferritin decorating external surface PM
(e) Exp 2: microinject ferritin-lectin into
cell cytoplasm
i) look with EM
ii) see no ferritin decorated membrane
(f) Exp 3: expose a fixed thin section of
cell to ferritin-lectin
i) look with EM
ii) see ferritin decorated external surface
PM & internal surface golgi and
secretion vesicles
5. membrane asymmetry
a. membranes are highly asymmetric
(1) outer monolayer of bilayer lipids has
different composition from inner monolayer
-eg: in RBCs there is more
cholesterol in outer monolayer
-eg: in plant membranes there is more
MGDG in inner monolayer
(2) protein domains exposed on outer
monolayer completely different from
protein domains exposed on inner
monolayer
i) see Fig 7-5 and Fig 7-16
ii) Note: proteins absolutely asymmetrical
-eg: glycophorin
(2) asymmetry created during
membrane synthesis
(3) asymmetry maintained during whole life of cell
(a) "flip-flop" of lipids and proteins
thermodynamically unfavorable
i) proteins DO NOT flip flop!
ii) large amount energy required to expose
hydrophilic head groups of lipid to
hydrophobic interior of membrane
iii) "flip-flop" = transverse diffusion
iv) "flip-flop" of lipids is slow
a) given molecule of lipid "flip-flops"
once in several hours (text)
b) Berg's experiments with model lipid bilayers
suggest that "flip-flop" as illustrated
in the cartoon is far slower
c) are probably proteins in biological membranes
that catalyze lipid exchange between monolayers
6. membrane fluidity
a. membrane hydrocarbon tails are "fluid"
when membrane is functional
b. hydrophobic interior has consistency of
salad oil when functional
(1) measure membrane fluidity with
fluorescently polarized, NMR and ESR
probes that partition into hydrophobic
interior of membrane
c. lipids/proteins diffuse laterally demonstrating
membrane fluidity
d. how to observe lateral lipid diffusion
(1) make bilayers with phospholipid (PL)
covalently labeled with fluorescent probe
(a) whole bilayer "glows"
(2) use tiny laser beam to photobleach
small patch of bilayer
(a) get a small dark patch on surface bilayer
(3) immediately see fluorescent-PL
diffusing back into bleached area
(4) bleached area gone in seconds
(5) see Fig 7-11
(6) very similar experiments done with proteins
i) see animation
ii) but diffusion back into bleached region much slower
e. how to see lateral protein diffusion
(1) clearly demonstrated by Frye and Edidin
(a) see Fig 7-28
(b) see animation of the Frye and Edidin Experiment
(c) study on your own (WILL BE EXAM QUESTION)
(2) clearly manifest in movement of
mitochondrial proteins under the
influence of an electric field
(a) see Fig 7-29
(b) study on your own (WILL BE EXAM QUESTION)
(3) see animation of protein diffusion
(photobleaching of fluorescent probes)
f. regulation of membrane fluidity
(1) membrane must be fluid to be functional
(a) to allow protein to collide and interact
(b) to allow special lipids to carry e- and H+ across
membrane
(c) to allow protein to "breathe"
(2) influences on membrane fluidity
(a) temperature
-temp to high => membrane get leaky
-contents enclosed in membrane leak out
-has been used in cancer chemotherapy
-trap toxic drug in liposome
-carefully control lipid composition
so that membrane becomes leaky
at about 105 F
-inject liposomes with enclosed drug
into blood
-liposomes are stable at 98.6 F
-elevate the temperature of the
tumor containing region to 105 F
-as liposomes enter the capillaries
close to the tumor they are warmed
and they leak the drug right on the
tumorous tissues
(b) protein composition
membrane fluidity
i) integral proteins tend to decrease
membrane fluidity especially
close to the transmembrane spans
ii) can be an important influence on
membrane fluidity when the
protein/lipid ratio is high as in
chloroplast thylakoid membrane or
in the inner mitochondrial membrane
(c) lipid composition
i) saturated hydrocarbon (HC) tails of
phospholipid produce membranes that
require higher temperature for fluidity
a) see Fig 7-13
ii) unsaturated HC tails of phospholipid
produce membranes that are more
fluid at lower temperature
a) see Fig 7-13
iii) cholesterol tends to decrease membrane
fluidity, especially at high temperature (Fig 7-15)
a) cholesterol used to regulate fluidity
in animal membranes
b) plasma membrane has large amount
cholesterol
-eg: RBC membranes have almost as much
cholesterol as phospholipid
c) can get TOO MUCH cholesterol in plasma membrane
-eg: spur cell anemia
-common in advanced alcoholics
with cirrosis of liver
-serum cholesterol gets very high
-extra (50% more) cholesterol goes into the OUTER
leaflet of RBC membrane expanding
the outer leaflet and producing spurs
-membrane loses flexibility
-cells become round and spur covered
-cells get trapped in spleen capillaries
-cells are destroyed
-total number of red cells goes way down
-anemia results
iv) bacteria can modify their lipid
composition to adapt to different
temperatures
a) hi temp
b) bacterial membrane has phospholipid
with more saturated hydrocarbon tails
c) lo temp
d) bacterial membrane has phospholipids
with more unsaturated hydrocarbon tails
v) plants modify membrane lipids too