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MB 207 – Molecular Cell Biology
Part III: Internal Organization of the cell
Membranes
Structure and Function
Main Functions of Membranes
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Membrane define boundaries of the cell and
its organelles
- To keep desirable substances in the cell but
also to keep undesirable substances out.
- Compartmentalization to contain the cell.
Membrane serve as permeability barriers
- Movement of molecules either into or out of
cell is regulated.
- Only very small uncharged molecules can
readily diffuse i.e.. O2, CO2 & water.
- Hydrophobic molecules pass through more
readily than hydrophilic ones.
Membrane possess transport proteins
- Facilitating and regulating the movement
of substances into and out of the cell and
its organelles against a concentration
gradient.
Small
hydrophobic
molecules
Small
uncharged
polar
molecules
Large
uncharged
polar
molecules
Ions
4) Membrane serve as sites for specific proteins such as enzymes and
receptors to detect external signals
- Receptors on the outer surface of the plasma membrane bind to specific
ligand (signaling molecule e.g. hormones, growth factors,
neurotransmitters).
- Binding of ligand is followed by specific chemical events in the inner
surface of the membrane that often lead to changes in gene expression
and cell function.
5) Membrane provide mechanism for cell-to-cell contact, adhesion and
communication
- During embryonic development, specific cell-to-cell contacts are critical and
often mediated by membrane proteins, cadherins.
→ Cadherins have extracellular sequences of amino acids that bind
calcium ions and stimulate adhesion between similar cells in a tissue.
- Other membrane proteins such as adhesive junctions which hold cells
together and tight junctions which form seals that block the passage of
fluids between cells.
- Intercellular communication is provided by gap junctions in animal cells and
plasmodesmata in plant cells.
All the above-mentioned functions are depending on the chemical composition
and structural features of the membranes.
Membrane structure
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Despite their differing functions, all biological membranes have a common
general structure: each is a very thin film of lipid and protein molecules, held
together mainly by non-covalent interactions.
- Plasma membrane: Separates the cytoplasm from the external environment.
- Membrane of the organelles: Maintain the characteristics differences between
contents of each organelle and cytosol.
In the early 70s, Singer and Nicolson proposed that a membrane is consists
of a mosaic of proteins in a fluid lipid bilayer.
This model has two key features, implied by its name. The model envisions
a membrane as a mosaic of proteins discontinously embedded in, or at
least attached to, a fluid lipid bilayer.
Three classes of membrane proteins:
i) Integral membrane proteins: embedded within the lipid bilayer.
ii) Peripheral proteins: located on the surface of the membrane.
iii) Lipid-anchored proteins: resides on the membrane surface but
covalently attached to lipid molecules that are embedded within the bilayer.
Recent findings further refine our understanding of
membrane structure
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Unwin and Henderson’s findings of the structure of bacteriordopsin , allows
cells to obtain energy directly from sunlight, further improved the
understanding of integral proteins.
Bacteriordopsin structure consists of a single polypeptide chain folded back
and forth across the lipid bilayer seven times.
→ Each of the seven transmembrane segments of the protein is closely
packed α-helix composed mainly of hydrophobic amino acids.
→ Successive transmembrane segments are linked to each other by short
loops of hydrophilic amino acids that extend into or protrude from the polar
surfaces of the membrane.
→ all transmembrane proteins are anchored in the lipid bilayer by one or
more transmembrane segments.
Membranes contain several major classes of lipids
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The main classes of membrane lipids are phospholipids, glycolipids and
sterols.
Phospholipids:
- The most abundant lipids found in membrane.
- Have a polar head and two hydrophobic hydrocarbon tails.
→ lipid molecules spontaneously aggregate to bury their hydrophobic tails
in the interior and expose their hydrophilic heads to water.
- Differences in the length and saturation of the fatty acid tails are important
because they influence the ability of phospholipid molecules to pack
against one another, thereby affecting fluidity of the membrane.
- Different kinds of phospholipids: phosphoglycerides (phosphatidylcholine,
phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol)
and sphingosine-based sphingolipids (sphingomycelin).
- Sphingomyelin is one of the main phospholipids of animal plasma
membrane but it is absent from the plasma membranes of plants and most
bacteria.
Phosphatidylcholine molecule
The kink resulting from the cis-double bond influence the packing ability of
phopholipid molecules
Four major phospholipids in mammalian plasma membranes
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Glycolipids:
- formed by adding carbohydrate group to lipids.
- some are glycerol based but most are derivatives of sphingosine
(glycosphingolipids).
•
Sterols:
- the main sterols in animal cell membranes is cholesterol which for
maintaining and stabilizing membranes in our bodies.
- the membrane of plant cells contain small amounts of cholesterol and
larger amount of phytosterols.
The structure of cholesterol
Fatty acids are essential to membrane structure and
function
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Are components of all membrane lipids except sterols.
Essential to membrane structure due to their long hydrocarbon tails which
form an effective hydrophobic barrier to the diffusion of polar solutes.
Most fatty acids in the membrane are between 12-20 carbon atoms in
length.
→ Chains fewer than 12 or more than 20 carbons are less able to form a
stable bilayer.
Fatty acids in the membrane lipids vary in the presence and number of
double bonds.
→ unsaturated fatty acids resulting in a sharp bend or kink in the
hydrocarbon chain at every double bond.
→ fatty acids with double bonds do not pack tightly in the membrane,
thereby influencing membrane fluidity.
Lipid molecules aggregate to bury their hydrophobic tails in the interior and
expose their hydrophilic heads to the water.
→ form spherical micelles with the tails inward or bilayer sheets with the
hydrophobic tails sandwiched between hydrophilic head groups.
Packing arrangements of lipid molecules in an aqueous
environment
Membrane asymmetry: Most lipids are distributed
unequally between the two monolayers
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Due to differences both in kinds of lipids present and in the degree of
unsaturation of the fatty acids in the phospholipid molecules.
Most of the glycolipids present in the plasma membrane of an animal cell
are restricted to the outer of the two monolayers.
→ their protruding carbohydrate group are involved in various signalling and
recognition events.
Phosphatidylethanolamine is more prominent in the inner monolayer.
→ involved in transmitting various kinds of signals from the plasma
membrane to the interior of the cell.
Membrane asymmetry is established during membrane biogenesis by the
insertion of different lipids or different proportions of the various lipids into
each of the two monolayers.
→ asymmetry is maintained because the movement of lipids from one
monolayer to the other requires their hydrophilic head groups pass through
the hydrophobic interior of the membrane, thermodynamically unfavorable.
How are phospholipids organized
in the Cell Membrane?
• Phospholipids constitute two
mirror – image oriented layers –
the lipid bilayer
• The hydrophilic heads are
exposed to the high content
water regions, while the
hydrophobic tails constitute a
barrier impenetrable to almost all
substances.
Three kinds of movement of membrane lipids
• Rotation about its long axis
• Lateral diffusion by exchanging places
• Transverse diffusion or ‘flip flop’ from one monolayer to the other.
→ catalyzes by flippases
The lipid bilayer is fluid
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Membrane lipids form a fluid bilayer that permits lateral diffusion of
membrane lipids as well as proteins.
→ not being fixed in place with the membrane.
Membrane fluidity changes with temperature.
→ decreasing as the temperature drops and increasing as it rises.
→ the change in the state of the membrane is called phase transition.
→ membrane must be maintained in the fluid state in order to function
properly.
Membrane fluidity depends primarily on the kinds of lipids it contains (length
of the fatty acid and their degree of unsaturation).
→ Long-chain fatty acids have higher transition temperatures than shorterchain fatty acids. Membranes enriched in long-chain fatty acids tends to be
less fluid.
→ Membranes containing many unsaturated fatty acids tend to have lower
transition temperatures and thus more fluid than membranes with many
saturated fatty acids.
Membrane fluidity is also affected by the presence of sterols.
→ cholesterol molecule orients itself in the layer with its single hydroxyl
group close to the polar head group of a neighboring phospholipid molecule.
→ intercalation of rigid cholesterol molecules into the membrane of animal
cell makes the membrane less fluid at high temperature and maintaining
membrane fluidity at low temperatures.
→ in addition, sterols also decrease the permeability of a lipid bilayer to ions
and small polar molecules.
The influence of cis-double bonds
in hydrocarbon chain
Cholesterol in a lipid bilayer
Lipid rafts are localized regions of the membrane lipids
that are involved in cell signaling
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Lipid rafts:
- dynamic structures that change in composition as individual lipids move in
and out of them.
- thicker and less fluid than the rest of the membranes due to tight packing
of cholesterol and the hydrocarbon tails of the glycosphingolipids and
phospholipids.
Cell signaling:
→ when receptor bind to their specific ligand, they move into particular lipid
rafts that are also located on the outer monolayer.
→ receptor-containing lipid rafts in the outer monolayer are thought to be
coupled functionally to specific lipid rafts in the inner monolayer which
contain specific kinases (generating second messenger).
The membrane consists of a mosaic of proteins
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Evidence freeze-fracture microscopy:
→ a lipid bilayer is frozen quickly and then subjected to a sharp blow from a
diamond knife.
→ nonpolar interior of the bilayer is least resistant through the frozen
specimen resulting fracture between the two layers of membrane lipid.
→ electron micrographs revealed that proteins are suspended within
membranes.
Membrane contains integral, peripheral and lipid-anchored proteins.
Membrane proteins differ in their affinity for the hydrophobic interior of the
membrane, therefore differ in the way they interact with lipid bilayer.
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Transmembrane protein with single  helix (note: covalently attached fatty acid
chain)
Transmembrane protein with multiple  helices
Transmembrane protein with rolled-up  sheet ( barrel)
Cytosolic proteins embedded with hydrophobic  helix
Membrane protein covalently attached to lipid bilayer through a fatty acid chain
or a prenyl group
Membrane protein covalently attached to lipid bilayer through a oligosaccharide
linker, to phosphatidylinositol in the noncytosolic monolayer
Peripheral membrane protein
Peripheral membrane protein
Integral membrane protein
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Hydrophobic regions are embedded within the membrane interior in a way
that makes these molecules difficult to remove from membrane.
Hydrophilic regions that extend outward from the membrane into an
aqueous phase on one or both sides of the membrane.
Protruding from one side of the bilayer: integral monotopic proteins
Protruding form both sides of the bilayer: transmembrane proteins
Transmembrane proteins:
- cross the membrane either once (singlepass proteins) or several times
(multipass proteins).
- most are anchored to the lipid bilayer by one or more hydrophobic
transmembrane segments, one for each time the protein crosses the
bilayer.
- singlepass membrane proteins have just one transmembrane segment
with a hydrophilic carboxyl terminus extending out of the membrane on one
side and a hydrophilic amino terminus protruding on the other side.
- multipass membrane proteins have several transmembrane segments
ranging from 2 or 3 to 20 or more segments.
Can only be released formt he membrane by disruption of the bilayer with
detergents.
Peripheral membrane proteins
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Lack discrete hydrophobic sequences and therefore do not penetrate into
the lipid bilayer.
Bound to membrane surfaces through weak electrostatic forces and
hydrogen bonding with the hydrophilic portions of integral proteins and
perhaps with the polar head groups of membrane lipids.
Can be released by changing the pH or ionic strength of the solution.
Lipid-anchored membrane proteins
• Located on one of the surfaces of the lipid bilayer but are covalently bound
to lipid molecules embedded within the bilayer.
• Proteins bound to the inner surface of the plasma membrane are attached
by covalent linkage either to a fatty acid or to an isoprene derivatives.
• Many lipid-anchored proteins attached to the external surface of the plasma
membrane are covalently linked to glycosylphosphatidylinositol (GPI), a
glycolipid found in the outer monolayer of the plasma membrane.
Membrane protein attachment by a fatty acid chain or a prenyl group.
Determining the Three-Dimensional Structure of
Membrane Protein
X-ray crystallography
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Used to determine the three-dimensional structure of the proteins.
Reconstructs images from the diffraction patterns of X-rays passing through
a crystalline or fibrous specimen, thereby revealing molecular structure at
the atomic level of resolution.
The difficulty of isolating integral membrane proteins in crystalline form
virtually excluded these proteins from crystallographic analysis.
Hydropathic analysis
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The number and locations of the transmembrane segments can be inferred
from hydropathy (or hydrophobicity) plot.
→ plot is constructed by using computer program to identify clusters of
hydrophobic amino acids.
→ the resulting plot predicts how many membrane-spanning regions are
present in the protein based on the number of positive peaks.
Using hydrophobicity plots to localize potential
membrane-spanning segments in a polypeptide chain.
Membrane proteins have a variety of functions
• enzymes to carry specific
functions to specific
membranes.
• transport proteins which
facilitate the movement of
nutrients across membranes.
• channel proteins which
provide hydrophilic
passageways through
otherwise hydrophobic
membranes.
• receptors in recognizing and
mediating the effects of specific
chemical signals.
• adhesion molecules for
holding cells to extracellular
matrix
Many membrane proteins are glycosylated
• Glycosylation is the addition of a carbohydrate side chain to a
protein which occurs in the ER and Golgi compartments of the cell
soon after synthesis.
• Glycosylation involves linkage of the carbohydrate either to the
nitrogen atom of an amino group (N-linked glycosylation) or to the
oxygen atom of a hydroxyl group (O-linked glycosylation).
• N-linked glycosylation are attached to the amino group on the side
chain of asparagine whereas O-linked carbohydrates are usually
bound to the hydroxyl groups of either serine or threonine.
• Carbohydrate chains attached to glycoproteins can be either straight
or branched and range in length from 2 to about 60 sugar units.
• Most common sugars used are galactose, mannose, Nacetylglucosamine and sialic acid.
• Glycoproteins are most prominent in plasma membranes where they
play important role in cell-cell recognition.
→ carbohydrate groups protrudes on the external surface of the cell
membrane.
Detergents can solubilize the membrane bound proteins
– Detergent consist of small amphipathic molecules
– Hydrophobic ends of detergents displace the hydrophobic ends
of phospholipids & bind to the hydrophobic domains of proteins
– Strong ionic detergents
eg. SDS (sodium dodecyl
sulfate) solubilize membrane
proteins as well as unfolding
proteins by attacking the
internal hydrophobic sites
– Mild non-ionic detergents
eg. Triton X-100 solubilize
membrane protein without
inducing protein
denaturation
Demonstration of the mobility of membrane proteins by cell fusion
• Sendai virus was used to fuse the human
and mouse cells.
•The fused cells were then exposed to the
red (anti-human) and green (anti-mouse)
fluorescent antibodies and observed by
fluorescence microscopy.
• When the fused cell were first exposed to
the antibodies, the GFP from the mouse cells
were localized on one-half of the hybrid cell
surface whereas RFP were restricted to the
other half.
• After 40 minutes, the separate regions of
green and red fluorescence were completely
intermingled.
• Intermingling of the fluorescent proteins had
been caused by lateral diffusion of the human
and mouse proteins through the fluid lipid
bilayer of the plasma membrane.