Chapter 7, Membranes
Cell and Molecular Biology
Cellular Membranes
• Membranes were “predicted” to surround cells
long before membranes were seen
• Membranes were predicted to be mostly lipid
based on the diffusion of lipid soluble
molecules into cells
• Other properties of membranes suggested they
also contained protein
• In the 1950’s, the EM allowed visualization of
membranes
Cellular Membranes
• In addition to the plasma membrane, which
separates the cell’s interior from the external
environment, the ER, nucleus, mitochondria,
chloroplasts, lysosomes, peroxisomes, and
transport vesicles are all surrounded by membrane
• The membrane isolates various “compartments”
within the cell
• Many processes in the cell occur either on, in, or
in association with membrane surfaces
Fluid Mosaic Model of
Membrane Structure
• The current understanding of membrane
structure and composition has led to the
formation of the “fluid mosaic model”
• In this model, the membrane is seen as a fluid
lipid bilayer (composed mostly of
phospholipids), with a mosaic of various
proteins “floating” on and in the lipid layer
• Some membranes, such as the mitochondrial
inner membrane, contain more protein than
lipid
Key Features of the
Fluid Mosaic Model
• 1) Membranes are arranged in the form of a
lipid bilayer, which is interrupted by
embedded proteins
• 2) The lipid bilayer is fluid
• 3) There are several ways in which proteins
interact with the membrane
– A) Integral membrane proteins
– B) Peripheral membrane proteins
– C) Lipid anchored proteins
Fluid Mosaic Model: Proteins
Floating in Lipid Bilayer
Membrane Proteins
• Integral membrane proteins: proteins which are
embedded within the lipid bilayer: these proteins are
held in the membrane by hydrophobic forces
• Peripheral membrane proteins: these proteins are
attached to the surface of the membrane, and can be
on either the outer or inner face of the membrane.
These are usually held in place by ionic forces with
the phospholipid head groups or other proteins
• Lipid anchored proteins: these proteins are outside the
layer of the membrane, but are covalently attached to
lipids which are within the membrane
Different Membranes Contain
Different Amounts of Proteins and
Different Lipid Compositions
Lipids of Membranes
• Most lipids in membranes are phospholipids,
glycolipids, and steroids
• The exact composition of a membrane varies
with the cell, the organism, and the organelle
• Membrane lipids are amphipathic, that is, they
have a polar or hydrophilic end (the head
group) and a hydrophobic (lipid) portion
Membrane Phospholipids
• Numerous
phospholipids are
found in membranes
• All have a
phosphate group
covalently attached
to the hydrophobic
lipid
Ended here monday
Membrane Glycolipids
• Numerous
glycolipids are found
in membranes
• These have a
carbohydrate
covalently attached
to the lipid
• Steroids (cholesterol)
represent a third
important class of
membrane lipids
Different Membranes have Different
Phospholipid Compositions
Different membranes in a cell, or in different cell types, have different lipid
compositions, giving the membranes different properties.
Membrane Transition
Temperature
• Altering the lipid type in a
membrane changes the
temperature at which the
membrane changes from a gel
to a fluid
• Cell membranes are generally
fluid at physiological
temperatures
• Cells maintain a fluid
membrane by changing the
lipids in the membrane
Transition Temperatures
• Long chain fatty acids: higher transition temps
(more solid)
• Unsaturated fatty acids: lower transition temps
(more liquid)
• Steroids: inflexible rings: more solid
• But Steroids prevent “packing” of fatty acid
chains, prevent gelling, more liquid
• Steroids (cholesterol) act as a structural
“buffer” to prevent changes in the gel state of
membranes
Transition Temperatures
• Organisms (Plants and Microbes) adjust to lower
temperatures by increasing the degree of lipid
unsaturation or decreasing average fatty acid
chain length
• This allows these organisms to maintain
membrane fluidity
• In animals, fatty acid composition of membranes
is influenced by diet
• Changing your diet can alter the membrane
makeup
Lipid Movement Within a Membrane
• Lipids are relatively unconstrained in lateral
diffusion
• Transverse diffusion (flip flop) occurs very
slowly
• In other words, lipids can move around on the
membrane, but don’t usually move from one
side to the other (from one leaflet to the other)
• Proteins called phospholipid translocators can
speed the movement of phospholipids from
one side of the membrane to the other
Techniques for
Studying Membrane
Composition
• Red Blood Cells (RBCs) can
be broken open and membrane
vesicles prepared.
• If Mg2+ ions are included in the
buffer, the membranes retain
their normal orientation.
• If no Mg2+ is included, the
vesicles are “inside out” : the
side of the membrane which
normally faces the inside of the
RBC facing out.
Determining the Composition of Inner
and Outer Membrane Leaflets
• Isolate either “right side out” or “inside out”
membrane vesicles
• Treat the vesicle with phospholipase, which
– A) digests phospholipids
– B) cannot pass through the membrane
• Determine which lipids were degraded
• Phosphatidylcholine and sphingomyelin are mostly
in the outer leaflet
• Phosphatidylethanolamine and phosphatidylserine
are mostly in the inner leaflet
“Inner” and “Outer” Leaflets
• Since transverse diffusion is limited, the inner and
outer leaflets of a membrane may have very
different compositions
• They may vary in the type of phospholipids present
• They may vary in the saturation level of the fatty
acids (less saturated outer membrane, less fluid)
• Glycolipids are seen only in the outer leaflet of the
plasma membrane
Function of Membrane Proteins
• Four major functions
of membrane proteins
– Transport: move
molecules across the
membrane
– Receptors: carry
signals across the
membrane
– Attachment: provide
structure and form
– Metabolism: enzymes
Labeling of Proteins on a Single
Surface of Membranes
• Selective labeling of proteins allows you to determine the
orientation of the protein in the membrane
Major Types of Membrane Proteins
• Membrane proteins are characterized by the
ways in which they interact with the membrane
Major Types of Membrane Proteins
• Peripheral membrane proteins interact with only
one side of the membrane (ionic interactions), and
are easily removed by high salt buffers
• Integral membrane proteins usually span the
membrane (can be labeled from either side) and
require strong detergent (SDS) extraction to be
removed
• Integral membrane proteins are described by the
number of “passes” through the membrane, and
often contain multiple subunits
Hydropathy Plot
• Plots of the
hydrophobic/hydrophylic
nature of a protein as you
“scan” along the protein’s
sequence are called
hydropathy plots
• Hydropathy plots are
used to predict regions of
a protein which will be
within a membrane
Movement of Proteins Within a
Membrane
• Various techniques
have been used to
examine the ability of
proteins to move
within a membrane
• Some are found to be
unconstrained in their
diffusion
Movement of Proteins Within a
Membrane
• Photobleaching is a technique to see the diffusion of a
single type of protein (labeled with a fluorescent marker)
Protein Movement
• Some proteins are relatively free to move
• Many membrane proteins are constrained in
their ability to move
• “Domains” exist within membranes: proteins
may be free to move, but only within a certain
region of the membrane
• Motion is limited by several mechanisms
– Aggregation
– anchoring to the cytoskeleton or extracellular matrix
– barriers such as tight junctions
RBC Proteins
• The Red Blood Cell
(RBC) has been a good
model for studying
membrane proteins
because it contains few
proteins
• SDS PAGE shows a
relatively simple
protein band pattern
Techniques for
Studying Membrane
Composition
• Red Blood Cells (RBCs) can
be broken open and membrane
vesicles prepared
• If Mg2+ ions are included in the
buffer, the membranes retain
their normal orientation
• If no Mg2+ is included, the
vesicles are “inside out” : the
side of the membrane which
normally faces the inside of the
RBC facing out
Major Types of Membrane Proteins
• Membrane proteins are characterized by the
ways in which they interact with the membrane
RBC Proteins
• Six major proteins in RBC membrane
• Extraction with high ionic strength solutions removed
spectrin, ankyrin, band 4.1, actin (what does this tell
you?)
• These 4 proteins labeled only in inside out vesicles (what
does that tell you?)
• Glycophorin and band 3 are only removed after SDS
extraction (what does this tell you?)
• “Periodic Acid- Schiff reagent” (PAS), which reacts with
(labels) carbohydrates, labels glycophorin and band 3
Quiz 6
• 1. A. What are the three types of lipids
which make up most of the membrane?
• B. What is the distinguishing characteristic
of each type?
• 2. A. What are the three types of proteins
associated with membranes?
• B. What is the distinguishing characteristic
of each type?
RBC Integral Membrane Proteins
• Proteins on “inside out” or “right side out”
vesicles were labeled with radioactive molecules
• Glycophorin and “Band 3” labeled in both types
of vesicles (what does that mean?)
• Protein isolated after labeling was digested (with
a protease) and the fragments separated by
electrophoresis
• Different fragments were labeled in the two types
of vesicles (what does that mean?)
Glycoproteins of RBC
• Treatment of right side out vesicles with
protease released carbohydrates into medium
• Similar treatment of inside out vesicles did not
release carbohydrate (what does this mean?)
• Carbohydrates could be enzymatically labeled
in right side out but not inside out vesicles
• Carbohydrates on proteins face the noncytoplasmic side of the membrane (the outside
of the plasma membrane, or the organelle
interior of other membranes)
Glycoproteins of RBC
• Labeling and digestion experiments have determined the
arrangement of glycophorin and “band 3” glycoproteins
Glycoproteins of RBC
• Glycophorin turns out to be a single pass
protein, 113 amino acids, with 16 carbohydrate
groups attached
• The N-terminal region (amino terminus) faces
away from the cytoplasm: this is where the
carbohydrates are attached
• The C-terminal region (carboxy terminus) faces
the cell interior: this is mostly hydrophilic
amino acids
• A hydrophobic alpha-helix spans the membrane
Glycoproteins of RBC
• Band 3 proteins is a dimer: two identical
polypeptide chains ( a homodimer)
• Both the N-terminus and the C-terminus of each
subunit face the cytoplasmic side
• Multiple (about 12) hydrophobic alpha-helical
segments span the membrane
• A single hydrophilic carbohydrate chain is
attached to the protein, on the non-cytoplasmic
side
N-Linked and OLinked Glycosylation
• There are two different
processing events which
attach carbohydrates to
proteins:
• N-linked glycosylation
attaches carbohydrate to
the nitrogen of asparigine
residues. This occurs in
the ER.
• O-linked glycosylation
attaches carbohydrates to
the oxygen of serine or
threonine. This process
occurs in the Golgi.
Glycosylation on Modified Amino Acids
• The amino acids (residues)
proline and lysine are
sometimes modified to
contain a hydroxyl group.
This occurs in the ER.
• These modified residues
can then be glycosylated
(O-linked)(in the Golgi).
Common Sugar Groups in Glycoproteins
• Galactose, Mannose, N-acetylglucosamine, and sialic
acid are the most common carbohydrate monomers in
the glycosylated proteins.
Oligosaccharides on Glycoproteins are often in
Complex, Branched Arrangements
Note that Sialic Acid is negatively charged.
RBC Peripheral Membrane Proteins
• Spectrin ( an intermediate filament protein)
and actin (actin microfilaments) are cytoskeleton proteins
• Band 4.1 links glycophorin to the cytoskeleton
• Ankyrin links (anchors) band 3 protein to the
cytoskeleton
• The interactions between these proteins appear
to give the RBC much of its characteristic “biconcave” shape
RBC Protein Arrangement
• “Cell and Molecular Biology” is an attempt to elucidate
the way in which cells work.
Some Proteins are
Complex
Structures
• The photosynthetic
reaction center of
Rhodopseudomonas
vividis contains 4
different subunits:
two are multipass
integral membrane
peptides, one is an
integral protein facing
the cytosol, and one is
a peripheral peptide
facing the exterior of
the cell