Lecture 7 - Université d`Ottawa

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Transcript Lecture 7 - Université d`Ottawa

Readings and Objectives
• Reading
– Russell : Chapter 5
– Cooper: Chapter 13
• Objectives
– Basic properties of plasma membrane
– Fluid mosaic model
– Transport of molecules across membrane
• Passive Diffusion
• Facilitated diffusion
• Active transport
– Endocytic pathways
Plasma membrane
• defines the boundary of the cell
• selective interface, determines the composition of the cytoplasm,
mediates interactions with environment
• Fundamental structure: phospholipid bilayer
• Proteins embedded in the phospholipid bilayer carry out specific
• Experimental evidence
• Bilayer property
– Electron microscopy
– Gorter & Grendel (1925)
monolayer of extracted membrane lipids of known
number of RBC spread on water produced 2x surface area
• Membrane contains proteins
– Chemical composition, 50% protein and 50% lipid
(1 protein per ~100 lipid)
– Asymmetric distribution: Freeze-fracture
followed by electron microscopy
Molecular organization of membranes
Membrane lipids
• Asymmetric distribution of lipids
• Phosphatidylcholine, glycolipids, Sphingomyelin on the outer leaflet
• Phosphatidylserine, phosphatidylinositol, phsophtidylethanoamine on the
inner leaflet, negatively charged head groups facing cytosol
• Cholesterol distributed equally in both layers
• Polar –OH end aligned with phospholipids and hydrophoibic ends with lipid
Two roles
• High temp: interferes with mobility of lipids preventing melt up and reduce
• Low temp: reducing the lipid tails interactions and maintains fluidity and
prevents membrane freezing
Lipid Rafts
• Cluster of Cholesterol, sphingomyelin
and glycolipids
• highly-ordered than most of the
phospholipid bilayer
• Glycolipid (GPI) anchored proteins
• Rafts involved in cell signaling
and endocytosis
Membrane proteins
Proposed by Nicolson and Singer (1972):
• Membrane integral proteins, traverse the membrane, N or C termini on either
side of membrane
• Peripheral proteins, loosely attached to one side by protein-protein interactions
Membrane proteins
Peripheral proteins
• protein-protein interactions
involve ionic bonds
• can be disrupted by polar
reagents (salts or extreme
pH); the proteins dissociate
from the membrane
Transmembrane proteins
• Contain hydrophobic
transmembrane domains (one
or more)
• Detergents, amphipathic
molecules, can solubilize
these proteins
Membranes are dynamic structures
• Proteins and lipids show dynamic lateral movement in membrane
• Frye and Edidin (1970)-provided experimental evidence
• Fused human and mouse cells, then analyzed for membrane proteins using
fluorescent antibodies.
Fluid Mosaic Model
• Singer and Nicolson (1972): Fluid mosaic model, accepted
paradigm for all biological membranes
• The bilayers are viscous fluids, not solid
• The unsaturated fatty acids make kinks in the chain, keep them
from packing together
• Desaturases: produce unsaturated fatty acids
• Regulation of desaturases controls amount of unsaturated fatty
acids, adjusting membrane fluidity
• Proper fluidity, maintained over broad range of temperatures
• Phospholipids and proteins freely diffuse laterally
• Membrane proteins of one half of the bilayer are structurally and
functionally distinct from the other half
Protein movements: How free is “Free”?
• Lateral diffusion of proteins is
restricted for some
• association with the
cytoskeleton, or with other
membrane proteins
• proteins on adjacent cells, or
with the extracellular matrix
• Local lipid composition, GPIanchored proteins localize to
• Polarized cells- apical and
basolateral membrane
Protein movements: How free is “Free”?
• Lateral diffusion of
proteins is restricted for
• association with the
cytoskeleton, or with
other membrane
• proteins on adjacent
cells, or with the
extracellular matrix
• Local lipid composition,
GPI-anchored proteins
localize to lipidrafts
• Polarized cells- apical
and basolateral
membrane domains
Transmembrane integral proteins
• membrane-spanning portions are usually α
helices of 20 to 25 hydrophobic amino acids;
they are inserted into the ER membrane during
• Carbohydrate groups are added in the ER and
Golgi apparatus
ER lumen
• The simplest mode of
insertion involves proteins
with an N-terminal signal
• Translocation halts at a
stop-transfer sequence;
• the protein exits
translococn laterally
• becomes anchored in the
ER membrane.
Internal signal sequences
• anchored in the ER
membrane by internal
signal sequences that are
not cleaved by signal
• No stop transfer sequence
• Internal signal sequences
act as transmembrane α
• exit the translocon and
anchor proteins in the ER
membrane, in either
Multipass proteins
• Proteins that span the membrane multiple times are thought
to be inserted by an alternating series of internal signal
sequences and transmembrane stop-transfer sequences
β barrel Transmembrane domains
• Some proteins have β
barrel transmembrane
• Porins are transmembrane
proteins in the outer
membrane of some
bacteria such as E. coli
• Porins cross the membrane
as β barrels.
• make the outer membrane
highly permeable to ions
and small polar molecules
• Carbohydrate portions of glycolipids, glycosylated proteins on the
outer face of the plasma membrane form a carbohydrate coat
known as the glycocalyx
• Protects the cell from ionic and mechanical stress and is a barrier
to invading microorganisms
• Oligosaccharides of the
glycocalyx participate in
cell-cell interactions
• White blood cells
(leukocytes) adhere to
endothelial cells of blood
• Involves transmembrane
proteins, selectins
• allows them to leave the
circulatory system
(diapedesis) and mediate
inflammatory responses
Transport of Small Molecules
• internal composition of the cell is maintained because the plasma membrane is
selectively permeable to small molecules
Transport of Small Molecules
• internal composition of the cell is maintained because the plasma
membrane is selectively permeable to small molecules
Mechanisms of membrane transport
• Passive transport:
> No chemical energy required
> molecules diffuse down their concentration gradient until
equilibrium reached
– Simple diffusion: O2, CO2, H2O and hydrophobic small molecules,
dissolve in membrane, slow rate
– Facilitated diffusion
– mediated by membrane protein
– allow polar and charged molecules (carbohydrates, amino acids,
nucleosides, ions) to cross the plasma membrane
– no chemical energy spent
Facilitated diffusion: Carrier proteins
• Facilitated diffusion- mediated by Carrier or Channel proteins
• Carrier proteins
– bind molecules on one side of the membrane (high concentration)
– undergo conformational changes that allow the molecule to pass
through membrane
– released on the other side
Facilitated diffusion: Channel proteins
• Facilitated diffusion
• Channel proteins
– form open pores through the
– allow free diffusion of any
molecule of the appropriate size
and charge
• Aquaporins (plant and animal ells)
• allow water molecules to cross the
membrane much more rapidly than
they can diffuse through the
phospholipid bilayer
• impermeable to charged ion
Facilitated diffusion: Ion channels
• Ion channels are well studied in nerve and muscle cells
• opening and closing of channels transmission of electric signals
• Transport through ion channels is extremely rapid: more than a
million ions per second
• Most have “gates” that open in response to specific stimuli
Ion channels: Voltage gated Na+ channel
• Ion channels are highly selective; specific channel proteins allow
passage of Na+, K+, Ca2+, and Cl–
• Voltage-gated channels open in response to changes in electric
potential across the plasma membrane
• Voltage-gated Na+ and K+ channels are selective
• Na+ (0.95 Å) is smaller than K+ (1.33 Å), and it is thought that the Na+
channel pore is too narrow for K+ or larger ions
Ion channels: Voltage gated K+ channel
• The 3-D structure of K+ channels was determined by X-ray
• Part of the channel pore is lined with carbonyl oxygen (C=O)
atoms from the polypeptide backbone
• Displace the water to which K+ is bound, and the K+ ion passes
• Na+ is too small to interact and remains bound to water
Ion channels: Ligand gated
• Ligand-gated channels open in response to the binding of
neurotransmitters or other signaling molecules
• neurotransmitters are released into the synapse, bind to receptors on
another nerve cell to open ligand-gated ion channels
• pore is blocked by side chains of hydrophobic amino acids.
• Binding of acetylcholine induces a conformational change, the
hydrophobic side chains shift out of the channel, which opens a pore
for positive ions
Active transport: Ion Pumps
• Molecules are transported against their concentration gradients
• coupled reaction to ATP hydrolysis
• The Na+-K+ pump (or Na+-K+ ATPase) uses energy from ATP hydrolysis to
transport Na+ and K+ against their electrochemical gradients
• The Na+-K+ pump operates by ATP-driven conformational changes
• Three Na+ are transported out of the cell and two K+ are transported into the
cell for every ATP used
Active transport: Symport and antiport
Active transport can also be driven by a Na+ gradient
Symport: solutes move in the same direction (Na+/Glucose)
Antiport: solutes move in opposite directions (Na+/Ca2+ antiporter)
The flow of Na+ down its electrochemical gradient provides energy for
transport glucose against its conc. gradient
Active transport: ABC transporters
• ABC transporters- have
conserved ATP-binding domains
or ATP-Binding Cassettes
>100 of this family have been
identified in prokaryotic and
eukaryotic cells
use energy from ATP hydrolysis
to transport molecules in one
In bacteria, transport nutrient
molecules into the cell including
ions, sugars, and amino acids
Eukaryotic cells: transport toxic
substances out of the cell
• Endocytosis allows cells to
take up macromolecules, fluids,
and large particles such as
• The material is surrounded by
an area of plasma membrane,
which buds off inside the cell to
form a vesicle containing the
ingested material
– Phagocytosis
– Pinocytosis
– Receptor mediated
Receptor Mediated Endocytosis
• first elucidated in studies of
patients with familial
• Cholesterol is transported in
bloodstream in the form of
low-density lipoprotein
• Macromolecules bind to cell
surface receptors in
specialized regions called
clathrin-coated pits
Receptor Mediated Endocytosis
• The internalization signals bind
to adaptor proteins, which in
turn bind to clathrin
• Clathrin assembles into a
basketlike structure that forms
invaginated pits
• Dynamin forms rings around the
necks of the pits, eventually
leading to the release of coated
vesicles inside the cell
• Pits bud from the membrane to
form small clathrin-coated
Receptor Mediated Endocytosis
• After internalization,
clathrin-coated vesicles shed
their coats and fuse with
early endosomes—vesicles
with tubular extensions at
the cell periphery
• Receptor is recycled to the
plasma membrane
• LDL remain in early
endosomes as they mature
to late endosomes and
lysosomes for degradation
• Cholesterol released for cell