Transcript Slide 1

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Cell biology 2014 (revised 21/1 -14), Note Lecture 2 handout.
Ester bond
Lecture 2:
Chapter 10 Chapter 11
Alberts et al
5th edition
617-626
628-636
651-664
Chapter 12
A lot of reading!
Focus on principles
and topics highlighted in
the lecture synopsis
695-699
704-710
Cell Biology interactive  media  ”video” or ”interactive”
Membranes are primary built from phospholipids
The major phospholipid:
Lipid bilayer
5 -8 nm thick
Variable
Phosphate
Glycerol
Fatty acid
Glycerides (acylglycerols):
esters formed from glycerol
and fatty acids
Fatty acid
Biological membranes are lipid
bilayers primary composed of
amphipathic phospholipids
Hydrophilic head Hydrophobic tails
Phosphoglyceride
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Packing of amphipathic lipids in water
- Wedge-shaped lipids form micelles in water
H2O is a dipole
Red: negative
Blue: positive
- Cylinder-shaped lipids form bilayers, followed by liposome
formation
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Amphipathic lipids will spontaneously form structures that
eliminate the exposure of hydrophobic parts to water
Movement of individual lipids within the bilayer
Rotational and lateral
movement (frequent)
Phospholipids can freely and rapidly
(mm/s) diffuse within the monolayer
Flip-flop (rare)
Spontaneous movements between the
two monolayers are rare
The lipid bilayer is a two-dimensional fluid
Similar viscosity as olive oil
video 01.2 crawling_amoeba.mov; 13.5 phagocytosis .mov
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Fatty acid length affects membrane fluidity
C=O C=O
C=O C=O
CH2 CH2
van der Waals
CH2 CH2
CH2 CH2
van der Waals
CH2 CH2
CH2 CH2
van der Waals
Long fatty acid tails
CH2 CH2
Short fatty acid tails
Weak interactions
Strong interactions
High fluidity
Low fluidity
Long aliphatic carbon chains promote van der Waals
interactions  decreased membrane fluidity
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Fatty acid saturation affects membrane fluidity
An unsaturated fatty
acid has a kink
Phospholipids containing only
saturated fatty acids
C=O C=O
CH2 CH2
CH2 CH
CH2
CH2
CH2
CH
Phospholipids containing a
unsaturated fatty acid
CH2
CH2
Unsaturation's results in steric hindrance  decreased van der
Waals interactions  increased membrane fluidity
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Effect of lipid composition on membrane fluidity
- Membrane thickness
- Interactions between fatty acid chains
- Membrane fluidity
Shorter fatty acid chains and an increased degree of
unsaturation make a thinner and more fluid lipid bilayer
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Anim. 09.1-laser_tweezer; Video 10.1- membrane_fluidity
Lipid rafts - clusters of strongly interacting lipids
< 100 nm
The phospholipid sphingomyelin have long saturated
fatty acid tails  strong van der Waals interactions
 Formation of a more static lipid environment
Lipid rafts are micro-domains of phospholipids with low fluidity
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Asymmetry of the plasma membrane
Outer monolayer
Inner monolayer (facing the cytosol)
Phosphatidylcholine
Phosphatidylethanolamine
Phosphatidylserine
Lipid raft
former
Sphingomyelin
50 40 30 20 10 0 10 20 30 40 50
Percentage of membrane lipids
Extracellular space
-
-
molecular_models 10.2-lipids.mov
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Phosphatidylinositol,
important for cell signaling
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Different types of membrane proteins
Peripheral
Integral
3.
1.
b-barrel
Multi-pass a-helix
Single-pass a-helix
Mono-topic protein
Integral membrane proteins are not
tossed into the membrane randomly,
but have a specific topology
2.
Associated to
1. Lipid
2. Integral protein
3. Glycolipid
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Dynamics of membrane proteins
Rapid movement of proteins
within the lipid bilayer
Original fluid
mosaic model
(Singer& Nicolson 1972)
Lipid raft
Lipid micro-domain
(Simons & Ikonen 1997)
~20 % of the 11
plasma membrane
Membrane permeability of different molecules
• Hydrophobic molecules
CO2
O2
Benzene
• Small uncharged polar molecules
H2O
Ethanol
• Large uncharged polar molecules
H
• Charged
molecules
H
+
N
R
C
-
Glucose
O
H+
C
H H
Amino acids
O
Na+ ClIons
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Two types of transmembrane transport proteins
Carrier Proteins
Channel proteins
From above
Creates a hydrophilic channel
Binds a “passenger” at one
side of membrane and deliver through the lipid bilayer that is
selective for a particular solute
it to the other side
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Ion channels
• Most channel proteins are involved in ion transport over the
membrane and are therefore called ion channels
• Ion channels are regulated and ion specific
Closed
Ion
Open
Ion
Ion
Ion A Ion B
Ion A
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Mechanisms behind membrane transport
Concentration gradient
Simple
diffusion
Facilitated
specific
diffusion
Energy independent
(down-hill)
Active
transport
Energy dependent
(up-hill)
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Different types of active membrane transport
Transport of molecules against a concentration gradient requires
energy. Cells uses two distinct strategies.
ATP-driven pumps
ATP
Coupled transporters
(symporters)
ADP + P
“Up-hill” transport
coupled directly to
hydrolysis of ATP
“Up-hill” transport of molecule
coupled to “down-hill” transport of
molecule
. The “down-hill” gradient
depends on a ATP-driven pump
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Example of active transport - Na+/K+ pump
Na+ 145 mM
K+ 5 mM
1.
2.
P
++
Na
Na
Na+
P
++
Na
Na
Na+
Na+ 10 mM
ATP
ADP
K+ 140 mM
3.
KK++
4.
1 cycle  10 milliseconds
P
KK++
Anim. 11.2-carrier_proteins , Anim. 11.1-Na_K_pump
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Using concentration gradients of
Na+ and
K+
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1. Active transport of Na+ and K+ creates concentration gradients
2. The Na+ gradient provides the energy for “up-hill transport”
3. Coupled transport of sucrose into the cytosol
K+
Na+ Na+
Na+ Na+ Na+
Na+
1.
K+
K+
K+
Na+
2.
Glucose
3.
Glucose
Glucose Glucose
The ATP driving the Na+/K+ pump is the energy source for
concentrating sugars and amino acids within cells
Example of trans-cellular transport by a symporter
Na+ Na+
Intestinal
lumen
2.
Na+ Na+
Glucose
Blood
vessels
Glucose
K+
Glucose
Glucose
Glucose
1. Na+/K+ pump establish Na+
gradient
2. Active transport: Na+ driven
3.
glucose symport (“cotransporter”)
3. Passive transport: facilitated
Glucose
“specific” diffusion of glucose to blood
Anim. 11.3-glucose_uptake
Glucose
K+
Na+
K+
K+
ATP
1.
K+
Na+
Na+
Na+
Na+ 19
Compartments/organelles of eukaryotic cells
Compartment
Main function
Cytosol
Protein synthesis, metabolism
Nucleus
DNA & RNA synthesis
Mitochondrion
ATP production
Endoplasmic
reticulum (ER)
Lipid synthesis, synthesis of proteins that
enters the secretory pathway
Golgi
Sorting and packaging
for delivery to cell
surface or lysosome
Lysosome
Protein degradation
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The nucleus – the instruction book of the cell
Nuclear processes: 1. DNA replication
2. Transcription  mRNA, rRNA and tRNA
3. Ribosome subunit assembly
1.
2.
rRNA + 3.
proteins
3-10 mm
Nuclear
pore
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One reason for a nucleus in eukaryotes
Prokaryote
Eukaryote
Transcription
Transcription
mRNA processing
Translation
Translation
In eukaryotes mRNA has to be processed prior to initiation of
translation, which requires spatial separation of transcription
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and translation (Note cloning of an ORF cDNA synthesis)
Transport in and out of the nucleus
1. Transcription
mRNA
1.
tRNA
Nuclear
Nuclear
rRNA
pore
pore
2.
2. DNA replication
Protein synthesis
in the cytosol 23
The nuclear pore complex (NPC)
A typical cell contains 3000-4000 nuclear pore complexes
120 nm
Outer nuclear
membrane
Inner nuclear
membrane
Annular subunit;
the gatekeeper
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Proteins less than 60 kDa
can diffuse ”freely” between
cytosol and nucleus
Nuclear import of proteins (>60kD)
Nuclear Localization Sequence (NLS) = sequence in a protein that
mediates nuclear uptake
N
NLS
N
C
NLS
C
N
N
N
L
N
N L S
C
Could be localized
anywhere in
the protein
NLS
C
S
C Even distant apart in
the primary structure
of the protein
Which becomes
adjacent in the
folded protein
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The process of facilitated nuclear protein import
1.
NLS
Nuclear import receptor (importin)
NLS
2.
3.
NLS
1. Association of target protein
and nuclear import receptor
in the cytosol
2. Binding to the nuclear pore
complex mediated by the
nuclear import receptor
3. ”Walking” through the
gate-keepers of the pore
NLS
4.
4. Dissociation of target
protein and nuclear import
receptor inside the nucleus
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The nuclear import cycle
Cytosol
NLS
Nucleus
1.
Importin
NLS
Importin
Importin
NLS
GDP
Ran
<60 kDa
4.
GTP
Ran
2.
NLS
GDP
Importin Ran
GTP
Importin Ran
+Pi
GTP
Importin Ran
3.
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The driving forces behind nuclear import
NLS
NLS
Cytosol
Nucleus
Importin
NLS
Importin
NLS
GDP
Ran
Importin
GDP
Ran
<60 kDa
GDP
Energy cost!
GTP
Importin Ran
Video 02.3-brownian_motion.mov
GTP
GTP
Ran
GTP
Importin Ran
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Directionality in nuclear import – the Ran cycle
Guanine-nucleotide
Exchange Factor (GEF)
GDP << GTP
GDP
G protein
GTP
G protein
GTPase Activating Protein (GAP)
GDP
Ran
GDP
Ran
Pi
Ran-GEF
Ran-GAP
GTP
Ran
GTP
Ran
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Cytosol
Nucleus
Nuclear export
Nuclear export of proteins is mediated by an intrinsic Nuclear
Export Signal (NES). Proteins with NES include:
NES
NLS
NES
Small protein that should
not be nuclear
Protein that shuttle between
cytosol and nucleus
Video 12.2-nuclear_import.mov
Export of mRNA is dependent on successful splicing
N
E
S
Proteins responsible for splicing
Splicing; removal of introns from mRNA
N ES
Spliced mRNA ready for nuclear export
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