Final Notes BIO2001

W1 BIO2001.......................................................................................................................1
Lecture 1: Membranes and membrane proteins..........................................................................1
1. What are cells?......................................................................................................................................2
2. Visualizing cells.....................................................................................................................................2
3. Cell membranes....................................................................................................................................3
4. Extracellular matrix (ECM)...................................................................................................................5
5. Membrane transport............................................................................................................................6
6. Membrane proteins..............................................................................................................................6
7. Protein translocation I..........................................................................................................................8

W2 BIO2001.....................................................................................................................14
Lecture 2: Intercellular Transport...............................................................................................14
1. Transmembrane transport: Protein translocation II..........................................................................14
2. Gated transport..................................................................................................................................16
3. Vesicle transport.................................................................................................................................18
4. Secretory pathway: transport b/w the ER and Golgi apparatus (COPII)............................................20
5. Transport b/w the trans Gogli network (TGN) and lysosomes...........................................................23
6. Endocytosis (vesicular transport).......................................................................................................23
7. Exocytosis (vesicular transport)..........................................................................................................24

W3 BIO2001.....................................................................................................................26
Lecture 3: Molecular switches and the cytoskeleton..................................................................26
1. Molecular switches.............................................................................................................................26
2. Cytoskeleton structure and function..................................................................................................26
3. Microfilaments (actin filaments)........................................................................................................28
4. Intermediate filaments- diverse proteins used to provide mechanical strength...............................30
5. Microtubules.......................................................................................................................................30
6. Molecular motors...............................................................................................................................32

W5 BIO2001.....................................................................................................................34
Energy........................................................................................................................................34
1. Introduction to energy conversion.....................................................................................................34
2. Energy carriers....................................................................................................................................35
3. ATP production by mitochondria........................................................................................................35
4. ATP production by chloroplasts (focus on light reactions).................................................................36
Signaling.....................................................................................................................................37
5. Signals and receptors..........................................................................................................................37
6. Receptors – GPCRs (15-6b).................................................................................................................38
7. How do signals signal? (15-7).............................................................................................................38

W6 BIO2001.....................................................................................................................39
Lecture – Cell Signaling...............................................................................................................39
1. G proteins which signal via cAMP.......................................................................................................39
2. G proteins which signal via phospholipids..........................................................................................39
3. Enzyme-linked receptors (15-6c)........................................................................................................40
W1 BIO2001
Lecture 1: Membranes and membrane proteins

1. What are cells?

a. Numbers
i. Cell types (200-400), total cells in the body (37 trillion), diameter of a
fibroblast (15 µm) , weight of one cell (2-3), lysosomes/cell (50-1000),
ribosomes/cell (1 million), proteins/cell (10 billion), proteins/µm3 (3
million)
b. Bacteria (basic cell)
c. Animal cell
d. Plant cell
2. Visualizing cells
a. The light microscope
i. Lenses – made of glass
1. Condenser – focuses light onto specimen
2. Objective lens, tube lenses, and eyepiece lenses – focuses
image (cone of light rays) in the eye
ii. Resolution
1. Depends on width of cone of illumination
2. R=0.61 λ /n sin θ
a. θ – Half of the angular width of cone of rays (objective).
b. n – refractive index of medium (ex. air, oil)
c. λ – wavelength of light (typically 0.53 μm for white
light)
iii. Numerical aperture
1. n sin θ
2. Affects light gathering ability
3. ↑ aperture = wider lens = ↑ resolution = brighter image
iv. Types
1. Brightfield (with contrast)
2. Stereomicroscope
3. Fluorescence microscopy
a. Confocal fluorescence microscopy – clear images of
small structures
i. Fluorescent specimen illuminated by focused
light from pinhole
ii. Emitted light (from in-focus point) is focused at
pinhole and reaches detector
b. Electron microscopy
i. Resolves smaller structures than light microscopes
 1. Filament/cathode – emits electrons as a source of illumination
2. Electrons are accelerated and passed through small hole to
form electron beam
3. Lenses – magnetic coils which focus electron beam
4. Specimen placed in a vacuum into path of beam
5. Electrons scattered by electron-dense material (staining),
remained focus to form image
6. Dense regions of specimen = reduced electron flux = darker
ii. Types
1. SEM microscopy
2. TEM microscopy
3. Cell membranes
a. Plasma membrane – selective barrier needed for passage of O2, nutrients,
and waste (determines volume of every cell)
b. Structure – bilayer of glycerophospholipids
i. Fluid mosaic model (1972)
1. Phospholipid bilayer is a 2D fluid matrix
a. Phosphoglycerates
i. Two long-chain fatty acids linked by ester bonds
to adjacent C atoms on glycerol
1. Only 1 typically contains cis-double
bonds
2. Influences packing and membrane
fluidity
ii. Third (remaining) C atom is linked to phosphate
group (which is linked to head group)
b. Lipid motions (extremely fast)
i. Lateral diffusion, flexion, and flip-flop (less
common)
ii. Flip-flop mechanism
1. Needed b/c phospholipid molecules are
synthesized in 1 monolayer (no new
bilayers)
2. Allows migration of new phospholipids
3. Can result in apoptosis
2. Fusion events regulated by fusion proteins (cannot condense
on its own)
3. Proteins
a. Peripheral/extrinsic
b. Integral/intrinsic
c. The lipid bilayer
 i. Phosphoglycerides – most abundant phospholipid in animal cells
1. Glycerol backbone – 3 carbons
2. Long-chain fatty acid (tails) – linked to adjacent carbons via
ester bonds
3. Phosphate group – linked to third C on glycerol and a head
group
ii. Sphingolipids
1. Built from spingosine (instead of glycerol)
a. Long fatty chain with two hydroxyl and an NH3 group
2. Sphignomyelin – most abundant
a. Fatty acid tail attached to amino group
b. Phosphocholine attached to one hydroxyl
iii. Mammalian plasma membranes
1. Phosphatidylethanolamine
2. Phosphatidylserine – carries net negative charge
3. Phosphatidylcholine
4. Sphingomyelin
d. Phospholipids are distributed asymmetrically
i. Cellular signaling – extracellular signals converted to innercellular
without opening up membrane
1. Inner (cytosol) – negatively charged phospholipids
2. Outer (extracellular space) – glycolipids, sphingomyelin, and
phosphatidylcholine
ii. Ex. PKC binds to negative changed phospholipids in cytosolic face
e. Fluidity
i. Temperature
1. Phase transition – gel phase to liquid crystalline as
temperature decreases
a. Occurs at transition/melting temp (Tm)
2. More unsaturated/cis chains = kinks in chains = harder to pack
= requires bigger drop in temp. to freeze = lower Tm
3. Shorter lipid = fewer interactions (for packing) = lower Tm
4. Organisms which cannot modulate their own temperature can
change the composition of their lipid bilayer (synthesizing cis
short chains when temp. decreases)
ii. Composition
1. Cholesterol – enhances permeability-barrier properties
a. Decreases mobility for CH2 groups in FA chain 
decreases permeability to small water-soluble
molecules
b. Tightens packing but does not decrease fluidity
 c. High concentrations (eukaryotes)  prevents
hydrocarbon chain crystallization
4. Extracellular matrix (ECM) – network of proteins and polysaccharides which are
produced and secreted by cell itself
a. Structure
i. Epithelial tissue – outer layer (ex. skin or gut lining) held together by
cell-to-cell contact
1. Cells bound in epithelia, form cell-cell junctions
2. Mechanical stress transmitted between cells via filaments
3. Linked to basal membrane via cell-matrix junctions
ii. Connective tissue – formed from ECM produced by cells which are
sparsely distributed
1. Matrix bears mechanical cells
2. Cell-matrix junctions link cytoskeleton to matrix
iii. Proteoglycans and collagen
b. Animal basal lamina
i. Surround cells (ex. skeletal muscle cells), underlie epithelia, and
interposed between cell sheets (ex. kidney)
ii. Components
1. Nidogen – glycoprotein
2. Perlecan – proteoglycan
3. Laminin – organizes sheet structure (self-assembles into
network)
4. Type IV collagen – assembles into flexible network to resist
tensile stress
a. 3 long protein chains which form superhelix
5. Integrin – transmembrane laminin receptor that holds cells in
place
c. Collagen synthesis and secretion (exocytosis)
i. Synthesis and modification occurs in ER and Golgi apparatus
ii. Leaves cell via exocytosis
iii. Undergoes modifications so that single fibrils can self-assemble into
fibers
d. Cells attach to ECM via transmembrane receptors
i. Occurs via integrins to keep cells attached and signaling
ii. N-terminal on integrin chains bond to extracellular protein
iii. C-terminal tail (on beta-subunit) binds to adaptor proteins
1. Interact with actin
iv. Adaptor protein (ex. talin) - regulates linkage with actin filaments
e. Plant cell wall components (similar to ECM)
i. Cellulose and pectin (secreted by exocytosis)
 ii. Pectin – resists compression
5. Membrane transport
a. Diffusion through a protein-free lipid bilayer
i. Hydrophobic molecules (fastest)
ii. Small uncharged polar molecules (smaller = faster)
iii. Large uncharged polar molecules (larger = slower)
iv. Ions (impermeable)
b. Passive transport (membrane transport proteins) – transfer polar molecules
i. Tend to be multi-pass transmembrane proteins
ii. Transporters proteins/carriers – bind to solute molecules and undergo
conformational changes
1. Solute binding site and ‘revolve’ to other side of membrane
2. Outward open  occluded  inward open
3. Passive – molecule flow depends on gradient
a. Concentration gradient
b. Electrochemical gradient – charged molecules attracted
to side with opposite charge
iii. Channel proteins – form pore through bilayer
1. Faster rate than for transporter proteins
2. Includes water channels to increase membrane permeability
iv. Kinetics of membrane transport
1. Simple diffusion (linear) – rate of transport dependent on
concentration of molecule
a. Limited by physical limits (SA, number of channels, etc.)
2. Transporter-mediated diffusion
a. Vmax – reached when transporters are fully saturated
i. Rate at which it can flip b/w conformational
states
b. Km (binding constant) – transport rate is half of Vmax
i. Affinity of transporter for solute
c. Active transport – requires energy to transport molecule against
concentration gradient
i. Coupled transporters – use energy in concentration gradients to
couple transport of one solute (uphill) to another (downhill)
ii. ATP-driven pumps – use hydrolysis of ATP to drive uphill transport
iii. Light-/redox-driven pumps – use energy from light or redox reaction
to drive uphill transport
6. Membrane proteins
a. Membrane proteins and their association with the lipid bilayer
i. Membrane proteins – amphiphilic structures which perform
membrane specific tasks, giving membrane characteristic functional
properties
1. Transmembrane proteins – extend through lipid bilayer (1, 2,
3)
a. Hydrophobic regions interact with hydrophobic tails of
phospholipid molecule s
b. Hydrophilic regions exposed to water on either side of
membrane
c. Covalently attached fatty acid chain increases
hydrophobicity
i. Inserts into cytosolic monolayer (1)
2. Located in cytosol (bound to cytosolic monolayer)
a. Attached by amphiphilic α-helix (4)
b. Attached by 1+ covalently lipid chains (5)
i. Made as soluble proteins in cytosol, anchored
to membrane by covalent bonding to lipid
3. Exposed at external cell surface (6)
a. Attached to lipid bilayer by covalent linkage to lipid
anchor (gray) in outer monolayer
b. Made as single pass proteins in ER
i. In ER – transmembrane segment cleaved off
and replaced by GPI anchor
ii. Leaves protein bound to non-cytosolic surface
of ER by anchor
iii. Proteins transported to plasma membrane by
transport vesicles
4. Membrane-associated proteins
a. Not found extended into interior of bilayer, instead,
bound to either face of membrane by non-covalent
interactions
b. “Peripheral membrane proteins”  easier to release

ii. Fig. 10-17

1. Single α-helix – fatty
acid chain inserted in
cytosolic lipid
monolayer
2. Multiple α-helices
3. β-sheet/barrel
 4. Exposed on one side of bilayer
5. Anchored to cytosolic side by amphiphilic α-helix
a. Partitions into monolayer by hydrophobic face of lipid
bilayer
6. Attached by a covalently bound lipid chain
a. Cytosolic monolayer
7. Attached by an oligosaccharide linker to phosphatidylinositol
a. Non-cytosolic
b. GPI anchor
b. Transmembrane α-helix
i. Unique orientation – asymmetric insertion and diff. functions in
cytosol and non-cytosol side
1. Membrane is hydrophopic ∴ proteins have hydrophobic
region (yellow/green amino acids)
2. Peptide bonds driven to form H bonds with each other
3. Maximized in alpha-helix structure
ii. Transmembrane sequence  recognized from amino acid sequence
1. Free energy needed to transfer segments of polypeptide chain
from non-polar solvent to water
2. Hydrophathy index
a. +ve – free energy required for transfer to water
(hydrophobic segment). Corresponds to alpha helices
iii. Singlepass – maximized hydrogen bonding in absence of water
1. Ensures polypeptide chain passes through entirely before
bending
iv. Multipass – contain regions which fold membrane from either side
1. Squeezes into spaces of alpha helices, w/out contacting
hydrophobic core of lipid bilayer
7. Protein translocation I
a. Protein synthesis
i. DNA in nucleus is transcribed to mRNA (ss molecule)
ii. mRNA enters cytoplasm where it goes through ribosome and is
translated (using tRNA) to protein via codons
b. Protein movement between cellular compartments (b/c needed throughout
cell)
i. Amino acid sequence – contains sorting signals to direct their
transport from cytosol into nucleus, ER, mitochondria, plastids, or
peroxisomes
1. No sorting signal – stays in cytosol
ii. Gated transport – cytosol to nucleus to nuclear pores
1. Proteins and RNA molecues
 2. Nuclear pores act as selective gates – support active transport
of macromolecules and allow free diffusion of smaller
molecules
iii. Transmembrane transport – protein translocation (active process)
1. Protein translocations directly transport proteins across
membrane (from cytosol)
2. Protein typically needs to unfold to pass through translocator
a. Integral proteins – translocated partically to become
embedded in membrane
3. Includes transport from ER lumen/mitochondria
iv. Vesicular transport – moving proteins in closed compartment
1. Loaded with cargo of molecules, and discharge by fusing with
membrane
2. Transfer of soluble proteins from ER to Gogli
c. Protein signal sequences
i. Found typically at end of protein sequence
1. Needs to be recognized by nuclear pore complex (gated),
translocator (transmembrane), or vesicle/organelle (vesicular)
2. Signal sequences - N-terminus of polypeptide chain
a. Can be removed by signal peptidases once sorting
completes
b. Recognized by sorting receptors
i. Function catalytically (reuse)
ii. Typically recognize protein classes
3. Signal patches – 3D arrangement of atoms on protein’s surface
a. Nuclear import and vesicular transport
ii. Sequence is necessary and sufficient
d. Cytosol  ER
i. ER – highly specialized
ii. Co-translational translocation (rER) – bacteria, archaea, eukaryotes
1. Co-translation – import of proteins into ER before complete
synthesis of polypeptide chain
2. Ribosome – synthesizing protein, attached directly
to ER membrane
a. One end of protein is translocated as rest of
polypeptide chain is synthesized
b. Translation by ribosomes creates signal
sequence
3. Operation
a. ER signal sequence and SRP directs
ribosome to a translocator or ER membrane
 i. Occurs using signal recognition particle (SRP)
ii. SRP is large complex which can accommodate
range of signal sequences (function related to
form)
iii. Recognition – binding of SRP to signal peptide
sequence and ribosome and creates pause in
translation (important to give ribosome time to
move to ER membrane)
iv. SRP-ribosome complex directed to Sec61
protein translocator
v. Targeting – using protein translocator
translation continues and translocation of
protein into ER lumen
b. Forms pore for polypeptide to be translocated
c. Signal peptidase cleaves signal peptide to produce
mature protein, which is released into lumen
d. Release - ribosomes leaves and translocator closes
(prevents leakiness)
i. Ensured by conformational changes b/c of GTP
and hydrolysis cycles
e. No additional energy needed
iii. Post-translational translocation – eukaryotes and bacteria
1. Post-translation – import of proteins into
mitochondria, chloroplasts, nuclei, and peroxisomes
2. Cytosolic/free ribosomes complete synthesis of
protein
a. Protein released before translocation
3. Operation
a. Eukaryotic cells - Sec62, 63, 71, 72 complex
i. Additional complex attached to Sec61
translocator
ii. Deposits BiP molecules on translocating chain
as it emerges from translocator in ER lumen
iii. BiP binding and release – ATP driven cycle
which pulls protein into lumen
b. Bacterial cells
i. Translocation motor protein, SecA ATPase
attaches to cytosolic side of translocator
1. Undergoes conformational changes b/c
of ATP hydrolysis
 ii. Each ATP hydrolysis – pushes segment of
protein through SecY complex
1. 20 amino acids per cycle through pore of
translocator
iv. Signal hypothesis
1. ER signal sequence on polypeptide chain emerges from
ribosomes
a. Directs ribosome to translocator on ER membrane
b. Forms pore in membrane
2. Signal peptidase (closely associated w/ translocator)
a. Removes signal sequence during translation
3. Mature protein released into lumen of ER after synthesis
4. Translocator closed until ribosome has bound
v. SRP – directs ER signal sequence to specific receptor in rER
e. Integration of single-pass transmembrane proteins into ER membrane
i. N-terminal signal sequence (post-translocation)
1. Initated by N-terminal ER signal sequence red)
a. Functions as start-transfer sequence
2. Protein contains stop transfer sequence (orange)
a. Interacts with binding site w/in pore
3. Translocator opens seam and discharges protein laterally into
bilayer
a. Anchored by stop-transfer
ii. Internal signal sequence (co-translocation)
1. Operation
a. SRP binds to signal sequence
i. Recognizes α-helix features
ii. Brings ribosome (making protein) to ER
membrane
b. ER signal sequence
acts as start-transfer
signal
i. Initiates proteins translocation
c. Mature protein
released from
translocator (lateral
gating)
d. Internal start
sequence stays in
lipid bilayer as α-
helix
 2. Orientation of internal sequence
a. Depends on distribution of nearby charged amino acids
b. C-terminus on luminal side (A)
i. More +ve amino acids preceding hydrophobic
core of start-transfer sequence
c. N-terminus on luminal side
i. More +ve amino acids follow hydrophobic core
of start-transfer sequence
ii. Can only occur after N-termal portion has been
fully synthesized b/c translocation must occur
at start-transfer sequence
f. Integration of multipass membrane protein
i. Polypeptide chain passes back and forth in lipid bilayer as α-helices
ii. Internal start-transfer sequence  stop-transfer  start-transfer
1. Sequences are hydrophobic
2. Lock into membrane as membrane-spanning α-helices
3. Distinction b/w start and stop depends on relative order
a. SRP scans unfolded polypeptide at N-terminus and
moves to C-terminus
b. Sets reading frame of hydrophobic region for
membrane integration
c. SRP initiates translocation
iii. Membrane proteins inserted from cytosolic side  same polypeptide
chains have the same orientation
1. Results in protein asymmetry
iv. Fig. 12-45
1. 7 hydrophobic regions  7 α-helices
2. Region nearest N-terminus (H2N) – start-transfer sequence
a. Protein passes into ER membrane
3. Alternate start- stop-transfer sequences (NB starts with start
again)
v. N-terminus – ER lumen
vi. C-terminus – cytosol
W2 BIO2001
Lecture 2: Intercellular Transport

1. Transmembrane transport: Protein translocation II

a. Cytosol to mitochondria
i. Mitochondrial precursor proteins – fully synthesized in cytosol
1. Translocated into mitochondrial by post-translation
2. Signal sequences direct precursor proteins into mitochondrial sub
compartment
a. N-terminus sequences removed by signal peptidase after import
b. Internal signal sequences not removed
c. Necessary for import and localization of proteins
d. Form amphiphilic α -helices – configuration recognized by receptor
proteins
ii. Protein translocators – mediate protein movement across mitochondrial
membranes
1. TOM complex – protein transfer across outer membrane
a. Import of nucleus-encoded proteins
i. Transports signal sequences into intermembrane space
 allows transmembrane protein insertion into outer
membrane
b. β -barrel proteins – passed onto SAM complex to be folded in
outer membrane
i. Abundant in outer membrane
2. TIM complexes (23 and 22) – protein transfer across inner membrane
a. TIM23 – transports soluble proteins into matrix space, and inserts
transmembrane proteins into inner membrane
b. TIM22 – mediates insertion of specific inner membrane proetins
i. Including transporter for ADP, ATP, and phosphate
3. OXA complex – mediates insertion of proteins synthesized within
mitochondria and for insertion of inner membrane proteins (first
transported by other complexes)
iii.

Protein import by mitochondria

1. N-terminal signal of mitochondrial
precursor protein recognized by
receptors on TOM complex
a. Inserted into membrane
2. Protein translocated through TIM23
to span both membranes
 3. Protein enters matrix, where signal sequence is cleaved by signal peptidase
a. Free signal sequence is degraded
iv. Role of energy in protein import
1. Signal sequence recognized by receptor proteins on TOM complex
2. Precursor protein has bound cytosolic hsp70 chaperone
a. Released from precursor protein via ATP hydrolysis
3. Insertion of protein through TOM complex, after which signal sequence
interacts with TIM complex
4. Signal sequence translocated into matrix space
a. Requires energy from inner membrane potential
5. Import ATPase complex
a. Mitochondrial hsp70 binds to regions of protein in matrix
b. ATP hydrolysis used to pull unfolded polypeptide chain through
translocation channel
v. Integration of porins (outer mitochondrial membranes and (Gram negative)
bacterial membranes)
1. Porin – pore-forming β -barrel proteins
a. Permeable to inorganic ions and metabolites (not proteins)
2. TOM complex unable to translocate porins through lipid bilayer
a. Transported (unfolded) into intermembrane space
b. Bind to chaperone proteins (to prevent aggregation)
c. Bind to SAM complex in outer membrane
i. Inserts protein into outermembrane
ii. Folds polypeptide
3. NB In bacteria, the SAM complex is replaced by a BAM complex
a. Catalyzes β -barrel protein insertion and folding
vi. Transport routes into inner mitochondrial membrane
1. A
a. N-terminal signal sequence initiates import into matrix
b. Hydrophobic sequence binds to TIM23 translocator – stops
translocation
c. TOM translocator pulls remainder into intermembrane space
d. Hydrophobic sequence anchors protein in intermembrane space
2. B
a. Protein completely translocated into matrix
b. Start-transfer signal cleaved
c. Adjacent hydrophobic region at new N-terminus
d. New signal directs protein to inner membrane, uses OXA pathway
to insert protein in intermembrane space
e. Hydrophobic sequence anchors protein in intermembrane space
3. C
a. Soluble proteins can have hydrophobic sequence cleaved by
second signal peptidase
b. Active site in intermembrane space
4. D
a. Soluble intermembrane proteins oxidized by Mia40 protein during
import
i. Forms covalent intermediate to pull protein through TOM
complex
 b. Mia40 is reduced, but can be reoxidized by ETC
5. E
a. Multipass inner membrane proteins – contain multiple signal
sequences
b. Chaperones bind to protein to direct it to TIM22 complex
i. Speacialized for insertion of multipass proteins
b. Cytosol to plastids/chloroplasts
i. Thylakoid precursor protein
1. N-terminal chloroplast signal sequence – initiates translocation into stroma
through TOC and TIC complexes
2. N-terminal signal sequence cleaved off
3. Thylakoid signal sequence – initiates translocation across thylakoid
membrane into thylakoid space
a. Sec pathway – mediate transportation
i. Requires ATP and H+ electrochemical gradient (across
thylakoid membrane)
b. SRP-like pathway – uses chloroplast homolog of SRP
i. Requires ATP and H+ electrochemical gradient
c. TAT pathway – Uses two arginines in signal sequence to direct
proteins
i. Requires ATP and H+ electrochemical gradient
d. Spontaneous insertion – no protein translocator required
i. None
c. Summary  mitochondria and chloroplasts
i. Import proteins from cytosol
ii. Proteins transported in unfolded state into matrix space or stroma
iii. Chaperone proteins (hsp70) – maintain proteins in unfolded state, and pulls
polypeptide into organelle
iv. Require specific signal sequence for translocation
1. Can be located at N-terminus (cleaved off) or internal (retained)
2. Sometimes requires second hydrophobic signal
v. Mitochondria – ATP hydrolysis and membrane potential
2. Gated transport
a. Cytosol to nucleus and vice versa
i. Nuclear pore complexes (NPCs) – composed of necleoporins
1. Contains aqueous passages for small water-soluble molecules (passive
diffusion)
2. Larger proteins pass through slower  allows nuclear compartment and
cytosol to have different protein compositions
ii. Nuclear localization signals (NLSs) – sorting signals responsible for selectivity of
active nuclear import
1. ½ sequences containing lysine and arginine
2. Located in amino acid sequence
iii. Arrangement of NPCs in nuclear envelope
1. Nucleoporins arranged with 8-fold rotational
symmetry
a. Transmembrane ring proteins – span
nuclear envelope and anchor NPC to
envelope
 b. Scaffold nucleoporins – form layered ring structures
1. Can help stability curvature of membrane where
nuclear envelope is penetrates
c. Channel nucleoporins – contains extensive unstructured regions
i. Blocks passive diffusion of large macromolecules
2. Fibrils facing nucleus – converge to form basket like structure
b. Active nuclear transport (Ran GTPase)
i. Import
1. Nuclear import receptors/importins – bind and transport cargo proteins
with correct nuclear localization signal
a. Soluble cytosolic proteins that bind to nuclear localization signal
(cargo protein) and FG repeats (channel nucleoporins)
2. Binding b/w import receptors and FG repeats = dissolve gel phase of
protein channels = passage of receptor-cargo complex
3. Import receptors dissociate from cargo
ii. Export
1. Nuclear export signals/exportins – bind to export signal and NPC proteins
to export cargo through NPC to cytosol
iii. Ran GTPase  export and import (12-12/13)
1. Import of nuclear proteins (through NPCs) – requires energy stored in conc.
gradients of GTPase Ran
a. Ran – molecular switch, can be bound to GDP or GTP
b. GAP/GTPase-activating protein – triggers GTP hydrolysis,
converting Ran-GTP to Ran-GDP
i. Located in cytosol
ii. Ran-GDP in cytosol
1. Imported by import receptor
c. GEF/guanine exchange factor – converts Ran-GDP to Ran-GTP
i. Located in nucleus (anchored to chromatin)
ii. Ran-GTP in nucleus
3.
Vesicle

transport

a. Facilitates endocytosis and exocytosis

b. Eukaryotic cells
i. Transport vesicles containing cargo continuously bud off of/fuse with membranes
 1. Must be selective in cargo and fuse w appropriate membrane
ii. Secretory and retrieval pathways determine traffic
c. Compartments involved in transport (fig. 13-3 in Alberts)
i. Compartments
1. Nuclear envelope
2. Endoplasmic reticulum
3. Golgi
4. Extracellular space
ii. Relies on endocytic, secretory, and retrieval (backflow for
membrane recycling) pathways
d. Coated vesicles – surrounded by cage of proteins covering cytosolic
surface
i. Function of coat
1. Inner coat layer – concentrates membrane
proteins giving rise to vesicle membrane
a. Selects appropriate membrane molecules (transport)
2. Outer coat layer – Assembles into lattice to deform membrane patch
(shape of vesicle)
ii. Clathrin-coated – mediate transport from Golgi
and plasma membrane
1. Cargo collected at cell membrane (by
cargo receptors)
2. Cargo receptors bind to adaptor
proteins
a. Adaptor proteins – select
specific set of transmembrane
proteins, and soluble proteins to be packaged
b. Forms coated bud (coated pit – plasma membrane)
3. Forms clathrin-triskelion
4. Membrane-bending and fission proteins found at neck of budding
(introduces curvature of vesicle)
a. Dynamin forms helix of neck of vesicle
i. PIP binding domain – tethers protein to membrane
ii. GTPase domain – regulates rate of vesicles pinching off
b. Contracts and brings phospholipid bilayer together to form vesicle
5. Coat is released (rapidly) after vesicle buds off
a. PIP phosphatase depleats PIP domains – weakens binding of
adaptor proteins
b. Hsp70 chaperone protein = uncoating ATPase – uses ATP
hydrolysis to remove clathrin coat
iii. COP-coated – mediate transport from ER and Golgi cisternae
1. COPI-coated – bud from Golgi compartments
2. COPII-coated – bud from ER
iv. Why are vesicles coated with proteins?
1. Phospholipid bilayer spontaneously forms vesicle  energetically favorable
2. Proteins form skeleton to stabilize structure of vesicle, with
help of another protein they are able to pinch off
e. Phosphoinositides (PIPs) – mark organelles and membrane domains
 i. Inositol phospholipids – found in cell membrane, important regulatory functions
1. Undergo rapid phosphorylation and dephosporylation – produces PIPs
i. Hydroxyl groups at 3’, 4’, and 5’ positions of sugar
(inositol group)
ii. Proteins involved in vesicle transport contain domains which bind to head groups on
PIPs
1. Controls binding of proteins to membrane (domain)
2. Regulates vesicle formation and transport
3. Controls vesicle traffic
iii. Intracellular location of PIPs
1. Secretory vesicles contain PI(4)P, which fuse with plasma membrane
a. PI5-kinase converts PI(4)P into PI(4,5)P2
b. Recruits adaptor proteins  formation of clathrin-coated pit 
clathrin-mediated endocytosis
c. Vesicle buds off  PI(5)P hydrolyzes to PI(4,5)P2
i. Weakens binding of adaptor proteins
f. Vesicle fusion w/ target membrane: Rab GTPases
i. Rab proteins and Rab effectors – direct vesicle to specific spots on target membrane
1. Rab proteins – GTPases associated with organelles in secretory or
endocytic pathway
a. Each organelle has >1 on cytosolic surface
b. Selective distribution = marker for identifying membrane type
c. Cycle b/w membrane and cytosol
2. Rab proteins in GDP-bound state – in cytosol in soluble state
3. Once Rab proteins are in a GTP-bound state – active and tight association
with membrane of organelles/transport vesicles
a. Activated by Rab-GEFs
4. Activated Rab proteins  bind to Rab effector proteins
a. Rab effectors – motor or tethering proteins
5. Tethering a transport vesicle
a. Rab effector proteins interact w Rab-GTPs (active)
i. On target or vesicle membrane
ii. Establishes connection b/w two fusing membranes
b. SNARE proteins on membranes pair to dock vesicle to target
membrane
i. Catalyzes fusion of lipid bilayers
ii. Rab-GTP hydrolyzes to Rab-GDP
1. Dissociates and returns to cystosol
ii. SNARE proteins and SNARE regulators – mediate fusion of lipid bilayers
1. Membrane fusion
a. Membranes brough 1.5 nm from each other
b. Energetically unfavorable b/c water needs to be displaced from
hydrophilic region
2. Complementary sets of proteins
a. V-SNARE – vesicle membranes
i. Single polypeptide chain
b. T-SNARE – target membranes
i. Three proteins
 ii. Associated w/ inhibitory proteins which need to be
released before T-SNARE functions
3. Trans-SNARE complex
a. Four-helix bundle which locks two membranes together
b. Catalyzes membrane fusion using energy of interacting helices
c. Rab proteins regulate availability of SNARE proteins  control
process
4. Untangling SNAREs
a. NSF – protein which catalyzes disassembly using energy from ATP
hydrolysis
b. NSF binds to SNARE complex
4. Secretory pathway: transport b/w the ER and Golgi apparatus (COPII)
a. Why?
i. Protein glycosylation in rough ER
1. Covalent addition of oligosaccharides to proteins  form glycoproteins
2. Precursor oligosaccharide (N-acetylglucosamine, mannose, and glucose) is
transferred to NH2 group (N-linked)
3. Catalyzed by oligosaccharyl transferase
a. Active site on luminal site of ER
membrane (does not occur for cytosolic
proteins)
b. Removes oligosaccharide from dolichol
and transfers to NH2/asparagine group
i. Dolichol – lipid molecule with
anchors precursor in ER
membrane
1. Linked by pyrophosphate bond  activation
energy to drive glycosylation reaction
ii. Folding check
1. Oligosaccharides – tags to mark state of protein folding
a. Proteins require N-linked glycolysation for proper folding
2. ER chaperone proteins
a. Calnexin – traps protein in ER
i. Binds to incompletely folded proteins (on N-linked
oligosaccharides)
ii. Glucosyl transferase determines if they are unfolded
b. Calreticulin  soluble ER resident protein
c. Bind to oligosaccharides on incompletely folded proteins, retained
in ER
i. Bind after 2/3 glucoses on precursor have been removed
ii. Prevent aggregation
iii. Promote association with other ER chaperone
3. How do they recognize folded/unfolded proteins?
a. Glucosyl transferase – adds glucose to oligosaccharides that are
losing last glucose
i. Oligosaccharide must be attached to unfolded proteins
b. Unfolded – undergoes cycles of trimming (glucosidase) and
addition (glucosyl transferase) until fully folded
iii. Misfolded proteins  activate unfolded protein response
 1. Recognized and targeted to translocator complex in ER membrane
2. Interact in ER lumen with chaperones (disulfide isomerases) and lectins
3. Exported into cytosol through translocator
4. Ubiquitylated, deglycosylated, and degraded in proteasomes
b. COPII coated vesicles
i. Inner coat – interacts w/ membrane proteins
ii. Outer coat – membrane curving
c. Recruitment of cargo
i. Membrane proteins packaged into budding vesicles
1. Interact w/ adaptor proteins of inner COPII coat
2. Proteins can function as cargo receptors (exit signal)
d. Exiting the ER
i. Protein glycosylation (rough ER)
1. Polypeptide enters ER lumen
2. Precursor oligosaccharide anchored in ER membrane by a lipid anchor
3. Precursor transferred from lipid anchor to asparagine
a. Catalyzed by oligossacharyl transferase enzyme
4. Function – verifies whether protein is folded correctly
a. Unfolded/incorrectly folded – remains in ER (requires ATP)
i. Targeted to translocator complex in ER
ii. Exported through translaocator in cytosol
iii. Ubiquitylated, deglycosylated, and sent to proteasome to
be degrades
b. Peptide chain  check cycle  protein exists
e. Vesicular tubular clusters – mediate transport from ER to GA
i. Move along microtubules to carry proteins
ii. Clusters bud off COPI coated vesicles
1. Mediate budding of vesicles which return to ER (from clusters and/or GA)
2. Function as retrieval pathway – ER proteins, cargo receptors, and SNAREs
3. Assembly initiated rapidly after COPII coats are shed
iii. Depends on ER retrieval signals
f. Golgi apparatus – carbohydrate synthesis, sorting and dispatching station
i. Cis FACE – vesicles enter
ii. Trans FACE – vesicles exit
iii. Protein maturation (by glycosylation)
1. Sorting – occurs via COPII vesicle
a. Phosphorylation of oligosaccharides
on lysosomal proteins
2. Removal of mannose
3. Removal of Man and addition of GlcNAc
4. Addition of Gal and addition of NANA
5. Sulfation of tyrosine and carbohydrates
6. Sorting – buds off as lysosome, plasma membrane, or secretory vesicle
iv. Glycosylation of proteins
1. General structure
a. Core region – derived from N-linked oligosaccharide added in ER
b. Contains two GlcNAc and
three Man
2. Complex oligosaccharide
 a. Core region
b. Terminal region – heterogenous structure
c. Three terminal branches
3. High-mannose oligosaccharide
a. Contain additional mannoses
v. Maturation in/of Goglgi cisternae
1. Cisternal maturation model
a. Each cisterna matures as it migrates outwards
b. Golgi resident protteins carried forward in maturing cisterna is
moved back via COPI vesicles
2. Vesicle transport model
a. Golgi certernae are static
i. Contain characteristic resident enzymes
b. Molecules moving from cis to trans occurs using forward moving
vesicles
3. Unclear if both or only one of these theories works
g. COPI – retrieval pathways (recycling)
i. ER retrieval signals – bind directly to COPI coats
1. Resident ER membrane proteins contain signals which allow them to be
packaged in COPI vesicles for transport back into the Er
ii. Resident proteins bind to receptor proteins (not directly to COPI coat)
1. KDEL receptor protein – multipass transmembrane protein
a. Binds to KDEL sequence
i. Retrieval signal on BiP proteins
b. Packages protein into COPI-coated retrograde transport vesicle
i. Travel back to ER
ii. Vesicles shed coat
c. Cycles between ER and GA
2. Binding affinity of receptor for KDEL sequence
a. High in vesicular tubular clusters and GA
i. Captures escaped soluble ER resident
proteins (in low conc.)
ii. Lower pH
b. Low in ER
i. Unloads cargo in high concentration of resident proteins
5. Transport b/w the trans Gogli network (TGN) and
lysosomes
a. Lysosomes – organelle containing soluble enzymes
i. Low pH – for acid hydrolases
1. Contents of cytosol protected against
attack by digestive system
a. Membrane
b. Enzymes denature at higher pH
of cytosol
2. Maturation
a. Late endosomes – contain material from plasma membrane
(endocytosis) and lysosomal hydrolases
b. Late endosomes fuse with preexisting lysosomes 
endolysosomes
 3. Resistant/slowly digestible residues remain  form lysosomes
b. Delivering material to the lysosomes
i. Endocytosis – macromolecules taken up by extracellular fluid
ii. Phagocytosis – engulfment of large particles/microorganisms to form phagosomes
iii. Macropinocytosis – non-specific uptake of fluids, membrane, and particles
iv. Autophagy – digests cytosol and degrading organelles
v. Final step  fusion with lysosomes
c. Mannose 6-phosphate receptor – sorts lysosomal hydrolases in TGN (unidirectional)
i. Enzymes (lysosomal hydrolases)  endosomes (from TGN)  endolysosomes 
lysosomes
ii. How are the enzymes recognized? Mannose 6-phosphate (M6P)
1. Added to N-linked oligosaccharides while passing through CGN
2. M6P receptor proteins in TGN recognize M6P groups
a. Bind to lysosomal hydrolases to membrane and adaptor proteins
(clathrin-coated vesicles)
iii. Hydrolases bud off in clathrin-coated vesicles from TGN
iv. Contents delivered to early endosomes (vesicles shed coat)
1. Lower pH causes hydrolases to dissociate from M6P receptors
2. Phosphate removed from M6P  lysosomal hydrolase precursor
v. Empty receptors retrieved in retromer-coated vesicles
1. Transported to TGN
6. Endocytosis (vesicular transport)
a. Process by which cells take up plasma membrane components, fluid, solutes,
macromolecules, and particulate substances.
b. Endosome maturation
i. Early endosome – fuses with endocytic vesicle fuses to sort internalized cargo
ii. Recycling endosome – returns cargo molecules to plasma membrane (can also
happen directly)
iii. Late endosome – delivers cargo destined for degradation
1. Undergo maturation
2. Protein composition of endosome membrane changes
a. Form intralumenal vesicles
3. Endosome itself moves closer to nucleus
4. Fuse with each other and lysosomes  endolysosomes
a. Degrade contents
iv. Bidirectional pathways to TGN
1. Insertion of new materials – ex. lysosomal enzymes from ER
2. Retrieval of components – ex. MP6 receptor
c. Receptor mediated endocytosis – importing selected extracellular macromolecules
i. Macromolecules bind to transmembrane receptor proteins (accumulate in coated
pits). Enter cell as receptor-macromolecule complexes in Clathrin-coated vesicles
ii. Ex. Transportation of cholesterol (as LDLs – a cholesteryl ester)
1. LDL inserted into membrane via transmembrane receptor proteins
2. Receptors diffuse until associate with forming clathrin-coated pits
3. Endocytosis signal in LDL receptor tail binds to AP2 (membrane-adaptor
protein)
4. AP2 recruits clathrin to initiate endocytosis (in coated vesicles)
5. By shedding the clathrin coats (once in cell), vesicles transfer contents to
early endosomes
 6. LDL and LDL receptors encounter low PH in early endosomes, LDL is
released and delivered to lysosomes (via late endosomes)
7. Cholesteryl esters are hydrolyzed to free cholesterol
d. Phagocytosis
i. Cell, debris, and bulky particulate matter undergoes endocytosis
1. Common in carnivorous cells (ex. amoeba or macrophages)
ii. Actin dependent
iii. Receptors activated by cargo/particles (ex. antibodies on surface of pathogens)
1. Facilitates polymerization of actin filaments  able to extend
pseudopods/cell body
iv. Neutrophil
1. Able to phagocytose a bacterium
2. Pseudopod extension is driven by actin polymerization and reorganization
3. Actin responds to accumulation of phosphoinositides in membrane
a. PI(4,5)P2 – actin polymerization (forming pseudopod)
b. PI(3,4,5)P3 – depolymerizes actin filaments at base
e. Transcytosis! – Vesicle transport
i. Transport of macromolecules across interior of cell
f. Pinocytosis
i. Vesicles form from coated pits in plasma membrane
7. Exocytosis (vesicular transport)
a. Cells separates proteins into those destined for lysosomes, secretory vesicles, and immediate
delivery for cell surface from TGN
b. Secretion of biomolecules and materials (secretory pathways)
i. Constitutive secretory pathway
1. Operates in all cells, no signal
needed
2. Supplies plasma membrane w/
new membrane lipids and
proteins
ii. Regulated secretory pathway
1. Proteins are concentrated and
stored in secretory vesicles
2. Extracellular signal required to
stimulate secretion
c. Pathways from TGN
i. Signal-mediated diversion to lysosomes via endosomes
1. Proteins with M6P marker diverted to lysosomes
2. Clathrin coated transport vesicles
ii. Signal-mediated diversion to secretory vesicles (regulated secretion)
1. Proteins directed to secretory vesicles
a. Uses signals
iii. Constitutive secretory pathway
1. Unpolarized cells – no markers
2. Polarized cells (ex. epithelial cells) – proteins directed to apical or
basolaterial plasma membrane domain
a. Signal needed to mediate pathway
d. Formation of secretory vesicles
i. Secretory proteins aggregate in ionic environment of TGN
 1. Aggregates concentrate  lumen acidifies
ii. Clathrin-coated vesicles retrieve excess membrane and luminal content
1. Found in immature secretory vesicles during maturation
e. Secretion of lipids for plasma membrane enlargement
i. Deliver more membrane to enlarge SA of cell’s plasma membrane
ii. Cytokinesis
iii. Phagocytosis
iv. Plasma membrane repair
v. Cellularization
W3 BIO2001
Lecture 3: Molecular switches and the cytoskeleton
1. Molecular switches
a. Microprocessors (input/output)
i. Protein function regulated by binding small molecules to amino acids
ii. Ex. Phosphorylation in eukaryotes
1. Phosphorylation – kinases
2. Dephosphorylation – phosphatases
3. Binding and hydrolyzing GTP (reversible process)
b. Phosphorylation – activating class of proteins (molecular switches)
i. Affecting the protein
1. Negative charges – induce conformational change,
reveals new binding sites on proteins
a. Comes from phosphate group (F
2. Phosphate group – partakes in binding, causes
association
3. Phosphate group – masks binding site, causes
dissociation
4. Fig 3-61a
ii. Via enzymes
1. Kinase – catalyzes transfer of phosphate on ATP to
hydroxyl group on a serine, threonine, or tyrosine
a. Amino acids which can be phosphorylated
2. Phosphatase – catalyzes reverse
a. Returns to circulation  recombined in
phosphate molecules
3. Occurs on serine, threonine, or tyrosine (O=P-O 3)
iii. Intracellular signalling proteins controlled by phosphorylation
can be protein kinases themselves  kinase cascades
c. GTPases (GTP binding proteins)
i. Phosphate is part of GTP (not directly attached to protein)
ii. GTP (on) hydrolyzed to GDP (off)
iii. Fig 15-7
1. RHS –GDP added = protein off, GTP bound = protein
on, hydrolysis = protein off
iv. Classes
1. Trimeric GTP-binding proteins/G proteins – relay
signals from GPCRs
2. Monomeric GTPases – relay signals from cell-surface receptors
2. Cytoskeleton structure and function
a. Cytoskeleton – network of fibers extending through cytoplasm
i. Organizes structure/activity of cell
ii. Anchors organelles
iii. Protein filaments
1. Microfilaments (actin) – cell surface shape, whole-cell locomotion,
pinching of cell
a. Thin helicopolymers of actin (protein)
b. Dispersed in cell, but highly concentrated in coretex (below
plasma membrane)
c. Can bundle into linear bundles, 2D networks, or 3D gels
i. I. TEM – single filament
 ii. II. Microvili in lumen of gut – expulsions/finger like
structures possible b/c of actin
iii. III. Stress fibers – terminate in focal adhesions
iv. IV. Striated muscle – actin (with myosin) work to
contract muscle cells on a small scale
2. Microtubules – positioning membrane-enclosed organelles,
intracellular transport, formation of mitotic spindle
a. Larger than microfilaments
b. Long hollow structures of tubulin (protein)
i. Provides rigidity
ii. Made of dimers and a centrosome (at end  MTOC)
c. II. Three cilia – work together to form triplets to form a
structure
d. III. Cell division (mitosis – interphase) – microtubules
organize organelles in cell
e. IV. Ciliae
3. Intermediate filaments – mechanical strength
a. Between microfilaments and microtubules
b. Form rope-like structures – many types of proteins
i. Ex. Nucleus – form cage around DNA. Cytosol –
cables for structure
ii. Formation of nails, hair, etc.
c. II. Neurons – Help to form long axon
d. III. Epithelial cell – form network in skin
e. IV. Nuclear lamina
b. Roles
i. Supports cell by forming specialized stable structures
1. Intestinal epithelial cells keep shape and polarity
a. Even under mechanical stress, shrinkage, stretching
2. Microvilli and cilia – maintain constant location, length, and
diameter over lifetime of cell
a. Actin bundles in cilia maintain organization b/c cells do not
turn over
ii. Large scale cell polarity – needed for cell to determine difference b/w
top/bottom
1. Maintained over lifetime of cell
2. Polarized epithelial cells – arrays of microtubulues, actin filaments,
and intermediate filaments
a. Adhere strongly to each other  physical barrier
3. Apical surface – faces intestinal lumen
a. Bundles of actin filaments form
microvilli  cell SA
4. Terminal web of actin – anchors cells together
via adherens junctions
5. Intermediate filaments – anchored to adhesive
structures (desmosomes and
hemidesmosomes)
a. Connects epithelial cells to sheet,
which connects to basal lamina
b. Chemical strength
 6. Microtubules – establish polarity from apical to basal side
a. Apical  neg.
b. Basal  pos.
c. Enables cell to direct newly synthesized components to
correct location
iii. Essential for (rapid) cell shape change and dynamics
1. Cytoskeletal organization  cell division (16-2)
a. Microtubules – rearrange to form bipolar mitotic spindle
i. Chromosomes align (for duplication)
ii. Chromosomes separate
b. Actin – form contractile ring which pinches cell
c. Microtubules and actin rearrange to pull cell apart
2. Rearrangement
a. Fig. ?
b. Actin polymerization – converts b/w small soluble subunits
and large filamentous polymer chains
c. Mechanism
i. Signal (ex. nutrient source)
ii. Disassembly of filaments – rapid diffusion of actin
subunits
iii. Reassembly of filaments at new site
d. Ex. Neutrophils – rapid actin cytoskeletal rearrangement
iv. Facilitates motility by interactions w/ motor proteins
v. Vesicle transport along monorails
vi. Regulates biochemical activities (signaling) – released which can be
transmitted
3. Microfilaments (actin filaments)
a. Actin subunits (G-actin)  Actin filaments (F-actin)
i. Regulating actin filament formation allows cells to control shape and
movement
b. F-actin filaments composed of G-actin molecules (globular/balls)
i. G-actin assembled head-to-tail and then wound to form helix
1. Acts as binding site for nucleotides (ATP or ADP at center of
molecule)
2. Plus end – fast-growing site
3. Minus end – slow-growing site
ii. Treadmilling
1. Difference in Cc (critical concentration) at each end (rate when other
side is capped)
a. Rate of subunit addition = rate of subunit loss
(concentration of free subunit)
b. C > Cc – both ends grow, C < Cc – both ends shrink
2. Treadmilling – net flux of subunits, but polymer remains at constant
length
a. C > Cc (plus end) and C < Cc (minus end)
iii. Fig 16-11
iv. Dynamic properties (fig 16-14a)
1. Soluble subunits bound to ATP in solution
2. Polymerization into filament – hydrolyzed into ADP
a. Occurs faster at +end
b. Rate of growth at +end = rate of diminishing at -end
c. Actin-bound proteins regulate dynamics and organization
i. Accessory proteins bind actin monomers or filaments
ii. Accessory proteins (examples)
1. Binding to subunits – thymosin (prevents assembly) and profilin
(speeds elongation)
2. Tropomyosin – stabilizes filament
3. Cofilin – accelerates disassembly (binds ADP-actin filaments)
4. Capping proteins – prevents assembly and disassembly at +end
iii. Formin – regulates F-actin at +end (unbranching)
1. Fig 16-17
2. Formin proteins form dimeric complex
3. Both subunits can bind to monomeric actin  nucleates actin
polymerization by acting as a mold for G-actin
4. Associated with +end
5. Nucleates growth of straight/unbranched filaments
6. Also indirectly connected to cell plasma membrane – insertional
polymerization of actin below membrane
iv. Arp2/3 complex – initiates actin polymerization of straight or branched
filaments
1. Structure
a. Top of Arp2 and Arp3 resembles plus end of actin
b. Differences on sides/minus end prevents complexes from
forming own filaments
2. Mechanism
a. Activating factor binds to inactive Arp2/3 complex
i. Inactive to ensure will not polymerize itself
b. Active Arp2/3 complex resembles plus end of actin
c. Actin subunits assemble onto complex
d. Bypasses rate-limiting step of filament nucleation
3. Nucleates actin growth from minus end, which allows elongation at
the plus end
a. More efficient when bound at 70 deg. to original actin
filament
b. Repeated branced nucleations  web of actin filaments
4. Fig 16-16b/c
d. F-actin architectures in the cell
i. Functions
1. Influence cellular mechanical properties and signaling
2. Linear/parallel/contractile bundles – form stress fibers
3. Gel-like network (criss cross) – form cell cortex
4. Dendritic network (branching via Arp2/3) – lamellipodium
5. Tight-parallel bundle (cross linked for strength) – filopodium
ii. Actin bundles
1. Bundling proteins cross-link filaments into parallel array
2. Fig. 16-23
3. Parallel bundle (tight)
a. Consist of closely packed actin filaments by fimbrin
b. Tight packing prevents myosin II from entering bundle 
not contractile
4. Contractile bundle (loose)
 a. Consist of actin filaments crosslinked with alpha-actinin
(homodimer)
b. Loose packing allows myosin II to enter  participates in
contraction
iii. Gel-like
1. Gel-forming proteins hold two actin filaments at large angle (looser
system)
2. Fig 16-24a
3. Cells use filamin protein (homodimer) – forms flexible linkages b/w
actin filaments at right angles
4. Needed for lamellipodia
4. Intermediate filaments- diverse proteins used to provide mechanical strength
a. Structure
i. Alpha helical region of polypeptide monomer  coiled-coil dimer 
staggered and antiparallel tetramer of two coiled-coil dimers  lateral
association of 8 tetramers  addition of 8 tetramers to growing filament
1. Half of each dimer points in each direction
ii. Monomers
1. Obs. Abnormal neurofilament assembly in
motor neuros can result in neurodegenerative
diseases (ex. ALS)
2. Different from actin/tubulin  many more
genes involved
b. Property: strength b/c of interactions b/w filaments
i. Fig. 16-9
ii. Staggered long subunits – lateral contacts most prevalent
iii. Rope-like properties – tolerate a lot of stretch and bending
1. Arise from hydrophobic interactions and non-covalent bonds b/w
subunits of cytoskeletal filament
c. Function: impart mechanical stability (animal cells) (16-68)
i. Keratins
1. Keratin filaments are made up of an equal mixture of type I and type
II keratin proteins
2. Form heterodimer filament subunit
3. Cross-linked by disulfide bonds
4. Function – mechanical strength in epithelial cells by anchoring
intermediate filaments (desmosomes/hemidesmosomes)
5. Mutations in keratin genes  Epidermolysis bullosa
a. Defective intermediate filaments
b. Weak skin connection to basal lamina and severe blistering
c. Results from defective keratin filaments (mutated) – keratin
network is disorganized causing cell rupture
ii. Laminins
1. Mesh like structure forming nuclear envelope
iii. Mechanoreceptive!
5. Microtubules
a. Structure
i. Composed of alpha- and beta-tubulin heterodimer (subunit of 2 monomers)
1. GTP binding site in both tubulins
a. Alpha – GTP is tightly bound, considered part of protein
 b. Beta – GTP is less tight, important for dynamics (can be in
GTP or GDP form)
2. Fig. 16-42
a. B. Formation of protofilament consisting of heterodimers
b. C. Microtubule formation by lining up 13 protofilaments in
parallel. Form hollow tube
ii. Hollow tubes formed by protofilaments (16-42)
1. 13 parallel protofilaments formed with high binding energy
a. Beta – plus end
b. Alpha – minus end
iii. Formation of microtubule (16-47)
1. Alpha and beta required in high conc.
2. Gamma tubulin  nucleation of microtubule growth
3. Growth starts at microtubule organizing center (MTOC) 
centrosome
a. Consists of two centrioles (short
microtubules)
b. Microtubules grow from gamma-
tubulin ring complexes (nucleating
sites) on centrosome
c. Located near nucleus
d. Have pair of centrioles embedded
b. Function of microtubule proteins is to regulate dynamics and
functions of microtubules
c. Undergoing dynamic instability
i. Why?
1. Alpha and beta subunits both have a GTP binding site
a. Only exchangeable in beta subunit, b/c too tight in alpha
b. Obs. Speed of polymerization in cell is faster than for
purified tubulin (in lab)
c. T form and D form
2. Rates of GTP hydrolysis and tubulin addition
a. Rate of tubulin addition high = new subunit added before
previous one has hydrolyzed = T form = GTP cap
b. Rate of tubulin addition low = D form
3. Mechanism
a. Rapid growth = GTP capped end on beta tubulin
b. If hydrolysis is faster than polymerization, microtubule
changes conformation and undergoes depolymerization
(shrinks)
c. GDP (after hydrolysis) can be exchanged by GTP,
recommences growth
ii. Dynamic instability (16-44a) for tubulin concentrations in solutions b/w C c
for T and D forms  SINGLE MICROTUBULE
1. Filament grows GTP-capped end (beta tubulin)
2. Catastrophe – GTP cap loss bc hydrolysis is faster than addition of
subunits
3. Rapid shrinkage
4. Rescue – regain of GTP cap (by GDP-GTP exchange)
5. Rapid growth reinstated
6. Modulated by several proteins
 iii. Structural consequences of GTP hydrolysis
1. Addition of T form  linear growth which can be packed into
protofilament cylinder
2. GTP hydrolysis into GDP weakens bonds  curved shape
3. Depolymerizes so that GDP-GTP exchange can take place again
d. Dynamic instability in protofilaments
i. Protofilament alignment
1. Less stable region of microtubule
containing GDP-tubulin dimers away
from GTP caps
2. Loss of GTP cap – rapid
depolymerization
e. Structure of protofilaments gives stability despite
dynamics
i. Single protofilament – thermally unstable
1. Breakage in middle bond equally likely to break filament as breakage
at end
ii. Multiple protofilaments – thermally stable
1. Multiple bonds need to be broken to break protofilament in 2
2. Removal from one end only breaks small number of bonds
iii. Facilitates dynamic behavior
6. Molecular motors
a. Motor proteins that orchestrate movement using chemical energy (ATP)
i. Chemical energy (ATP) to mechanical energy (movement)
ii. Linear – move along a lattice
1. Myosins
2. Kinesins
3. Dyneins
iii. Rotating – consist of rotor and stator
1. Flagellar motor
2. ATP synthase
b. Myosin II – linear motor that produces muscle contractions (with help from actin)
i. Structure (Fig. 16-26)
1. 2 heavy chains with heads responsible for ATPase activity which
drives contraction
2. 4 light chains
ii. Contraction (Fig. 16-29)
1. Attached in rigor configuration
a. Myosin head locks onto actin filament in rigor configuration
b. Head lacks bound nucleotide  no energy involved
c. Short-lived state for active muscle (bc terminated with ATP
binding)
2. Released (from actin)
a. ATP binds to cleft on back of head (furthest from actin
filament)
i. Conformation changes in actin-binding site
b. Reduced affinity b/w head and actin  release  freedom
to move along actin
3. Cocked
a. Cleft on myosin head closes around ATP molecule (with
ATPase activity)
 b. Triggers lever arm to move (head is displaced along filament
by around 5 nm)
c. ATP hydrolysis occurs, ADP and Pi remain bound to protein
4. Force generating
a. Myosin head binds (weakly) to new site on actin filament
b. Releases Pi concomitantly with tight binding b/w myosin
head and actin
c. Triggers power stroke
i. Head regains original confirmation
ii. Loses bound ADP
5. Attached
a. Myosin head locked in rigor configuration (at new position)
iii. 16-82
1. Actin-mediated potrusions push cell forward when there are strong
cell-cell adhesions b/w actin and focal points to link cell to ECM
2. Transmembrane integrin proteins form focal adhesions
3. Disengaged – no interactions b/w actin filaments and focal
adhesions
a. Actin filament moves rearwards w/ help of myosin motors
4. Engaged – Actin-binding proteins link actin and integrins
a. Myosin contractile function generates traction
b. Actin polymerization  leading edge forms a protrusion
c. Microtubule based transport using motor proteins
i. Kinesins move organelles away from nucleus (- to +) (16-57)
1. Structure - dimer of two motor heads connected by a coiled coil tail
2. Mechanism
a. Kinesin step – rear head (at negative end) detaches from
tubulin and passes leading head
b. Rear head undergoes hydrolysis to drive motion
c. Pi is bound to leading head  ATP  new rear head
3. Small movements at nucleotide-binding site regulates docking and
undocking of motor head domain
4. Walk along microtubule
ii. Dyneins move organelles towards nucleus (+ to -) in axons (
1. Structure
a. Large dynein motor head - ATPase activity
b. Coiled-coil stalk with microtubule binding site at end
2. Mechanism
a. ATP bound state – stalk detaches from microtubule
b. ATP hydrolysis causes stalk microtubule attachment
c. Release of ADP and Pi – power stroke by rotation of head
and stalk relative to the tail
d. Flagellar motor
i. Fig. 14-35
ii. Stator proteina
Rotor proteins
W5 BIO2001
Energy
1. Introduction to energy conversion
a. Cells work against disorganization  requires energy (total entropy
increases)
b. Energy production by ATP
i. Related to structural organization of cell
ii. Cell organelles for energy production
1. Oxidative phosphorylation – Mitochondria (all cells of animals,
plants, and fungi)
2. Photosynthesis – Chloroplasts (plants and green algae)
iii. Fundamental mechanism – chemiosmotic coupling (stage 1 and stage
2)
1. Performed by protein complexes in a membrane
a. Chemical bond-forming reactions generate ATP
b. Membrane transport processes rely on osmosis
c. Stage 1: Generating an electrochemical gradient
i. High energy electrons are transferred along an electron-transport
chain
ii. Each electron transfer releases energy
iii. Energy pumps H+ across the electrochemical energy
iv. Stored as potential energy

v. Mitochondria
1. Conversion of energy from fuels through citric acid cycle
2. Citric acid cycle produces electrons, CO2, and H2O
3. Connects to ETC
a. NAD+ to NADH
i. Takes up two electrons and one H+
b. H+ pumps create gradient
c. Electrons combine with O  O2
vi. Chloroplasts
1. Light interacts with photosystems
2. CO2 undergoes carbon-fixation cycle
 3. Electrons extracted from water through photolysis
4. Connects to ETC
a. NADP+ to NADPH
i. Takes up two electrons and one H+
b. H+ create gradient
d. Stage 2: Using the gradient to produce chemical energy (ATP)
i. Protons flow down electrochemical gradient through ATP synthase
ii. ATP synthase – complex of membrane proteins
1. Catalyzes production of ATP from ADP
2. Uses H+ in a turbine like motion
2. Energy carriers
a. ATP – adenosis triphosphate
i. Synthesized in energetically unfavorable reaction
ii. Carries inorganic phosphate group in high-energy linkage
iii. Hydrolyzed ATP  ADP
b. GTP – guanosine triphosphate
c. UTP
d. CTP
e. NADH, NADPH, FADH2 – carries electrons and hydrogens in high-energy
linkage
i. NAD(P)H – electron carrier found in mitochondria/chloroplasts
1. Specialized molecules
2. Interplay with NADH and NAD+, or NADPH and NADH
ii. FADH2
1. Two energy carrying P atoms
2. FAD + 2H+ + 2e-  FADH2
3. ATP production by mitochondria
a. General knowledge
i. Sugars go through glycolysis to produce pyruvate
ii. Fatty acids are oxidized in the fatty acid cycle, until
they are degraded into acetyl CoA. Used to produce
FADH2
iii. NAD+ + H+ + 2e-1  NADH
b. Citric acid cycle reduces NAD+ to NADH
i. NADH enters respiratory chain  start of energy
production
c. Three respiratory-chain proton pumps (14-18)
i. Found in inner mitochondrial membrane
ii. Protein pumps driven by electron transport
1. 1. NADH dehydrogenase
a. Accepts two electrons from each
NADH and brings them to
ubiquinone/Q (electron carrier)
b. Three/four proton-pumping
molecules
c. Structure
i. Electron transfer – occurs in matrix arm
 ii. Proton pumping – occurs in membrane arm
d. Mechanism
i. NADH docks near tip of matrix arm
ii. Transfers electrons via flavin mononucleotide to
iron-sulfur clusters
iii. Electron transfer to quinone triggers proton
translocation in membrane arm  connected
via alpha helix
iv. Pumps 4 protons for the 2 electrons from NADH
2. 2. Succinate dehydrogenase
a. Does not pump protons
3. 3. Cytochrome c reductase
a. Accepts reduced Q, and offers them to cytochrome C
4. 4. Cytochrome c oxidase
a. Accepts electrons one at a time, and passes them on to
oxygen molecules to produce water
iii. Summary – NADH (from citric acid cycle)  Complex I  ubiquinone
(Complex II)  Complex III  cytochrome c  Complex IV 
molecular oxygen  water
1. 14-19
a. Redox potential increases as electrons flow down ETC
to oxygen
b. Oxygen has highest redox potential  wants electrons
(end of chain)
iv. FADH2 (2-57)
1. Generated by fatty acid oxidation and succinate
dehydrogenase in citric acid cycle
2. Each turn = 1 FAD  1 FADH2
d. Protons drive ATP synthase (14.10)
i. ATP synthase (14-30a)
1. Works in forward and reverse directions
2. Driven by electrochemical gradients (ATP from ADP and Pi)
3. Composed of rotor and stator
ii. Mechanism
1. Inner membrane –
2. Inner boundary membrane –
3. Crista space – contains x and ATP synthase
a. ATP synthase dimers – stabilize curvature
4. Electron transport chain pumps protons from inner membrane
into cristae space (proton sink) – enable ATP synthase to
generate ATP efficiently
5. Converts mechanical force into chemical energy by joining ADP
and P  ATP
a. Protons flow rapidly
b. 3 ATP molecules per turn
4. ATP production by chloroplasts (focus on light reactions)
a. Net result: 6CO2 + 6H2O  C6H12O6 + 6O2
 b. 14.46/52
c. Antenna complex – contains chlorophyll, traps excitation from sunlight
d. Photosystem II – withdraws an electron from water
i. Produces 4H+
ii. Supplied by photolysis?
e. Electron passes via plastoquinone and cytochrome b6-f to photosystem I
i. Cytochrome – protons pumps from stroma to thylakoid space using
energy from electron movement
ii. Plastoquinone – electron carrier
f. Photosystem I – uses another light-driven change separate reaction to
produce NADPH
i. Low energy electrons are reenergized
ii. NADP+ to NADPH
iii. Transferred to ferredoxin NADP+ reductase
g. ATP Synthase
Signaling
5. Signals and receptors
a. Cells change in response to signals
i. Monitor intracellular and extracellular environments
ii. Emit and receive signals
b. Signals
i. Hydrophilic signals (most signalling)
1. Membrane impermeable
2. Bind to cell-surface receptors
ii. Hydrophobic signals
1. Membrane permeable
2. Bind to intracellular receptors (ex. steroid or thyroid
hormones)
iii. Selective and specific in interactions (first messengers)
iv. Ex. Stimulators, hormones, growth factors, local regulators,
neurotransmitters
c. Types of signaling (15-2)
i. Contact dependent – short
ii. Paracrine – medium-short
iii. Synaptic – specific for signaling between nerve cells or nerve-muscle
cells
iv. Endocrine – hormone travels over long distances in blood
d. Cells respond to specific combinations of extracellular signals
i. Different receptors = machinery
ii. Ex.
1. Survival
2. Growth and division
3. Differentiation
4. Die
iii. Neurotransmitter – Ach elicits various responses (15-5)
1. Decreased firing rate in heart pacemaker cells
2. Secretion in salivary gland cells
 3. Contraction in skeletal muscle cells
e. Classes of cell-surface receptor proteins
i. Ion channel coupled receptors – involved in rapid signalling in cells
which can be electrically excited
ii. G-protein coupled receptors – reactivating enzyme channels?
iii. Enzyme coupled receptors – activate enzymes, used to further signals
6. Receptors – GPCRs (15-6b)
a. Key players
i. Inactive receptor binds to inactive G protein (figure 15-22?)
1. Alpha, beta, and gamma subunits
2. Lipid anchors  alpha and beta
3. When binding occurs, alpha subunit releases GDP and GTP
moves in
ii. Inactive enzyme
1. Activated when bound to alpha?
iii. General
1. 7 transmembrane protein
2. Extracellular domain – binding of signals
a. Protein ligand binds to domain
b. Mechanism (15-23)
i. G proteins relay signals from GPCRs
ii. GPCR activation leads to G protein activation
1. Alpha subunit undergoes conformational change
a. GDP leaves (dissociates), GTP moves in (abundant in
cytosol)
b. Activation - GTP causes conformational change in G-
alpha subunit
c. Dissociation - releases G protein from receptor, and
dissociates G-alpha from G-beta-gamma
2. Effector activation
a. Both pairs can interact with enzymes and/or ion
channels in plasma
7. How do signals signal? (15-7)
a. 1. OFF  signaling by phosphorylation  signal induces kinase to
catalyze transfer of P from ATP to ADP  ON  signal out
b. 2. OFF (GDP state)  signaling by GTP binding  ON  signal out
 (Back off through hydrolysis)
c. Binding signaling protein or second messenger
i. Small intracellular chemicals
ii. Generated in large amounts in response to receptor
activation (first messenger)
d. Signal amplification (15-41)
W6 BIO2001
Lecture – Cell Signaling
G protein signaling – secondary messengers
1. G proteins which signal via cAMP
a. Key players (15-26/27)
i. Signal  activated GPCR  activated G protein
ii. Gs – the G protein that stimulates adenylyl cyclase (AC)
iii. AC – catalyzes cAMP synthesis
1. cAMP – synthesized from ATP
2. 15-25
3. Two phosphate groups cleaved off from ATP  adenosine
monophosphate
4. Made cyclic by AC
iv. Gi – the G protein that inhibits AC (and formation of cAMP)
v. cAMP – activates cAMP dependent protein kinase A (PKA)
1. Inactive PKA – consists of 2 regulatory subunits and 2 inactive
catalytic subunits
2. cAMP – binds to several sites on regulatory subunits
3. Subunits undergo conformational change so that catalytic
subunits cannot bind to regulatory subunits
4. Active catalytic subunits released – kinases
a. Can phosphorylate other molecules
vi. Activated PKA
1. Transported through cytosol  nucleus via nuclear pore
2. PKA activates CREB by phosphorylation
a. Inactive CREB  activated CREB
b. Activated CREB binds to the coactivator CREB-binding
protein (CBP)
3. Complex of activated CREB and CBP – binds to cAMP response
element on DNA (CRE)
a. Upon binding to regulatory region, DNA opens up and
allows RNA polymerase to binds  gene transcription
b. Mechanism
i. Signal molecules activates GPCR (7-domain membrane)
ii. GPCR recruits alpha-beta-gamma complex of G protein
iii. Activated alpha-subunit of Gs binds to AC to activate it
iv. ATP  cAMP  Activated PKA moves into nucleus
v. CREB  transcription
2. G proteins which signal via phospholipids
a. Key players
i. Gq – the G protein that activates activated
phospholipase C-beta
1. Plasma mebmrane bound
ii. Activated phospholipase C-beta cleaves PI(4,5)P2
into  I(1,4,5)P3 and DAG
1. Secondary messengers!
b. Mechanism (15-28/29)
 i. Phospholipase hydrolyzes PIP2 into IP3 and DAG
ii. IP3 – releases Ca2+ from endoplasmic reticulum b/c of electrochemical
gradient
1. Soluble in water that leaves plasma membrane
2. ! Moves through cytosol  ER membrane
3. In ER – binds to calcium channel (IP3 receptors)
a. Concentration of calcium is higher in ER than cytosol
b. Ca2+ flows out towards cytosol
iii. Moves through protein kinase C (Ca2+ dependent)
1. Rise in cytosolic calcium ions (from IP3) alters PKC to move
from cytosol to cytoplasmic side of plasma membrane
iv. DAG (diacylglycerol) – activates PKC with help of neg. charged
membrane phospholipid (phosphatidylserine)
1. Activated PCK targets proteins by phosphorylation
2. Remains in membrane, Ca2+ dependent
3. DAG can also release arachidonic acid
a. Used as own signal or for making small lipid signaling
molecules
c. Function – muscle contraction, platelet aggregation
3. Enzyme-linked receptors (15-6c)
a. Enzyme coupled receptor - intrinsic enzymatic activity
i. Single-pass membrane proteins
ii. Mechanism
1. Signal molecule in dimer form
2. Activates catalytic domain
iii. Receptor tyrosine kinases (RTK) – kinase that phosphorylates the
amino acid tyrosine
1. Important for adult animals – growth, survival, etc.
2. 60 types in humans – mostly growth factor receptors
3. Mechanism – RTKs act as scaffolds for effector enzymes (15-
44)
a. Start with 2 inactive RTK monomers (containing
tyrosine kinase domains)
b. Signal protein dimer – binds the RTK monomers
i. Phosphorylate each other (1 site in cytosol
(inside) and 1 site on outside)
ii. Phosphorylated sites becomes binding sites for
signaling proteins
c. Activated signaling proteins (by binding sites) relay
signals downstream
4. Ex. RAS signaling pathway (15-47)
a. Superfamily of GTPases
b. Cell proliferation and differation
c. Mechanism
i. Ligand binds two monomers  dimer
ii. Phosphorylated on medial and lateral sides
iii. Activated RTK
 iv. Grb2 – adaptor protein that binds to activated
RTK using SH2 domain
v. Grb2 allows for binding to Ras-GEF (Sos)
vi. Inactive Ras protein (GDP)  Activated Ras
protein (GTP)
1. GDP phosphorylated by Sos
vii. Downstream signaling  MAP kinase
5. Ras activates MAP kinase ERK ½ cascade
a. Activated Ras protein  MAP kinase kinase kinase
(Raf)
b. Phosphorylates to MAP kinase kinase (Mek)
c. Phosphorylates to MAP kinase (Erk)
i. First discovered
d. Phosphorylates transcription regulatory proteins 
changes in protein activity or changes in gene
expression
b. Receptor with associated enzyme
i. Mechanism
1. Signal molecule binds to enzyme, activating receptor
ii. PI-3-kinase signaling  cell survival (15-53)
1. Dimer formed by binding
2. PI 3-kinase is bound  activates PI 3-
kinase
3. PI 3-kinase catalyzes phosphorylation
of PIP2  PIP3
4. PIP3 has docking sites for PDK1 and
Akt
a. PDK1 – phosphorylates Akt
b. Akt can be further
phosphorylated by mTOR
i. Undergoes conformational change  active Akt
5. Akt dissociated from PIP3
a. Phosphorylates target proteins
b. Bad – protein needed for inhibiting apoptosis
c. Phosphorylation of Bad – undergoes conformational
change
d. Releases inactive apoptosis inhibitory protein
e. Active apoptosis inhibitory protein  inhibition of
apoptosis
CLEAVING VS ADDING !

c. Signaling pathways interact !

d. Insulin receptor
i. 2 monomers
ii. Insulin binds to monomers  dimer which can be phosphorylated
1. Activation of Grb  SOS  RAS
2. OR Gluc4  cell membrane  glucose enters cell