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Cell Bio Exam 1

Lecture 1 Notes:
❖ Central Dogma Review:
⮚ DNA is SYNTHESIZED in the nucleus
⮚ RNA is TRANSCRIBED and PROCESSED in the nucleus (finishes processing
before leaving nucleus)
⮚ RNA is TRANSLATED in the cytoplasm
⮚ DNA Polymerase is responsible for DNA Replication
⮚ RNA Polymerase is responsible for RNA synthesis (transcription)
⮚ Spliceosomes are responsible for RNA Splicing
⮚ Ribosomes are responsible for translation
❖ Microscopy
⮚ Light microscopy is insufficient to resolve many cellular structures
⮚ Fluorescence microscopy is more modern
▪ Standard (light bulb) and confocal (beam)
▪ Works by manipulating the position of electrons
● Light (excitation light) hits the fluorophore molecule
● Causes one electron to jump to a higher energy orbital than normal
● When electron falls BACK onto the orbital, fluorophore emits light (emission
light) of a longer wavelength

▪ High to low energy waves: V🡪I🡪B🡪G🡪Y🡪O🡪R
❖ Clicker Question: You need to examine a fluorophore on the microscope. It
requires Orange excitation light. What wavelength do you expect the fluorophore
to emit?
A. Green light B.Yellow light C. Red light D. Blue light E. Microwaves
▪ Types of Fluorphores:
● Chemical “dyes”🡪DAPI (DNA dye)
♦ Naturally targets DNA; fluoresces blue when inside a DNA strand
● Chemical “tags”
♦ Need help in order to target substrate; must be tetheres to another protein
to direct their position
❖ Clicker Question: What excitation light should be used to make these
fluorescently labeled microtubules emit green?
A)UV B) Blue C) Green D) Yellow E) Red
❖ Antibodies were used to attach fluorophore to the microtubules
⮚ Antibodies bind to specific protein targets (antigens)
▪ Primary Antibodies
● Inject SPECIFIC antigen (microtubules) of what you want to highlight into
animal (mammal or bird); we are using bunny
● Extract antibodies (rabbit anti-tubulin) created against antigen
● Sticks to the target
▪ Secondary antibody
● Target animal (goat) gets injected with A LOT OF bunny protein (antigen to
● Makes bunny antibodies and sticks to anything that is bunny
● DOES NOT recognize original antigen (microtubules)
● Extract antibody (goat anti-rabbit antibody), attach fluorophores to antibodies
▪ Primary and secondary antibodies injected into human🡪 microtubules light up
● The Rabbit anti-tubulin sticks to human microtubule and the fluorescent goat
anti-rabbit antibody attaches to the rabbit anti-tubulin, making the microtubule
❖ Clicker question: Which type of primary antibody is best for labeling actin
filaments in human cells?
A) Rabbit anti-tubulin
B) Goat anti-actin
C) Rabbit anti-goat
D) Answers A and B are both correct
E) Answers A, B, and C are all correct.
▪ Zika virus
● Injected mice with Zika, used antibody staining to find out if you can see Zika
in neurons
● Stained with DAPI (blue) and zika antibody (mouse anti-zika and goat anti-
mouse with green fluorescence)
● No controls (no picture of mouse without Zika)
● Also used goat anti-mouse IN mouse; this means that fluorescence will also
stain other mouse cells that AREN’T zika
● Controls should NOT be stained to validate claims
1. Be able to explain similarities/differences between eukaryotic and prokaryotic cells
2. Be able to explain the central dogma and name the key players.
3. Why is fluorescence microscopy an important research tool?
4. How/why does excitation light make a fluorophore glow?
5. Why is the emission light of a fluorophore always a longer wavelength/lower energy
than excitation light?
6. What kind of synthetic fluorophores are there, and how are they targeted? i.e.
how/why does a DNA dye only stain DNA?
7. How does a label like FITC get targeted to other things like microtubules?
8. What is the “indirect immunofluorescence” technique?
9. Can you comfortably design an indirect immunofluorescence experiment? (i.e. if you
wanted to label Zika proteins red, how would you do that?)
10. How does the immunofluorescence technique inform disease scenarios? i.e. how did
today’s paper investigate Zika interaction with adult brain cells?
11. What controls need to be run to make correct interpretations?

Lecture 2 Notes:
❖ Membranes:
⮚ Phopholipids are “amphipathic”: one end “water-loving”, one end “water-fearing”
▪ Polar head (compatible with water), non-polar hydrocarbon tails (oily)
⮚ Lipid bilayer is a boundary for the cell; top and bottom of bilayer are polar heads and
center is hydrophobic tails
⮚ Water tends to cluster hydrophobic clusters together; being pushed by water (not
attracted to each other)
▪ Creates a linear structure
▪ The structure of a phospholipid is always going to spontaneously push it into a
sheet and then into a ball
❖ Clicker question: If you could poke a small hole in a lipid bilayer, what would you
expect to happen next?
A. The bilayer will re-seal to close the hole.
B. The hole will remain as a permanent membrane defect.
C. The whole membrane will unravel and fall apart
⮚ Lipid bilayers are semi-permeable
▪ Membranes repel
● All charged compounds
● Most polar compounds
● Exceptions: small polar molecules (H2O) and gases (CO2 and O2)
❖ Clicker Question: If you expose live cells to anti-tubulin antibodies, will their
microtubules (inside the cell) be labeled?
A. Yes, because the antibodies are specific for microtubules, which are found in the cytoplasm.
B. No, the antibodies will not get through the membrane. (Nearly all cells labeled by antibodies
have been “fixed” and “permeabilized”; they aren’t injecting these antibodies into real
animals, just manipulated cells)
⮚ Bilayer vs. Micelle
▪ Micelle’s are unstable because too much hydrophobic content is exposed
▪ Micelle: have huge polar head groups and shorter fatty acid chains (single chain)
▪ Bilayer: small polar head, long or several fatty chains
▪ Amphipathic molecules tend to form micelles (cylindrical shape)
▪ Movement within the bilayer reflects the extent of lipid saturation
⮚ Fatty Acid Chains
▪ Saturated: straight; all carbons have two hydrogen’s (more solid)
▪ Unsaturated: bent; more mobility; creates more space; double bond (more liquid)
▪ The extent of lipid saturation affects membrane fluidity
▪ Long lipids group together to shield each others’ hydrophobic tails (less areas are
accessible to water)
▪ Cholesterol extends the range in which lipids can be fluid (saturated fat)
▪ Unsaturated fat falls apart on its own (too loose)
▪ Unsaturated fat and cholesterol combined makes a more stable association.\
▪ Cholesterol helps hold membranes together
❖ Clicker Question: Hibernating frogs are somehow able to avoid freezing to death.
What membrane lipid(s) must these frogs carry LESS of during winter?
A. Saturated fat B. Unsaturated fat C. Micelle (saturated fats tend to crystallize more; to
prevent that, you need something unsaturated or cholesterol to make it more fluid)
Lecture 3 Notes:
❖ In some cell types, protein comprises ~50% of the membrane WEIGHT (not content)
❖ Membrane proteins:
⮚ Integral membrane proteins🡪 physically embedded in the bilayer (1,2,3,4)
⮚ Peripheral membrane proteins🡪 associated via electrostatic interactions; not in the
bilayer at all (7,8) can wash right off when rinsed with salt water
⮚ Lipid anchored proteins🡪covalently attached fatty acid holds protein at bilayer (5,6)

⮚ Multiple types of anchoring are possible in one protein
❖ Integral membrane proteins
⮚ Alpha helix: looks like a telephone cord
▪ Amino acids (N=H): proton donators (+); Carboxyl (C=O): protons acceptors (-)
● Proteins are ALWAYS polar; through the entire length of the protein
▪ Proteins fold up into a secondary structure to hide it’s polar character of the
▪ The “R” groups stick outward from an alpha helix.
● If all the “R” groups are non-polar, they are compatible with hydrophobic
interior of bilayer
▪ NON-POLAR amino acids: cluster in the middle of a bilayer to shield themselves
from aqueous
● F-Phenylaniline, L-Leucine, I-Isoleucine, P-Proline, M-Methionine, A-Alanine,
G-Glycine, V-Valine, W-Tryptophan
▪ All nonpolar R groups
▪ Multipass alpha helical proteins zig-zag through the membrane (clustered in a
● About 20 amino acids that happen to have hydrophobic side chains
♦ Hydrophobic exterior allows it to be compatible to the hydrophobic interior
● Gap in between group of helices: enable movement of specific molecules
from one side of the bilayer to the other
❖ Clicker Question: How would you expect this molecule to get across a lipid
A.It will diffuse easily through the bilayer B. It must pass through the middle of a membrane
❖ Clicker Question: Why does this alpha helix embed “sideways” in the bilayer
(instead of standing straight up and down)?
A.) Because this alpha helix is too long to span across the bilayer.
B.) Because the alpha helix is made of all polar and charged amino acids.
C.) Because only one side of the helix has nonpolar residues exposed.
D.) Because the protein has large unstructured regions outside the bilayer.
● Some proteins have an “amphipathic” alpha helical structure where all polar
AA’s are on one side and non-polar AA’s on another side of the helix. The
hydrophobic side will embed in the middle of the bilayer and the hydrophilic
side will stay in the aqueous solution.
⮚ Beta sheet: looks like a solid wall, shown here as a cylinder
▪ Beta barrels (beta sheet in cylinder shape) structures are also integral membrane
▪ Secondary structures are similar to alpha helix secondary structure
● Beta sheet also can have hydrophobic AA sticking out, allowing it to stay
inside membrane
▪ Polar character of backbone is masked because of H bonds between O in
carbonyl and H in amine
▪ R groups alternate up and down
▪ Can have different character on either side of sheet
● One side mainly hydrophobic = hydrophobic character
● One side mainly hydrophilic = hydrophilic character

▪ Range in size:
● 1. 8-stranded OmpA: Acts as an ion channel
● 2. 12- stranded OMPLA: NOT a channel; enzymes that hydrolyzes lipids
● 3. 16-stranded porin: passage for specific sugars
● 4. 22-stranded FepA: passage for iron; most of barrel is plugged; VERY
selective (that’s why it’s huge)
▪ Beta barrels don’t have to be in membrane
❖ Clicker Question: Do all beta barrel proteins = transmembrane proteins?
A.) No. Many beta barrels have hydrophilic residues exposed on their outer surface. This
makes their structures soluble in the cytoplasm.
B.) Yes, because all beta barrels have hydrophobic residues on the outside.
❖ Synthesis of learning
⮚ Green fluorescent protein (GFP)🡪 beta barrel protein in the cytosol
⮚ Jellyfish scare predators with GFP
▪ Calcium release (elicited by startle response) binds Aequorin, changes its shape.
Aequorin emits a blue photon, absorbed by GFP (which glows green).

⮚ Chimeric genes are made to create GFP-fusion proteins

⮚ Tweaking GFP structure changes its fluorescence
⮚ GFP-tagged proteins have been used to test for mobility of proteins and structures in
living cells
▪ Normal case:
● Exciting a fluorophore causes one of its electrons to jump into a higher energy
▪ Special case (Photobleaching):
● Intense light is used
● The excited electrons are lost
● Fluorophores no longer glow
▪ If bleached area persists, indicates GFP is stationary
▪ If bleached area fills in, indicates the GFP is mobile
⮚ Lipid bilayers generally work as a fluid mosaic model (mosaic because its not
▪ Protein and lipid content assumed to be mobile unless something specifically
restrains motion of the molecules
▪ 1) Protein is bound to intracellular structures
▪ 2) Protein is bound to extracellular structures
▪ 3) Protein is bound to surface proteins on other cells
▪ 4) Protein is in large complexes
▪ 5) Tight junctions prevent diffusion of membrane proteins between apical and
basal (but allow free movement within each)
❖ Outcomes:
⮚ Can you describe integral, peripheral and lipid-anchored membrane proteins?(What
distinguishes these types from each other?)
⮚ What secondary structures are seen in transmembrane proteins? (Describe?)
⮚ Why can’t a linear/unfolded protein reach the whole way through a lipid bilayer?
⮚ What makes it possible for alpha helices and beta barrels to embed in a bilayer?--
how is polar character of protein backbone masked by these structures?-- what
makes the surface of these proteins compatible with bilayer interior?
⮚ Related: Learn your amino acids. (non-polar AAs = FLIP MAG VW).
⮚ How does a single-pass differ from a multi-pass transmembrane protein?
⮚ How do amphipathic alpha helices embed within a lipid bilayer?
⮚ Which molecules diffuse directly through a bilayer? Which ones don’t? Note that
selectivity defines what passes thru a membrane protein vs. not.
⮚ How does a protein get “tagged with GFP”?
⮚ How does the FRAP technique inform the movement of fluorescent proteins?
⮚ What is the “Fluid mosaic model”?
⮚ Under what circumstances do proteins fail to diffuse around freely within a lipid
Lecture 4 Notes:
❖ Warm-up:
⮚ Membrane fluidity depends on its content

❖ Clicker Question: Of the simplified choices below, what kind of bilayer content is
a living organism most likely to have?
❖ A. All saturated B. Mix of saturated + unsaturated C. All unsaturated
❖ Clicker Question: If you used genetic engineering to GFP-tag this protein, where
would you want/expect the GFP part to be located?

⮚ GFP is a beta-barrel and aqueous so it has to be either outside the cell or in the
⮚ GFP is polar or charged
⮚ Beta-barrel is polar (C’s beta-barrel is nonpolar)
⮚ Location of beta-barrel is determined by what kind of AA are facing out:
▪ Non-polar AA🡪in bilayer (nonpolar = oily and greasy🡪 hydrophobic)
▪ Polar or charged AA🡪 in cytosol
❖ Clicker Question: Which type of lipid bilayer do you expect to be MORE permeable
to passage of small molecules?
A. Mostly saturated B. Mostly Unsaturated
▪ Mostly unsaturated allows more molecules to sneak through
❖ Movement of Small Molecules: Protein Types
⮚ Channels🡪 passive only
⮚ Transporters🡪 uniports are passive; symporters and antiporters are active
⮚ Pumps🡪 active only
❖ Channels
⮚ “Passive transport” proteins allow transit of solute to cross the bilayer, as per
concentration gradient (high🡪low)
⮚ Channel protein forms a water-filled pore in the lipid bilayer
⮚ Channels are selective about which substrates pass thru
▪ Gated opening; physical “selectivity filter” in the channel
⮚ Potassium specific channel ONLY allows potassium through; if sodium tried to go in,
because of its smaller size, it can’t get a good grip with all of the carbonyl’s and pops
⮚ Some classes of toxic molecules are very effective in blocking ion binding sites
▪ Tetrodotoxin plugs the “selectivity filter” of sodium channels very effectively
● Produced by: Pufferfish (i.e. Fugu), blue-ringed octopi, certain toads, etc.
● Neurotoxic because control of these ion channels is essential for proper
function of the nervous system
⮚ Some channels can open and close (accept different molecules) in a regulated
❖ Clicker Question: Does plasma membrane lipid content affect opening/closing of
K+ channels?
A. Yes, it does B. No, it doesn’t
❖ Clicker Question: Can a beta barrel open and close by changing its width?
A. Yes B. No
▪ The H-bonds between strands rigidly attach the strands.
▪ To adjust that, you’d have to break ALL H-bonds at the SAME TIME, then
❖ Transporters
⮚ can also operate in a passive manner
⮚ actually changes as solute goes through; limits the rate of entry
⮚ These binds solute on one side of membrane, change shape, then release solute on
the other side of the membrane
⮚ Active Transport:
▪ proteins drive solute across the bilayer, against concentration gradient
● REQUIRES ENERGY; in many cases is provided by strategic use of
concentration gradients
▪ Symporters: active transport protein (2+ things enter in one direction)
● Na+/Glucose symporter:
♦ Eukaryotic cells need import glucose against its concentration gradient
♦ (Steep) Na+ gradient “pushes” glucose into the cell.
♦ Result: 2 sodiums come in, bringing 2 glucoses in with them (same
❖ Clicker Question: What will happen to the hydration of a person’s gut epithelial
cells after drinking a salt/sugar mix? (Gatorade)
A.The gut epithelium will be more hydrated. B.The gut epithelium will be less hydrated.
⮚ Salt and sugar together helps you take in the sugar faster
⮚ As sugar goes in, so does salt, water follows
⮚ Because increasing solute uptake will bring in water also by osmosis
▪ Antiporters: active transport protein (2+ things enter in opposite direction)
● Na+/Ca+ antiporter:
♦ Sodium gradient is used to expel excess intracellular calcium (Na+ enters
cell and makes Ca+ leave)
♦ Note: this can run in reverse if the cytosol gets too salty (Cytosol expels
Na+ and allows Ca+ to enter)
♦ Result: Sodium and calcium move in opposite directions.
● Na+/K+ ATPase pump (sodium potassium pump)
♦ Uses the energy of ATP hydrolysis to import K+ and export Na+
♦ BOTH ions are being moved against their (strong) electrochemical
♦ Result: 3 Sodiums go OUT while 2 Potassiums come IN
❖ Multiple Transporter Proteins

❖ Clicker Question: Why doesn’t this diagram show Na+/glucose symporters on the
lateral or basal sides of the cell?
A.) The placement of these proteins is driven by random chance.
B.) There probably is a even distribution of that transporter all over the cell, just not shown for
C.) The Na+/glucose transporters are physically restricted to the apical side.
❖ Outcomes:
⮚ What kind of conformations (shapes) are possible for an unsaturated lipid?
⮚ How well does cholesterol “fit” into the irregular structure of those lipids?
⮚ Why do most living cells carry a mixture of saturated and unsaturated membrane
⮚ What experimental procedures are using for immunofluorescence staining? i.e. how
do antibodies get into the cell to bind and label antigens of interest?
⮚ Practice: What are the nonpolar amino acids? (single letter abbreviations ok to use.)
⮚ What decides if a beta barrel will be in the cytoplasm, or embedded in a lipid bilayer?
⮚ What governs movement of solute across the lipid bilayer?
⮚ Which molecules are able to diffuse thru? Which can’t?
⮚ What are the basic properties of an ion channel?
⮚ How do they choose which ion goes through?
⮚ What are the similarities/differences between: channels vs. transporters and passive
vs. active transport?
⮚ What is an example of a symporter, antiporter, and pump?
⮚ How do cells import substrates that don’t fit through a channel or transporter?
Lecture 5 Notes:
❖ LDL cholesterol particles
⮚ 1500 cholesterol molecules
⮚ Need particles like this to be able to deliver cholesterol all over the body
⮚ Cholesterol is mostly nonpolar; can’t go into blood bc blood is aqueous
❖ Clicker Question: What sort of lipid structure is an LDL particle?
A. a micelle B. a vesicle
▪ Though huge, LDL interior is all hydrophobic; lipid monolayer outside
⮚ Cholesterol rich plaques can clog up arteries
▪ Thought to be caused by cholesterol high diets
▪ NOT proven
▪ Hypotheses:
● High glycemic diet stresses out liver, causing it to make more LDL particles
● Gingivalis bacteria appear in cholesterol rich plaques
❖ Clathrin-mediated endocytosis- allows cell to take up large substrates
⮚ Seen through Transmission Electron Microscopy (TEM)- 2D image

▪ Sample Preparation for TEM:
● 1. Preserve tissue
● 2. Permeabilize and fix (crosslinks the sample)
● 3. Embed in a resin block
● 4. Section with a diamond knife (ultra-thin sections)
● 5. Take pictures
⮚ Scanning Electron Microscopy (SEM)- 3D image
▪ Sample Preparation for SEM:
● 1. Sample on slide i.e. pollen grains.
● 2. “Sputter” coat with metal
● 3. Silver coated sample
● 4. Shine electron beam at sample
⮚ Import of LDL
▪ When an LDL particle binds to its receptor, this cross-links LDL with the adaptins
(and thus also to clathrin), enabling endocytosis
❖ Clicker question: What would happen if the LDL receptor acquired a mutation that
prevented it from binding to Adaptin?
A. Reduced B. No impact on LDL uptake C. Increased
▪ Dynamin is required to finish clathrin-mediated endocytosis (ATP-powered
protein that pinches off the new vesicle)
● Squeezes the water out so bilayer areas can fuse together
❖ Hydrophobic Clustering
⮚ 1.) Drives key binding interactions between proteins (protein homodimer)

⮚ 2.) Complementary hydrophobic surfaces tend to bind (protein heterodimer)

⮚ 3.) Features around the binding site restrict access (only heterodimer is possible)

❖ Caveolin-mediated endocytosis
⮚ Special, curved lipid rafts (containing Caveolin) can also be endocytosed
⮚ Vesicle is pinched off from membrane by Dynamin (same as for clathrin-mediated
❖ Clicker Question: How is blowing a bubble completely different from
A) Soap is made of amphipathic molecules, whereas lipid bilayers are not.
B) Soap bubbles are blown in, whereas endocytic vesicles are pulled in.
C) Soap lipids are mobile, but vesicle lipids are structured and stay in place
❖ Endocytosed content (after endocytosis)
⮚ 1. LDL receptor in cell membrane binds to an LDL particle (The Receptor is a single
pass, alpha helical transmembrane protein.)
⮚ 2. Receptors bind to Adaptins. (Adaptins are also bound to Clathrin.)
⮚ 3. Clusters of Clathrin form round baskets, containing LDL-bound receptors
⮚ 4. Dynamin pinches off the “neck” of membrane to separate away a new clathrin-
coated vesicle away from the plasma membrane.
⮚ 5. New vesicle is called “early endosome”.
⮚ 6.) Vesicle uncoats, then fuses with the “sorting endosome.”
⮚ 7.) Recycling endosome sends LDL Receptor back to the plasma membrane.
⮚ 8.) Cholesterol is directed to the lysosome.
❖ Membrane fusion
⮚ 1.) Rab protein docks with its effector on the target membrane
⮚ 2.) v-SNARES interact with proteins on the target membrane (t-SNAREs)
▪ vSNARE/tSNARE interactions squeeze the water out so the membranes can
❖ Outcomes:
⮚ What is clathrin-mediated endocytosis?
⮚ How does the stepwise mechanism of clathrin-mediated endocytosis work?
What are the different proteins and how do they work together to make this possible?
⮚ How does clathrin actually deform the membrane to create a sphere?
⮚ How does the dynamin protein help to detach a vesicle from the plasma membrane?
⮚ After content is endocytosed to create a new endocytic vesicle, where does it go?
How is the content of that vesicle directed on to other places in the cell?
⮚ How do vesicles fuse with other bilayers (destination compartments) in a cell?
Can you describe the role of Rabs/Rab effectors and SNARE proteins in that
⮚ How do lipid rafts contribute to endocytosis?
⮚ What do the mechanisms of clathrin- and caveolin-mediated endocytosis have in
⮚ How do hydrophobic clustering forces affect binding between molecules?
i.e. protein-protein interactions, or binding of a small molecule to a protein
⮚ How is binding specificity between molecules controlled?
What makes it possible for proteins bind to a specific target?

Lecture 6 notes:
❖ Soap normally exists as micelles
❖ Vesicles after endocytosis
⮚ Vesicle does not diffuse well inside cell
▪ The cell is packed full of solute (proteins, ions, water); thick gel
▪ Doesn’t float, needs to be directed to other places that undergo fusion events
⮚ Newly formed vesicles called early endosomes
▪ Can fuse together to form sorting endosome (large body)
▪ Some of the content could be redirected back to the surface of the cell or directed
elsewhere for degradation or redistribution
⮚ Early endosome🡪sorting endosome
▪ Specifying who fuses with who
● Using proteins that are associated with the membrane content
▪ 1.) Rab protein docks with its effector on the target membrane.

● Bilayer🡪dark gray; vesicle lumen🡪 light gray
● Rab protein helps decide what vesicle will and won’t fuse to
♦ Function of Rab🡪 to bind to a protein (Rab effector) that is on the target
♦ Specific match between the vesicle and the membrane it is supposed to
fuse with (Rab and effector); lock and key
● Rab protein functions are controlled by the GTP-binding status of the Rab
♦ GTP binding protein changes Rab shape; this allows Rab to bind to the
effector protein
⮚ GEF allows GTP to bind to Rab
⮚ GAP makes Rab hydrolyze (get rid of) the GTP

● The GTP-binding status of Rab proteins directs Rab association with
♦ GTP binding makes it possible for Rab to bind to effector
♦ After GTP hydrolyzed, Rab lets go of effector
♦ A lipid carrier can recycle the GDP-Rabs from target membrane back to
previous, donor membrane
♦ The way you determine where Rab is active is where the GEF is
♦ GTP allows the hydrophobic parts of the vesicle to stick out, making it
stick to the GEF
♦ Rab1-GEF1; Rab2-GEF2; etc.
● Different Rabs are found on different subsets of membrane
♦ There are 30-70 Rabs used to direct targeting specificity in a cell
♦ In some cases:
⮚ Many Rabs per organelle
⮚ Same Rab on diff organelles
❖ Clicker Question: What determines the site where a Rab will be active?
A. The size of the vesicle
B. Where the Rab-GEF is
C. Where the GTP is
D. Answers A, B and C are all correct.
▪ 2.) v-SNARES interact with proteins on the target membrane (t-SNAREs)

● Lock and key as well
● Membrane fusion driven by vSNARE/tSNARE interactions
♦ SNARE’s twist together, membrane content in close proximity, and
pushes all of the water out
⮚ Allows for interaction between the bilayers and ultimately dry fusion
⮚ Fusion event of one contiguous large object instead of two separate

❖ Protein Folding
⮚ Protein folding is also driven by the hydrophobic clustering force
⮚ Won’t work unless folded into glob
▪ Hydrophobic character helps it stay in glob
⮚ Types of folding:
▪ SECONDARY STRUCTURES🡪alpha helix, beta sheet
▪ TERTIARY STRUCTURE🡪“globular” protein (May include secondary structures
as components)
● Co-translational folding
♦ Protein spontaneously folds up (correctly) as it’s coming out of the
ribosome; the same way every time
♦ Sometimes this works well to generate a functional protein
♦ Doesn’t work with large hydrophobic domains
⮚ Leads to misfolded protein product
⮚ Hydrophobic bits in a big blob that doesn’t form into the intended
shape🡪 it’s non-functional
● Post-transla0onal folding (Hsp70-assisted)
♦ Solves problem
⮚ Hsp70 Chaperonin protein binds to exposed hydrophobic regions;
keeps it unfolded
▪ Does this AS hydrophobic region leaves the ribosome
▪ Now the ribosome has time to make more protein
⮚ Remains unfolded until ribosome is done being synthesized
▪ Pop Hsp70 chaperonin off and stick the hydrophobic areas
together to make a FUNCTIONAL protein
♦ How chaperonins stick to a target protein
⮚ Chaperonins are bound to ATP
▪ When in contact with a hydrophobic area, it drives them to
hydrolyze their GTP and grab on
▪ Over time, they pick up new ATP
● Binding new ATP makes them let go
❖ Clicker question: What causes Hsp70 chaperonins to bind to a target protein?
A) Hsp70 binds any protein that has reached a certain length.
B) Hsp70 binds at regular intervals throughout a protein.
C) Hsp70 binds random sites within a target protein.
D) Hsp70 binds to groups of non-polar amino acids.
E) Hsp70 can bind to any unfolded protein region.
❖ Clicker Question: You saw last week that an Hsp70 homolog (Hsc70) helps
Clathrin baskets fall apart. How/why does that happen?
A) Hsc70 provides ATP to Clathrin, causing Clathrin to shake.
B) Hsc70 makes the Clathrin basket too heavy, so It falls apart.
C) Hsc70 loosens hydrophobic interactions between Clathrins.
D) Hsc70 re-folds hydrophilic portions of the Clathrin protein.
⮚ Temperature is a common stressor; alters how it folds and its function
⮚ Misfolded proteins can be detrimental to the cell
⮚ Cells can re-fold misfolded proteins (Hsp60-assisted)
▪ Hsp60 chaperonin proteins bind together to form a large complex (GroEL)
● 14 sub-units
● One side of the chamber is used at any given time and the other side is

❖ Clicker Question: Cancer cells are known to carry lots of mutations. Do you
expect Hsp60 to generally help or hurt cancer cells?
A) Hsp60 helps cancer cells survive because it re-folds proteins that would otherwise be
misfolded/toxic to the cells.
B) Hsp60 is detrimental to cancer cells because it re-folds proteins that would normally prevent
over-replication of the cells.
❖ Consequences of protein misfolding
⮚ Can take on toxic attributes
▪ Prion proteins
● Normal: PrPC; Misfolded: PrPSC
● Misfolded prion proteins are not toxic due to “loss of function”
● A single misfolded prion goes on to induce the same folding defect in all the
other (initially normal) prion proteins.
❖ Clicker Question: You read in an article that the center of an amyloid fibril
“excludes water.” What does that mean?
A.Prion proteins are composed entirely of hydrophilic amino acids.
B.Amyloid fibrils are repelled by the polar content of the cytoplasm.
C.Amyloid fibrils represent a specialized form of alpha helix.
D.Hydrophobic amino acid side chains are facing the center of the fibril.
❖ Clicker question: Many secreted proteins like insulin are thought to be packaged
as amyloids prior to release from the cell. Why/how are natural amyloids NOT
A.The fibril-like structure of some natural amyloids is reversible.
B.Since no proteins are secreted in the brain, no amyloid toxicity.
C.The immune response aggressively attacks natural amyloids before damage occurs.
⮚ Amyloids from insulin are not toxic, unlike the Alzheimer’s ones
⮚ Crystal dissolves; structure is reversible
❖ Outcomes:
⮚ Where does a vesicle “go” after it is endocytosed?
⮚ How does one vesicle fuse with another? Why is a special mechanism needed?
⮚ What is a GTPase, and how are Rab proteins an example of that?
⮚ In a folded protein, where are most of the nonpolar vs. polar residues located?
⮚ How is tertiary structure different from secondary structure?
⮚ What is co-translational folding? What are possible shortcomings of that?
⮚ How does Hsp70-mediate correct protein folding?
⮚ How do Hsp70-like proteins affect existing interactions between proteins?
⮚ How does Hsp60 enable protein re-folding? Be able to explain the mechanisms for
these, including usage of ATP, etc.
⮚ Do you expect Chaperonins to contribute to cell health? If so, how?
⮚ What is the structural difference between normal and misfolded prion proteins?
⮚ Through what process does a misfolded prion corrupt the normally folded ones?
⮚ Why are disease-related amyloid fibrils so structurally stable?
⮚ Why are most natural amyloids regarded as harmless?
Lecture 7 Notes:
❖ Ubiquitin-Proteasome System
⮚ Misfolded proteins can be identified and eliminated
⮚ Ubiquitin pathway
⮚ Use ubiquitin as a backup mechanism for how cells can manage protein content so
they don’t get overloaded with toxic, malfunctioning amyloids
⮚ Usually destroy misfolded proteins before they cause a problem
▪ Main players:
● Ubiquitin
● E1: the Ubiquitin-activating enzyme (teal blue)
● E2: the Ubiquitin-conjugating enzyme (dark blue)
● E3: the Ubiquitin Ligase (medium blue)
⮚ Main points:
▪ 1.) Ubiquitin is conjugated to the Ubiquitin-activating enzyme (E1)
▪ 2.) Ubiquitin is transferred to Ubiquitin-conjugating enzyme (E2)
● Note: E2 is bound to E3 in a complex

▪ 3.) The E3 subunit of the complex binds to a protein to be degraded.This brings
the protein target close to E2 and the Ubiquitin tag.
▪ 4.) E2 transfers the ubiquitin to a lysine residue in the target protein.

▪ 5.) E2 dissociates from E2/E3 complex.
▪ 6.) New E2-ubiquitin binds to E3, adds another
ubiquitin to target.
▪ 7.) The whole cycle repeats to create a poly-
ubiquitin chain.
● A 4+ Ubiquitin chain is needed to target a
protein for degradation.
⮚ One ubiquitin doesn’t really do anything

❖ Clicker question: How do you think E3 ligase recognizes mis-folded proteins?

A.Unfolded proteins are unusually bumpy.
B. E3 binds to Ubiquitin that got stuck inside the unfolded protein.
C. E3 has hydrophobic areas that recognize the misfolded protein.
D. None of the above: E2 binds to the mis-folded protein, not E3.
⮚ There are multiple types of E2 and E3
▪ Each E2/E3 combination ubiquitinates a different target protein
▪ binding site will only bind certain misfolded regions
▪ High specificity
⮚ Many ubiquinated proteins are targeted to the proteasome
▪ The proteasome is a large complex made from > 10 different
▪ The cap complex (red) unfolds proteins.
▪ The central cylinder (blue), destroys protein with protease
❖ How the Proteasome recognizes and processes ubiquitinated proteins
⮚ the proteasome cap has a binding site for the polyubiquitin chain.
⮚ Ubiquitins are released from the target protein.
⮚ ATPases in the cap (chaperone-
like) unfold the protein, feed it
into the core.
⮚ Protein is degraded by proteases
in 20S core of the proteasome.
⮚ Even very large proteins (i.e.
cytoplasmic dynein) are normally
recycled in this way.
▪ the ubiquitin never gets
destroyed; it’s recycled
❖ Clicker question: What happens if a protein to be degraded is a transmembrane
A. The proteasome has to integrate into the lipid bilayer so it can access/destroy the
target protein.
B. The misfolded protein has to be pulled completely out of the bilayer so that the
proteasome can digest it.
▪ Cell has to use ATP to extract protein in the membrane
▪ Bilayer friendly E3 Ligase used to recognize misfolded transmembrane protein
❖ Targeting a protein to the proteasome requires a specific type of linkage within the
Ubiquitin chain
⮚ A ubiquitin chain that = a degradation signal is linked at either Lysine 11 or Lysine
❖ Ubiquitin chains can act as a regulatory mark
⮚ Ubiquitin connected via other Lysines
▪ (like Lysine 63, = other consequences)
⮚ This chain can bind proteasome also, but usually doesn’t. (bound to epsins, etc.)
⮚ Chain drives non-degradative functions, such as EGF receptor endocytosis
⮚ Ubiquitin does a lot of things
❖ Clicker question: Many proteins, like histone proteins that bind DNA, are known to
get mono-ubiquitinated. What do you think the consequence of that might be?
A) This drives histone degradation
B) This drives histone endocytosis
C) This changes histone function.
D) Answers A and B are both correct.
E) Answers A, B and C are all correct.
❖ Nucleus
⮚ Ribosome production factory
⮚ small and large subunits are built here and sent into the cytosol
▪ assembled after they get to the cytoplasm
⮚ Where DNA is housed
⮚ mRNA created here (made by RNA polymerase)
⮚ 5’ cap and poly A tail confirm stability of mRNA in nucleus
⮚ modified mRNA is sent out of the nucleus
▪ ribosome reads it to assemble a polypeptide according to the code
⮚ completely full
▪ unwound DNA fills up a lot of the space
● thin and easy to break
⮚ Nucleus serves to preserve the physical integrity of the DNA and give an
environment to separate transcription and translation
❖ DNA compression
⮚ Chromatin→ DNA + EVERYTHING that is attached to it; large
macromolecular assembly
▪ RNA poly, transcription backers, histones, etc.
⮚ Structural proteins
▪ DNA is packaged around proteins called “Histones”
● both together = nucleosome
● keeps DNA organized
● can be used to compact down regions that are not useful
▪ Phosphate sticks out (-)
▪ histone tails are positively charged
● DNA spontaneously wraps around histones because of charge
❖ Clicker Question: Which of these histones are you more likely to find associated
with an expressed gene?

❖ A) The histone on the left.

❖ B) The histone on the right.
⮚ Lower affinity of histone tails for DNA = looser packaging = more gene expression
❖ Chromatin structure influences gene expression
⮚ As a general rule:
▪ Highly condensed DNA: inactive for transcription; tightly wound
▪ Less condensed DNA: actively transcribed region of the chromosome; loose
● expressed part is dimmer because it isn’t densely packed

❖ Clicker question: How

much gene expression do you expect from the centromere and telomeres?
A. Lots of expression
B. Not much expression
❖ Clicker question: Ubiquitin binds to well-expressed areas of chromatin. Where
would ubiquitin be found on this chromosome?
A. At the light gray parts
B. At the dark parts
❖ Outcomes:
⮚ What are the main players in the ubiquitin-proteasome system (UPS)?
What is the function of each component, and how do they work together?
⮚ How are misfolded proteins recognized and tagged by the E2-E3 ligase complex?
⮚ How/why does a proteasome degrade just ubiquitin-tagged proteins? (not all
⮚ Why don’t all proteins with a ubiquitin chain get degraded by the proteasome?
⮚ What other outcomes might occur for a ubiqutin-tagged protein?
⮚ What are the basic structural, organizational, functional features of the nucleus?
⮚ What types of proteins can associate with DNA to form “chromatin”?
⮚ Why is it important to “package” DNA? (why bother?)
⮚ What is a histone octomer made of? What is the nucleosome is made of?
⮚ How does modifying histone charge affect DNA affinity for the octomer?
⮚ How do histone modifications affect chromatin compaction?
⮚ What is the relationship between chromatin compaction and gene expression?
⮚ Why/how is chromatin state (DNA packaging) relevant to human health?