Membranes (PD) Flashcards
What do intracellular membranes do?
Maintain the different environments of intracellular organelles and prevents the components of intracellular organelles mixing with each other (e.g. ER and mitochondria have different pH environments)
Why do cells need a plasma membrane? (3)
- To provide a boundary between the intracellular and extracellular environments
- To receive and transmit external signals to/from the cell
- For energy conversion processes such as maintaining ion conc. gradients to make ATP
What are the 2 components of membranes and what do they do?
Phospholipids - prevents water and water soluble (hydrophilic) molecules from crossing the membrane, by forming a hydrophobic environment with their tails
Proteins - transport molecules across the membrane
Why can phospholipids be described as “amphipathic”?
They contain hydrophobic tails and hydrophilic, polar heads
What is the difference between saturated and unsaturated fatty acid chains, and how does this affect membrane fluidity?
What state are saturated and unsaturated fatty acids in at room temp, and where are they commonly found?
Saturated = no double bonds
Unsaturated = one or more double bonds, causing “kinks” in the tail
More unsaturation = lipids are less closely packed due to having the kinks in the chain - makes the membrane more fluid
Saturated = solid at room temp, found in meat and dairy products
Unsaturated = liquid at room temp, found in plants
What are the 3 ways which phospholipids spontaneously assemble and describe each of them?
- Bilayer
- Micelle = single layered circle of phospholipids with tails point to middle of circle
- Liposome = a circle of a bilayer (heads pointing in and out) (see summary sheet for diagrams)
What does a tear in the lipid bilayer cause?
What is the difference between how small and large tears are fixed?
Creates a free edge with water (part of the bilayer exposed to water)
Only way to fix this is to form a closed compartment (a round, liposome structure)
Small tears = self-seal into a single liposome structure
Large tears = split into 2 separate liposomes
What are the 4 different types of movement of lipids?
- Lateral diffusion = lipids swapping places with each other
- Flip-flop = swapping of lipids from one side of the bilayer to the other
- Flexion = movement of the tails
- Rotation = lipid spins around
See summary sheet diagram
What are the 2 different states a lipid bilayer can exist in?
What is Tm?
What does the Tm depend on?
2 states:
- Ordered, rigid state
- Relatively disordered, fluid state
Tm = melting temp, where phase transition occurs (where it goes from being solid like to fluid like)
Depends on chain length and degree of saturation:
- Longer chains pack closer, as have stronger interactions, so longer chains stay solid at higher temps
- Unsaturated fatty acids pack together less easily, so more likely to stay fluid over lower temps
What 2 things control membrane fluidity and how?
Unsaturation - explained previously, more unsaturation = more kinks = less closely packed = more fluid like
Cholesterol = inserts itself between phospholipids.
OH group of cholesterol aligns with the polar heads of the phospholipids
The steroid ring part of cholesterol stiffens the upper part of the hydrophobic chain of the phospholipid
At high concentrations of cholesterol, this prevents the fatty-acids from becoming fluid-like (prevents phase transition) (see diagram on summary sheet)
What are the 4 types of membrane proteins and what are their functions?
- Transporters = transport molecules across the membrane
- Anchors = help in signalling pathways and maintaining membrane structure
- Receptors = relay signal from a binding event and turn it into an intracellular signal
- Enzymes = catalyse internal reactions
What are the 2 classes of membrane proteins and how are they different?
- Integral = found inside the phospholipid bilayer (usually transmembrane) - hydrophobic
- Peripheral = attached to the outside of the bilayer - are hydrophilic and so are attracted to the polar heads of the bilayer (Also interact with the surfaces of integral proteins)
What are hydropathy plots and how can they be used to identify transmembrane domains?
They identify stretches of hydrophobic residues, which would suggest a transmembrane domain
A positive peak on the hydropathy plot indicates a stretch of amino acids that are hydrophobic, as it means that energy is required for the amino acids to be transferred to water (As the amino acids are hydrophobic, this is unnatural so occurs non-spontaneously, like having a positive delta G)
What are the 8 hydrophobic amino acids?
F - Phenylalanine
A - Alanine
M - Methionine
I - Isoleucine
L - Leucine
Y - Tyrosine
V - Valine
W - Tryptophan
How can the use of detergents be used to solubilise membrane proteins?
What 2 types of detergents can be used, how are their structures similar, and how are they different in the way they interact with the protein?
Detergent is added to membrane protein in the lipid bilayer
This forms a water-soluble protein-detergent complex, where the membrane is surrounded by detergent monomers
It also forms water-soluble lipid-detergent micelles, with the phospholipid bilayer and surrounding detergent monomers (See summary sheet for diagram)
Types of detergent:
Both detergents have a hydrophobic and hydrophilic group
- SDS, strong ionic detergent, causes the protein to denature
- Triton x100, mild non-ionic (has a non-charged polar group), doesn’t cause the protein to unfold
Which detergent is used depends on the protein we want to study
What are the 2 typical features of a transmembrane protein, and what side of the membrane are they both present on?
- Glycosylated = sugar residues added
- Disulphide bonds between folded parts to stabilise the structure
Both present on the non-cytosolic side (Side facing away from the cell
How can the structure of membrane proteins be visualised?
By freezing them and then cutting into the frozen cell with a knife. Fractures will occur at points of weakness in the bilayer (usually the hydrophobic core) to expose the membrane proteins. Electron microscopy can then be used to visualise the structure of the proteins.
How can the movement of membrane proteins be measured?
Using FRAP - Fluorescence recovery after photobleaching
Area of the membrane is bleached, which denatures the proteins in that area and forms a gap within the membrane.
Proteins surrounding the missing proteins then diffuse to fill the gap. All proteins will be labelled with a fluorescence label to track the movement of them.
We can then use this to monitor how fast the fluorescent proteins are moving and how fast they diffuse to recover the gap created (recovery time) - allows us to track the lateral mobility of individual protein molecules
Which molecules is the membrane permeable to and which molecules is it impermeable to?
Small hydrophobic gases such as O2, CO2, and N2 and steroid hormones can diffuse freely across the membrane.
Small, uncharged polar molecules such as H2O and ethanol can also diffuse freely across the membrane (only slightly permeable to H2O)
Impermeable to ions (e.g. Na+, K+), charged polar molecules, and larger uncharged polar molecules such as glucose, nucleotides and amino acids (all require transmembrane proteins to cross the membrane)
What are the 2 types of transport protein and what is the difference between them?
Do transport molecules through passive or active transport?
- Carrier proteins = bind the molecule to be transported and moves the molecules across the membrane by undergoing a conformation change - based on conformation of the binding site and transported molecule
- Channel proteins = form hydrophilic pores through the membrane to allow molecules to pass through - based on their size and charge
Channel proteins = always use passive transport
Carrier proteins = can be either passive or active transport
What is the difference between passive and active transport?
- Passive = movement of molecules down their concentration/electrochemical gradient
- Active = movement of molecules against their electrochemical gradient - requires energy (ATP)
What is the electrochemical gradient?
Net driving force of ions, composed of concentration and voltage
Moving down conc. gradient if going from high to low area of conc.
Moving down electrical gradient if moving to an area of opposite charge (e.g. positive ions moving into the cell = down electrical gradient as they are going from a positive area outside the cell to a negative area inside the cell) (See summary sheet for diagram)
What are the 3 types of transporter proteins that transport solutes against their electrochemical gradient and how do they work?
- Coupled transporter = Uses a molecule moving down their electrochemical gradient to provide energy to move another molecule against their electrochemical gradient (e.g. glucose symport)
- ATP-driven pump = Hydrolyses ATP to get energy to move molecules against their electrochemical gradient
- Light-driven pump = Uses energy from light to move molecules against their electrochemical gradient
What does the sodium potassium pump do and how does this create an electrochemical gradient, and what can this electrochemical gradient be used for?
What is an example of an inhibitor of the sodium potassium pump and how does the drug inhibit it?
Hydrolyses ATP to provide energy for the active transport of Na+ out of the cell and K+ into the cell
Maintains a high conc. of Na+ outside the cell and a high conc. of K+ inside the cell (Can be put in reverse, so that sodium and potassium ions move down their gradients)
Can be used to drive the active transport of another molecule
OUABAIN - binds to the K+ binding site, preventing K+ ions from binding and therefore preventing the pump from working
What are the 5 steps to the sodium-potassium pump? (include number of sodium and potassium molecules transported in/out of the cell)
- Na+ binds from inside the cell
- ATP hydrolysis occurs, phosphorylating the pump
- Phosphorylation causes a conformational change, causing 3 Na+ to be release outside the cell
- 2 K+ bind from outside the cell
- Dephosphorylation occurs, causing a conformational change and 2 K+ is released into the cell
What is a coupled transporter?
Moves ions down their electrochemical gradient to release free energy to be used to drive the active transport of another molecule
What are the 3 types of protein carrier transport? (1 single and 2 coupled)
Uniport = simple transport of one soluble molecule from one side of the membrane to the other
Symport = coupled transport with both molecules moving in the same direction (both moving into/out of the cell)
(e.g. glucose symport - transports glucose and sodium ions into the cell, sodium down its gradient, glucose against its gradient)
Antiport = coupled transport with the 2 molecule moving in opposite directions (one going into the cell, one going out)
What do Gut villi do in terms of glucose transport?
What are the 2 types of glucose transporter involved in intestinal epithelial cells?
Absorb glucose from the gut and release it into the bloodstream on the other side
Apical = glucose symport, allows the uptake of glucose even when the conc. of glucose in the cell is already high (active transport)
Basal = passive glucose transporters, releases glucose into the cell when it’s needed
What is endocytosis and what are the 3 types of endocytosis?
Endocytosis = actively transporting molecules into the cell by engulfing them with its membrane
Types:
- Phagocytosis = cell “eating”, for large molecules
- Pinocytosis = cell “drinking”, for small molecules
- Receptor mediated endocytosis (RME)
What is phagocytosis?
What are macrophages and what do they engulf? (5, include what they are)
Process where the cell membrane changes shape to engulf relatively large particles, like bacteria
Macrophages are specialised white blood cells that circulate the blood and engulf invading microorganisms via phagocytosis.
Also engulf dead cells (from apoptosis), senescent cells (cells that have stopped multiplying, but won’t die off), cell debris, damaged cells
How does phagocytosis work? (in macrophages) (3)
- Cell extends pseudopodia (membrane arm-like projections) which surround and engulf the cell target - closed form formation (pseudopod) is driven by changes in the cytoskeleton
- Once engulfed, the cell forms a phagosome
- The phagosome fuses to a lysosome and it’s contents are digested
What is pinocytosis?
How does it work? (including what are endosomes?)
How is the vesicle membrane then recycled?
“Cell drink” = Soluble material is taken up from the immediate surroundings
Mechanism:
- Curved coated pit is formed via clathrin recruitment on the cytoplasmic face of the membrane
- Membrane curves more and more as more clathrin accumulates until part of the membrane buds off to form a clathrin vesicle
- The clathrin coat on the vesicle is lost (shed) and the uncoated vesicle fuses to the endosome - where the molecules get sorted out to be transported elsewhere in the cell
Recycled via exocytosis - empty intracellular vesicle goes back up to the cell membrane and fuses with it to maintain the overall cell size
What is receptor mediated endocytosis (RME) and what is an example of this?
What are the 5 steps to RME, using the example to explain?
Endocytosis (similar to pinocytosis) using specific receptors
Example = cholesterol intake into cells, carried in the blood to cells by LDLs
Steps: (diagram in jotter) (practice by writing out?)
- LDL carrying cholesterol binds to LDL receptor on plasma membrane
- Clathrin coated pits form and bud off into clathrin coated vesicles containing the LDL-LDL receptor complex
- The clathrin layer un coats to form an uncoated vesicle with the LDL-LDL receptor complex inside
- This vesicle then fuses with the endosome. Low pH environment in the endosome separates the LDL receptor from the LDL, allowing the receptor to be recycled (along with the vesicle)
- The LDL then buds off from the endosome in another vesicle to be transferred to a lysosome. Enzymes in the lysosome breakdown the LDL into cholesterol in the cell
What happens in RME when LDL receptors are defective?
What is the name of this?
Why are proteins such as LDL and insulin taken into the cell by RME and not through channel/carrier proteins?
Causes hypercholesterolaemia
Receptors can still bind to LDL, but can’t bind to the clathrin
Results in high circulating cholesterol levels as the LDL can’t be taken into the cell and so it circulates the blood instead (see diagram in jotter)
Too large to be passed through a protein
How is the fate of endocytic vesicles followed?
What are early and late endosomes?
What is the role of an endosome?
How do endosomes release the molecule off of it’s receptor and what helps maintain this?
Via tracers such as radioactive/fluorescent markers
Early endosome = endosomes located near the plasma membrane
Late endosome = endosomes located further into the cytosol
Role = to sort endocytosed molecules
pH of cytoplasm = 6.8, pH of endosome = 5/6, more acidic pH in endosome releases molecule from it’s receptor
Proton-motive ATPases pump protons into endosomes to keep the endosome pH low
What are the 3 possible fates of endocytosed receptors?
- Recycled to the plasma membrane
- Degraded by lysosomes
- Transcytosed to the opposite side of the cell to be released into the bloodstream and travel to another cell
How does transcytosis work in epithelial cells of the gut when transporting antibodies from milk? (4)
- Antibodies from milk bind to receptors on the apical surface of the gut (bottom surface)
- They are then internalised and delivered to endosomes
- Retrieved from transport vesicles which bud off from the early endosome
- These vesicles then fuse with the basolateral domain (opposite side of the cell, top side) of the plasma membrane. The antibody is released into the bloodstream from here.
(diagram in jotter)
What are the 3 types of coats coated vesicles can have and what is each coated vesicle involved in?
What 2 diseases is the last type linked to?
- Clatherin coated = RME and pinocytosis
- Non-clatherin coated = mediates vesicular transport between the ER and Golgi
- Caveolar (protein similar to clatherin) coated vesicles = regulate cellular signalling pathways and are linked to diseases such as muscular dystrophy and cardiomyopathies
What is the name for the shape of clathrin molecules and what is this shape?
What structures do clathrin molecules assemble into?
Shape = Triskelion = 3 armed shapes (see summary sheet for diagram)
Spontaneously assemble into basket like structures (as they curve)
What is intercellular signalling and why is it important?
What are the 5 different types of intercellular signalling and what happens in each of them?
Intercellular signalling = signalling between cells
Important as cells have to exchange signals to determine whether it will survive, divide, or grow
Types:
- Endocrine = hormone from an endocrine cell travelling through bloodstream to receptor of another cell
- Paracrine = signalling cell releases molecules that act on all nearby cells with the receptor
- Neuronal = Neurotransmitter diffuses across the synapse and binds to postsynaptic cell receptors
- Contact-dependant = one cell has membrane bound signalling molecule (can’t move) and other cell has membrane bound receptor. 2 cells have to be close together in order for signalling molecule and receptor to bind
- Autocrine = cell releases a signalling molecule that binds to receptors on its own surface
What is signal transduction?
What are the signal transduction steps? (2)
What does amplification of a signal lead to?
Signal transduction = extracellular signalling molecule binds, which triggers intracellular signalling pathways
Steps: (diagram in jotter)
- Extracellular protein binds to its receptor, which triggers an intracellular protein which passes the signal onto another intracellular protein etc. in a chain
- This signal is then passed onto multiple target proteins which produce a response to receiving the signal
Amplification leads to more than one effector protein being targeted (allows multiple target proteins instead of one), leading to complex responses
What are the 2 different types of cell receptor and what type of molecules do they bind?
- Cell surface (extracellular) = larger, hydrophilic molecules
- Intracellular (in the cytoplasm/nucleus) = smaller, hydrophobic molecules that can pass through the membrane
What is an example of an intracellular receptor and how does it work?
Steroid hormone receptors
Steroid hormones like cortisol, oestrogen, or testosterone can easily pass through the plasma membrane
Steroid hormone binds to its receptor found in the cytoplasm to form a steroid hormone-receptor complex, which then travel to the nucleus
The complex then directly binds to DNA, to a promotor regulating region, upstream from the target gene (just before the target gene) to activate the expression of the target gene
What are the 3 different classes of cell-surface receptors?
- Ion channel coupled receptors
- G-protein coupled receptors (GPCRs)
- Enzyme-linked receptors
How do ion-coupled receptors work (ligand gated ion channels)? What type of molecules bind to them and give and example?
What is curare and what does it do?
What are 2 examples of psychoactive drugs and where do they bind?
Ligand binds to receptor on channel, which changes the channels conformation to an open form to allow the ions to pass through the membrane
Often the target for neurotransmitters like acetylcholine
Curare are poison darts. They bind to acetylcholine receptors, blocking the receptors and preventing acetylcholine from binding
Psychoactive drugs like barbiturates and Valium bind to neurotransmitter receptors (blocking them)
What are G-protein coupled receptors (GPCRs)?
What type of molecules bind to them and what are 3 examples?
What is a G-protein?
7-pass transmembrane receptors (have 7 folds in the membrane)
Molecules that bind = hormones and neurotransmitters like serotonin, adrenaline, and noradrenaline
G-protein = trimeric (3 subunits) GTP-binding regulatory proteins
What is the signalling pathway of G-protein coupled receptors (GPCRs)? (4)
What are 3 examples of secondary messengers?
- Signalling molecule binds to the GPCR, activating both the GPCR and G-protein
- G-protein binds to GPCR forming a receptor-G protein complex, which activates a separate effector protein (e.g. enzyme)
- Effector protein generates a secondary messenger
- Messenger moves away from the effector protein and binds to target proteins, causing a response in the cell
Diagram in jotter for this, and steps drawn out in summary sheet
Secondary messengers = cAMP, cGMP, Ca2+
What is an enzyme-linked receptor?
What are the 3 functional domains of an enzyme-linked receptor?
Either the receptor itself is an enzyme, or it activates an associated enzyme
3 functional domains:
- Ligand binding domain (extracellular)
- Single transmembrane domain
- Enzyme catalytic domain (intracellular)
See diagram in jotter
What is an example of an enzyme-linked receptor and what are the 2 examples for each type of enzyme-linked receptor?
What are 3 other examples of enzyme-linked receptors?
Tyrosine kinase receptor (itself is an enzyme) = insulin/EGF receptors
Receptors linked to intracellular tyrosine kinases = interferon/erythropoietin receptors
Receptors also linked to:
- serine/threonine kinases
- Phosphatases
What is a phosphorylation cascade?
Series of kinases, where each kinase acts on multiple other kinases to phosphorylate and activate them. These kinases then go onto act on more kinases and activate them etc. in a chain reaction
Leads to a massive and rapid amplification of the signal
(see summary sheet diagram)
What is the complete signalling pathway for a GPCR acting through gas? (from membrane to nucleus, example) (5 steps)
- GPCR alpha subunit activates adenylate cyclase (AC) (effector protein)
- AC converts ATP into cAMP (secondary messenger)
- cAMP activates Protein Kinase A (PKA) (target protein)
- PKA phosphorylates and activates transcription regulators (response)
- Transcription of target gene occurs (response)
(drawn out on summary sheet)
Where in the cell does protein synthesis begin?
What directs the proteins to the correct subcellular location?
How do proteins enter the nucleus?
Cytoplasm
Sorting signals
Enter the nucleus through hydrophilic, nuclear pores. Allows small, water soluble molecules through and prevents larger molecules from crossing the membrane
What is the sorting signal called which directs the protein to be imported into the nucleus and what 2 residues is the signal mainly made of?
At what stage in their synthesis do proteins enter through the nuclear pore and what are the 2 steps for entry?
Nuclear localisation signal (nls)
Cluster of small basic (+vley charged_ residues - K and Rs
Post translational proteins - fully synthesised
Steps:
- Nls recognised and the protein binds to a nuclear transport receptor
- Nuclear pores are wide enough to allow the protein-receptor complex to pass through
What 2 types of proteins are synthesised in ribosomes on the ER?
What is the ER?
What is the structure of the signal sequences for these 2 proteins?
Secretory (cell surface) and transmembrane
ER = Network of tubules found in the cytoplasm
Found at the N-terminus, contains one or more +vely charged amino acid followed by a stretch of hydrophobic residues
How does the signal sequences of ribosomes translating secretory and transmembrane protein locate them into the ER? (3)
What happens to the signal sequence once the protein has entered the ER lumen?
- A signal recognition particle (SRP) binds to the signal sequence and causes a pause in translation
- SRP-bound ribosome then moves to the ER and attaches to the SRP receptor on the ER membrane
- Protein synthesis starts again (translation starts again), and the newly synthesised protein is co-translationally (at the same time as translation is occurring) inserted into the ER lumen through a channel
(diagram in jotter)
Signal sequence gets cleaved and rapidly degrades
What 4 things happen to the secretory protein once in the lumen of the ER?
What happens to transmembrane proteins?
- Polypeptide is folded into the correct tertiary structure via chaperone proteins
- Glycosylation = addition of one or more carbohydrate side chains
- Specific proteolytic cleavages occur (e.g. signal peptide sequence)
- Assembly of polypeptide into multimeric complexes (joining more than one polypeptide together to form a protein complex)
Transmembrane proteins don’t enter the lumen of the ER, but instead become integrated into the ER membrane during synthesis (during translation)
What is the Golgi apparatus?
How are proteins moved between the ER and Golgi?
On what face to proteins leave/enter the Golgi network?
Series of membrane discs
Vesicles
Vesicles fuse with the Cis face of the Golgi and bud off from the trans face of the Golgi
What are the 2 main functions of the Golgi apparatus? (include what the default and sorting pathways are)
- Post-translational modification of proteins
- Protein solving (directing to correct compartment)
Default pathway = protein goes to cell surface for secretion
Sorting pathway = protein goes to an intracellular compartment (e.g. the nucleus or mitochondria)
What is constitutive secretion?
What is regulated secretion?
Constitutive = continuously secretes proteins to the outside of the cell from the Golgi
Regulated = proteins stored in vesicles and are only secreted out of the cell when a signalling molecule binds to a membrane receptor, causing an intracellular signal for the vesicle to fuse with the membrane
What is the difference between protein signalling sequences for proteins being directed into the nucleus and proteins being directed into the ER?
ER = one or more +vely charged residues followed by a long stretch of hydrophobic residues
Nucleus (nsl) = contains a cluster of K and R residues (+vely charged)
