Lecture 4

Question 1

Movement of Molecules Across the Membrane

Answer 1

• Endocytosis/exocytosis
• Simple diffusion
• Osmosis
• Membrane transporters

–Passive transporters

–Active transporters

(slide 3)

Question 2

Movement of Molecules Across the Membrane

Image

Question 3

Endocytic Vesicles and Membrane Turnover

#1

Answer 3

•Endocytosis involves the folding inward of the membrane to create an intracellular vesicle. Each event removes a bit of membrane from the surface of the cell.

Question 4

Endocytic Vesicles and Membrane Turnover #2

Answer 4

•Different cells endocytose at different rates.

–Each time a vesicle is formed, some plasma membrane is lost.

–Since cells do not shrink, they must replace that membrane somehow.

–We can measure this as membrane turnover.

Question 5

Endocytic Vesicles and Membrane Turnover #3

Answer 5

• At the high end: macrophages ingest 25% of their fluid volume every hour and turn over 100% of their membrane every 30 min.
• At the low end: Fibroblasts endocytose at a rate of about 1% per hour.

Question 6

**Vesicles are Often Formed at Clatherin Coated Pits **

Answer 6

• In early electron microscope (EM) studies of cells, inward folding of the membrane would often correlate with a thickening of the membrane at that location.
• Eventually, the material which caused membrane thickening was identified and named clatherin.

Question 7

Vesicles are Often Formed at Clatherin Coated Pits

Answer 7

• The multi-subunit clatherin protein forms regular polyhedral structures which provide the curvature to form the vesicle.
• There exist other vesicles known as caveoli that do not use clatherin. Their function is less well understood***

Question 8

There exist multiple “vesicle cage” proteins

Answer 8

The purpose of this figure is to demonstrate the different traffic patterns that vesicles can take using different “caging” proteins.

Note how the endoplasmic reticulum (ER) uses COPII while the Golgi uses COPI.

You are only responsible for clatherin (the green), which is involved in endocytosis and exocytosis.

Question 9

Snare Proteins Guide Transport

Answer 9

•Snare proteins provide critical targeting information that get the vesicle to its correct destination.

–Snare proteins bind to partners found on the destination membrane.

–The vesicular snare is known as v-snare.

–The target membrane snare is known as t-snare.

•Cells have approximately 20 different snare proteins.

Question 10

Clatherin associates with the Membrane via Adaptin

Answer 10

• Adaptin is a multi-subunit protein that binds both to clatherin and to integral membrane proteins.
• The amino acid motif for adaptin binding is found on the cytoplasmic side of a number of integral membrane proteins.
• Through adaptin, clatherin begins to induce the clustering of these integral membrane proteins.
• As clatherin multimerizes, curvature is introduced into the membrane and the inward folding begins.

(see slide 9)

Question 11

Receptor Mediated Endocytosis #1

Answer 11

•In some cases the adaptin binding membrane protein is a receptor

–During the time it takes to assemble the clatherin skeleton and invaginate, the receptors are binding whatever ligand is available in the extracellular space.

–The vesicle is now loaded with ligand at a concentration much higher than that seen in the extracellular space.

Question 12

Receptor Mediated Endocytosis #2

Answer 12

•Dynamin is a protein that binds to the neck of the nascent vesicle.
–It recruits other proteins to form a complex that cleaves the vesicle from the membrane.

Question 13

Receptor Mediated Endocytosis #3

Answer 13

• Other proteins strip clatherin from the vesicle .
• This is an ATP dependent process.
• Somehow it is inhibited until after dynamin cleaves the vesicle.

Question 14

Receptor Mediated Endocytosis Image

Answer 14

This figure shows the sequence of events during receptor mediated endocytosis.

The things to pay attention to are:

(1) the ligand is quite dilute in the extracellular space but is quite concentrated in the vesicle. This is because receptor binding concentrates the ligand.
(2) Adaptin plus clatherin restricts the mobility of the receptor (discussed in lecture #1).
(3) A number of other proteins are needed to close the final gap and release the vesicle into the cytosol (only dynamin is shown here).
(4) The release of clatherin from the vesicle requires energy (ATP) and involves other proteins (not shown).
(5) this process does not require ligand to bind to receptor. In theory, a vesicle could form with absolutely no ligands. While a vesicle could be empty of ligands it must, by definition, have many receptors.

Any of this is fair game on the exam.

Question 15

Fusion with Endosomal Compartments

Answer 15

• The early endosome is an important sorting point for incoming biomolecules.
• There are 3 general paths that can be followed.

–Recycling to the plasma membrane

–Transport to the other side of the cell (transcytosis)

–Metabolism within the lysosome

Question 16

Fusion with Endosomal Compartments #2

Answer 16

•Endosomal compartments are acidic

–The acid environment is created by a proton pump (H+ ATPase)

–Early endosomes maintain a pH of approximately 6

–Late endosomes maintain a pH of approximately 5

•The acid environment causes receptors to release their ligands.

–Receptor and ligand may then follow different processing pathways.

Question 17

Cholesterol Uptake

Answer 17

An important clinical example of receptor mediated endocytosis

Question 18

Dietary cholesterol is packaged into LDL

Answer 18

•Low density lipoproteins (LDL) derive from chylomicrons

–Large lipoprotein complexes that deliver fats and cholesterol to tissues.

–Chylomicrons are reduced in size as material is extracted to become very low density lipoproteins (VLDL) and LDL.

Question 19

Dietary cholesterol is packaged into LDL #2

Answer 19

•LDL structure:

–LDL contains one copy of the protein; ApoB100 complexed with cholesterol and phospholipids in its outer shell.

–The core contains cholesterol molecules esterified to a lipid (often linoleate)

–Typically an LDL holds about 1500 esterified cholesterol molecules

The function of LDL is to deliver cholesterol to tissues

Question 20

LDL is Recognized by the LDL Receptor

Answer 20

• Newly synthesized LDL receptors diffuse to a newly forming clatherin coated pit.
• The adaptin binding motif (coated pit binding site) on the cytoplasmic tail of the LDL receptor is available in the absence or presence of LDL
• Since the association of LDL receptor with the coated pit is passive, other receptors and membrane proteins are also incorporated into the vesicle.
• It has been estimated through labeling experiments that approximately 1000 membrane proteins may be included in the endocytic vesicle

(see slide 14)

Question 21

LDL is released in the Endosomal Compartments

Answer 21

• When the vesicle fuses with the endosome the LDL receptor becomes exposed to an acid environment.
• LDL is released from the receptor
• The receptor (associated with the membrane) is recycled to the membrane via another vesicle.
• LDL is sent to the lysosome where it is broken down to its components

–The cholesterol ester is converted to free cholesterol and is made available to the biosynthetic pool.

Question 22

Familial hypercholesterolemia

Answer 22

• Familial hypercholesterolemia (FH) is a monogenic disease which leads to early cardiovascular disease.
• The disease was mapped to a series of mutations in the LDL receptor which disrupt receptor mediated endocytosis of LDL.
• Disruption of the LDL uptake pathway will result in increased cholesterol in the blood.
• Cholesterol accumulates in blood vessels forming atherosclerotic plaque.

(see slide 16)

Question 23

Summary of cholesterol uptake

Question 24

Transferrin

Answer 24

•Transferrin represents another example of receptor mediated uptake.

Question 25

Transferrin

Answer 25

•Transferrin binds iron ions and brings them to the cell.

–Transferrin binds to the transferrin receptor which, like the LDL receptor, associated with clatherin coated pits and is taken up into endocytic vesicles.

–Unlike the LDL receptor the transferrin receptor does not release transferrin in the endosome.

–The iron is released into the cell but both receptor and ligand are recycled.

Question 26

Maternal Antibodies

Answer 26

• The uptake of maternal antibodies by an infant is an example of transcytosis.
• Antibodies with the mother’s milk are endocytosed by endothelial cells within the lumen of the infant’s gut.

–The antibodies are transferred from the endosome to transport vesicles which deposit them on the other side of the cell.

–From this side of the tissue, the antibodies can now enter the bloodstream and provide immunity to the child.

Question 27

Constitutive and Regulated Exocytosis Pathways

Question 28

Constitutive and Regulated Exocytosis Pathways

Answer 28

•Transport vesicles leave the trans Golgi in a steady stream.

–Membrane proteins and lipids resupply the plasma membrane.

–Soluble material is secreted

• Constitutive Exocytosis
• Regulated Exocytosis

Question 29

Constitutive Exocytosis Pathway

Answer 29

•Constitutive exocytosis provides both membrane constituents as well as components of the extracellular matrix.

–All cells have this pathway (also called the default pathway)

–Vesicles travel from the Golgi directly to the plasma membrane where they fuse and deliver their contents.

Question 30

Regulated Exocytosis Pathway

Answer 30

•Regulated exocytosis represents a separate secretory pathway

–Only specialized cells use this method of secretion.

–Here secretory vesicles may be stored until needed.

–A signal such as a hormone or ion influx will promote vesicle fusion.

Question 31

Transport from Golgi to Lysosomes

Answer 31

Important concept: cells have evolved a simple means to route proteins to a specific location by including the sequence for mannose-6-phosphate addition.

Question 32

Transport from Golgi to Lysosomes

Answer 32

•Lysosomes require proteins for function.

–Lysosomes must be supplied with acid hydrolases and H+ pumps as well as other proteins in order to function.

–The Golgi tags proteins to get them to go to the right place

• Proteins are tagged for lysosomal transport by adding a special phosphorylated sugar: mannose-6-phosphate (M6P).
• The Golgi have a M6P receptor that clusters M6P containing proteins to be placed in clatherin coated vesicles.

–The vesicles are transported to endosomes and from endosomes to the lysosomal compartment.

•Proteins are modified with M6P by means of a simple protein sequence.

Question 33

Lysosomal Storage Diseases

Answer 33

•There exist approximately 50 different rare inherited disease that affect lysosomes.

–Tay-sachs is one such disease. The mutation which disrupts G2M ganglioside biosynthesis results in lysosomal dysfunction.

–Other lysosomal storage diseases include; Niemann-Pick, Gaucher, and Fabry

•Lysosomal storage diseases have markedly different presentations.

–Some, such as Tay-Sachs, affect development of nervous system (most common)

–Others affect specific organs such as liver or spleen.

•Cause of disease usually involves loss of specific acid hydrolase.

–Suggests that these hydrolases are all performing different functions.

•Several drugs have been developed to replace the missing enzyme function and crucially, they use the mannose-6-phosphate receptor targeting system to get to the lysosome.

Question 34

Membrane Transporters

Answer 34

• Govern the transport of solutes (nutrients & xenobiotics) into and out of the cells
• Major determinants of pharmacokinetic, safety, and efficacy profiles of drugs
• More than 400 membrane transporters (approx. 5% of all genes!)

Question 35

Membrane Transporters #2

Answer 35

•Two major superfamilies

–Solute carrier (SLC) superfamilies – Influx of solutes

–ATP-binding cassette (ABC) – Efflux of xenobiotics

•Transport mechanism

–Passive transporters

–Active transporters

•Molecules transported

–Bicarbonate, Ions: Na+, K+, Ca+, Cl-, Organic cations, and anions

–Amino acids, glucose, fatty acids, cholesterol, urea

Question 36

Membrane Transporters #3

Answer 36

Membrane transporters are transmembrane proteins that goes from one side of the membrane to the other side.

They basically function as loading docks to deny or permit the transport of specific molecules across the membrane to get into the cell or out of the cell.

Question 37

Passive Transporters

Question 38

Passive Transporters #2

Answer 38

• Transfer solute molecules across the membrane.
• This sort of transporter does not require energy.

–Downhill process - The movement of the solute is driven solely by its concentration gradient.

–We call this passive diffusion.

•It transports the solute molecule by undergoing a reversible change in conformation.

–The solute binding site is first exposed on one side of the membrane.

–Then the solute binding site is exposed on the other side of the membrane.

Question 39

Passive Transporters #3

Answer 39

Membrane transporters act like ‘receptor” that bind to specific substrates.

Note how the binding sites for the solute molecule are accessible only from the outside of the cell. When the binding sites are filled, the protein is induced by the presence of the substrate molecules to change conformation.

That conformational change just so happens to change the relative position of the binding sites so that now the solute is accessible to the inside of the cell.

Since the concentration of solute inside the cell is low, the molecules eventually release from their binding sites and diffuse away. The protein then assumes its original conformation and can now accept more solute molecules.

Note how this is one way transport. The yellow solute molecule cannot be transported out of the cell by this mechanism – only inwards. Another protein is needed that does things in reverse to move the same solute molecule out of the cell. This could very well happen.

Consider the blood brain barrier. Molecules need to move fully across the cell to enter the brain. Thus one side of the cell might have influx transporters and the other side of the cell have efflux transporters!

Question 40

Ion Driven Active Transport

Answer 40

•Coupled carriers have binding sites for two separate molecules.

–One molecule is the solute (transported molecule, yellow circle)

–The other molecule is a co-transported ion (blue square).

–An ATPase is used to generate an electrochemical gradient for the ion (not shown here).

Question 41

Ion Driven Active Transport #2

Question 42

Ion Driven Active Transport #3

Answer 42

• The co-transported ion can move in the same direction as the solute (symport) or in the opposite direction (antiport).
• The energy stored in the electro-chemical gradient is harvested (by co-transport) to pump the solute against its concentration gradient.
• The ion is often sodium (Na+)

–The sodium-potassium ATPase creates the gradient.

Question 43

**Coupling an ion gradient **

Answer 43

• Consider the sodium dependent glucose transporter of the kidney as an example.
• The binding of Na+ and glucose to their respective sites on the transporter is cooperative.

–When Na+ occupies its site, the affinity for glucose is greatly increased.

–This is far more likely to happen on the extracellular side of the cell since Na+ is prevalent on this side of the membrane (state A)

(see slide 30)

Question 44

Coupling an ion gradient #2

Answer 44

•The change in transporter conformation is spontaneous and does not require energy.

–Sodium releases from its site because its relative concentration is low inside the cell.

–Glucose must also release because its affinity decreases markedly upon the loss of sodium

•Thus even though glucose levels are higher within the cell, glucose is transported against its electrochemical gradient.

Question 45

Transporters: Location

Answer 45

Take home message is that transporters are present in all the organs important for absorption/metabolism/clearance. You are not responsible to remember the location of each and every transporter.

Question 46

A few Important SLC Subfamilies

Answer 46

• OAT (organic anion transporters)
• OCT (organic cation transporters)
• ABC (ATP binding cassette transporters)
• PEPT (peptide transporters)
• These carriers are often able to transport a variety of similarly shaped molecules – i.e. specificity is often relaxed.

Note: We will use the words carrier and transporter interchangeably here so consider them the same things

Question 47

“Through” versus “Into/Out of”

Answer 47

• Obviously, cells want to import nutrients and export wastes.
• However, we sometimes find transporters on both sides of the cell.

–Thus a cell may actively import a waste product….why?

•The reason is to move a molecule completely through the cell from one side to the other.

–Here cells are organized into a semipermeable barrier to create this effect.

–Example 1: the renal tubules of the kidney move molecules either into or out of the body depending on need and context.

–Example 2: the blood brain barrier actively pumps out waste products into the blood stream.

–Example 3: the retinal pigment epithelium remove waste products secreted by photoreceptors.

Question 48

“Through” versus “Into/Out of” #2

Answer 48

A crucial concept here is that cells seldom act alone.

They are usually part of tissues and tissues part of organs. Thus the business of the cell is to enact what is necessary for organ function.

This may require taking in a waste product so that it can be disposed of on the other side of the tissue.

Alternatively, nutrients may be taken in and transported out again on the other side of the tissue – to feed internal regions.

We saw that retinal pigment epithelia feed photoreceptors as an example of this.

Question 49

OAT and OCT

Answer 49

•Transporters play an active role at the surfaces of rate-limiting physiological barriers to exclude molecules (like toxins or drugs)

–Small intestine, liver, kidney, and brain

•Example of drug interaction with a transporter

–OAT1 transports ACE inhibitors, diuretics, statins, antibiotics, H2 inhibitors, immune suppressants, uricosuric agents.

•Example of a beneficial drug-drug interaction

–Penicillins can cause renal toxicity in some people.

–Probenecid interacts with the OAT3 transporter in the kidney.

–Administration of penicillin with probenicid results in decreased kidney levels of penicillin thus decreasing renal toxicity!

Question 50

OAT and OCT #2

Answer 50

Thus the dose of any of these drugs is going to be dependent on how much or how fast the drug gets pumped out by transporters such as OAT1.

By the way there are many transporters and drugs may be acted on by several simultaneously. This can get quite complicated.

If you have one drug that inhibits or otherwise gets in the way of one of these transporters a second drug will suddenly be much more effective.

The example shown here is of probenecid and penecillin. An essential point to remember in this example is that (as mentioned earlier) the cell does the business of the organ. The purpose of the kidney is to excrete waste. OAT3 transports substances INTO kidney cells such that they can be routed on through to the other side where they will enter the renal tubule and be excreted. Penecillin is toxic to these cells at a certain dose and OAT3 is the mechanism by which it gets in. Unfortunately, you might need a whopping dose to kill the infection. Probenecid is a drug used to treat gout that works by messing with various transporters in the kidney to increase uric acid secretion. It happens to inhibit OAT3. By combining probenecid with penecillin, one can increase the dose of the penecillin safely.

Question 51

Clinical relevance of transporters

Answer 51

• Drug-drug interactions
• Exclusion of drugs from their site of action
• Aiding drug transport

Question 52

ABC (ATP Binding Cassette) Family

Answer 52

• This family is of great clinical interest
• Structure: The protein has two ATP binding domains “cassettes” on different cytoplasmic loops.

–Binding of ATP causes the two domains to dimerize

–Dimerization enforces a conformational change onto the protein resulting in the opening of a different face of the transporter.

–Removal of the ATP results in the opening of the other face of the transporter.

Question 53

Some (in)famous ABC transporters: MDR

Answer 53

•The multi-drug resistance protein (MDR)

–Also called P-glycoprotein

–This protein pumps out a large number of drugs from the cell.

–Its physiological role is likely the pumping out of toxins.

–Up to 40% of cancers overexpress this protein and have become drug resistant simply by keeping drug levels low within the cell.

Question 54

Some (in)famous ABC transporters: Plasmodium

Answer 54

•Plasmodium falciparum is the cause of malaria

–It expresses an ABC transporter that does a very good job of removing chloroquine – thus creating a chloroquine resistant malaria.

Question 55

Some (in)famous ABC transporters: Cystic Fibrosis

Answer 55

•Cystic fibrosis is caused by the disruption of yet another ABC transporter; called CFTR (cystic fibrosis transporter).

–This one is somewhat unique in that it does not actually transport anything itself.

–The protein is associated with a chloride channel in the plasma membrane of airway epithelial cells.

Question 56

Cystic Fibrosis

Answer 56

• Cystic fibrosis affects approximately 70,000 people world wide. The average lifespan for a CF patient is about 37 years (with intensive intervention).
• The main characteristic of the disease is the presence of exceptionally thick mucus in the airways as well as other organs such as the pancreas.

Question 57

Cystic Fibrosis #2

Answer 57

• Normally mucus traps pathogens. Cilia move the mucus out of the airways thus clearing pathogens. However, in cystic fibrosis the mucus is too thick. Bacteria such as Pseudomonas aeruginosa are able to live within the mucus and progressively destroy the lung.
• The mucus also acts to block exocrine ducts within the pancreas blocking the secretion of digestive enzymes. Bile ducts are similarly clogged leading to liver damage.
• Inactivation of CFTR leads to reduced chloride secretion and increased sodium absorption. The consequence of this is reduced water and thickened mucus.

Question 58

The PEPT1 example

Answer 58

• PEPT1 is an amino acid transporter which pumps amino acids into cells that need to make proteins (which is all cells).
• Serendipitously, PEPT1 has been found to play a major role in the absorption of drugs such penicillin class antibiotics and ACE inhibitors.
• More recently, groups have chemically modified drugs to attempt to take advantage of PEPT1 transport properties to increase bioavailability.
• Valacyclovir: the L-valyl ester of acyclovir

–Placing a valine onto the molecule via an ester linkage increases bioavailability 2 to 3 fold by promoting uptake via intestinal PEPT1.

Question 59

Summary

Answer 59

• Cells bring material in via endocytosis and release material via exocytosis.
• Clatherin is one of several vesicle coating proteins that works to give shape and promote vesicle formation.
• Receptor mediated endocytosis concentrates imported ligands such as LDL and disruption of this process can cause disease.
• Exocytosis can be regulated or unregulated.
• Transporters can be passive or active.
• Many transporters interact with drugs either increasing or decreasing their effective dose.
• Some transporters are associated with important pathologies such as p-glycoprotein (cancer) and CTFR (cystic fibrosis).
• Some drugs have been engineered to take advantage of the properties of transporters.