Lysosome Transport

Question 1

What is the pH of lysosomes?

Answer 1

~5

Lysosomes contain a variety of hydrolytic enzymes that are active at acidic pH.

Question 2

How is the acidic pH of a lysosome maintained?

Answer 2

by a proton pump in the membrane which uses the energy of ATP hydrolysis to move H+ into the lysosome against a concentration gradient.

Question 3

What is the major function of lysosomes?

Answer 3

site of intracellular degradation of macromolecules

Question 4

Vesicles destined for lysosomes from the Golgi contain what coat protein and adaptor?

Answer 4

Clathrin and GGA adaptor

Question 5

Many proteins targeted to the lysosome possess what modification?

Answer 5

mannose 6-phosphate modification

Most soluble hydrolases are targeted to the lysosome by this modification

Question 6

Where is the mannose 6-phosphate tag added to proteins bound for lysosomes?

Answer 6

Cis Golgi

Question 7

What does the mannose 6-phosphate attach to on proteins bound for lysosomes?

Answer 7

the N-linked oligosaccarhride

Question 8

The addition of mannose 6-phosphate is dependent on which enzymes?

Answer 8

N-acetylglucosamine phosphotransferase and an uncovering enzyme (for what?)

Question 9

How are lysosome proteins tagged with mannose 6-phosphate transferred from the golgi to the lysosome?

Answer 9

Lysosomal proteins with a mannose 6-phosphate residue bind a mannose 6-phosphate receptor in the trans Golgi network. These complexes become incorporated into specialized vesicles that are transported to the late endosome and then to the lysosome.

Question 10

What is I-cell disease?

Answer 10

This disease is due to a deficiency in multiple lysosomal hydrolases.

It is often due to a defect in N-acetylglucosamine phosphotransferase, which prevents the addition of mannose 6-phosphate tag. In the absence of the tag, many (but not all) of the hydrolases are secreted instead of being targeted to the lysosomes

Question 11

What is Tay Sachs disease caused by?

Answer 11

due to the absence of the lysosomal hydrolase, β-hexosaminadase A, which results in the accumulation of GM2 ganglioside.

Question 12

What is autophagy?

Answer 12

mechanism of targeting intracellular organelles (old or damaged) and cytosolic proteins to the lysosome for degradation

Glucose degradation also occurs this way

Question 13

Extracellular material brought into the cell through engulfing in called what?

Answer 13

Phagocytosis. Bacteria/ foreign particle is enveloped in a phagosome

Question 14

Phagocytosis occurs primarily in what kinds of cells?

Answer 14

White blood cells (macrophages, neutrophils, etc). These mediate phagocytosis

Question 15

What does phagocytosis require to work?

Answer 15

Requires binding to specific receptors on cell. Binging to receptors triggers formation of a pseudopod that engulfs the particle

Also requires coating of the bacterium or particle with an antibody (FC receptors on the membrane can recognize antibodies on the foreign object) or complement

Following internalization, the phagosome (the vesicle enveloping the particle) fuses with lysosomes

Question 16

Psuedopod formation requires what?

Answer 16

actin filaments just under the membrane. Remember, actin is always involved when deformation of the cell membrane occurs

Mechanism is not sure

Question 17

How do bacteria like Mycobacterium, Salmonella, Chlamydia disrupt lysosome function?

Answer 17

These pathogens, when phagocytosed by cells prevent phagosome-lysosome fusion. This allows for growth of the organism in the phagosome in a protective niche. Eventually it is released from the cell to infect another cell

Question 18

What is pinocytosis?

Answer 18

The nonspecific uptake of extracellular fluid into a cell. Material dissolved in the fluid is taken up at a rate proportional to its concentration. Non-saturable.

No binding at the membrane is necessary

Question 19

Is receptor mediated endocytosis saturable?

Answer 19

Yes

Question 20

What is receptor mediated endocytosis?

Answer 20

allows for the selective uptake of extracellular proteins and small particles. Ultimate fate of such proteins or particles may be degradation, utilization or storage in the cell, or transport across the cell. The process is saturable.

Question 21

Pinocytosis and receptor mediated endocytosis usually occur where?

Answer 21

at coated pits - pits in the membrane with a protein layer made up of clathrin on the cytoplasmic side of the membrane.

Question 22

What are the steps of receptor mediated endocytosis?

Answer 22

i. Cell surface receptors bind extracellular ligands
ii. The cytosolic tail of the receptor then binds adaptins (AP2), which recruit clathrin to the cytoplasmic face of the plasma membrane
iii. Clathrin drives membrane budding and pinching off of the vesicle from the plasma membrane (requires the dynamin GTPase)
iv. Following budding from the plasma membrane, the clathrin coat dissociates from the endosome and the cell surface receptor buds off to return to the cell surface
v. Certain ligands, such as LDL, dissociate from their receptor in the acidic pH of the early endosome (pH 6.0-6.2)
vi. In the case of LDL, the receptor then recycles back to the cell surface, and LDL is sorted first to late endosomes then to lysosomes where it is degraded. Free cholesterol is then released into the cytoplasm
vii. Certain cell surface receptors, such as the EGF receptor, do not dissociate from their bound ligand in the acidic pH of the early endosome. Rather, the receptor and ligand are delivered to lysosome where they are both degraded. This forces new receptors to be synthesized and thus uptake can be mediated in time

Question 23

What is Familial hypercholesterolemia?

Answer 23

affected individuals can not take up LDL (containing cholesterol) from the blood due to mutation in LDL receptor tail so binding of AP2 and recruitment of clathrin cannot occur

• Affected individuals have high levels of serum cholesterol and develop atherosclerosis leading to plaque in the arteries (thus, prone to myocardia)
• Some cases of inherited familial hypercholesterolemia are due to a mutation in the LDL receptor which can not bind adaptins, and therefore can not be internalized via clathrin
• Heritable mutations are also found in specific adaptins for the LDL receptor. Mutations in these adaptins result in autosomal recessive hypercholesterolemia

Question 24

What is the strategy for treatment of individuals with

lysosomal storage disorder?

Answer 24

“Enzyme-replacement therapy”

1) Deliver recombinant enzyme with mannose 6-phosphate
tag to the cells of individuals with the disease (there are receptors for this on the cell surface).

2) Surface mannose 6-phosphate receptors bind and mediate
the uptake of the enzyme by receptor-mediated endocytosis (with clathrin and adoption 2).

3) The enzyme is delivered to the lysosome where it degrades its substrate.

Blood-brain barrier may disrupt this therapy

Question 25

T or F. Many viruses and toxins enter cells by receptor mediated endocytosis

Answer 25

T.

Question 26

How does Diphtheria toxin enter the cell?

Answer 26

B chain binds to a receptor on the membrane and the toxin is endocytosed by clathrin-dependent endocytosis. In the acidic environment of the endosome, the toxin dissociates from its receptor. The B chain makes a pore in the membrane of the endosome which allows the A chain to move into the cytoplasm and inhibit protein synthesis (eef-2).

Question 27

How does Cholera toxin enter the cell?

Answer 27

binds GM1 ganglioside on the cell surface and is thought to be internalized via caveolae.

Question 28

How does HIV enter cells?

Answer 28

Does not require endocytosis (they undergo cell-surface fusion instead). It uses HIV fusion protein and Gp 120. Gp120 binds with CD4 on cell surface, causing a conformation. Domains of the fusion protein can mimic snares and initiate fusion.

*The virus binds then fuses with the membrane. Fusion occurs at neutral pH

Question 29

COPII uses which motor protein?

Answer 29

Dynein. It is an anterograde process that moves from the periphery of the cell to the perinuclear golgi (i.e. from the + end of the microtubule to the - end)

Question 30

The H+ pump of the pump for lysosomes is driven by what?

Answer 30

Hydrolysis of ATP

Question 31

COPI uses which motor protein?

Answer 31

Unsure evidence. This is a retrograde grade process

Question 32

What is Pompe disease?

Answer 32

Defect in ability to break down glucose by a-glucosidase in the lysosome (1 in 20,000 births)- primary target organ is muscle

Question 33

T or F. In transfer of endocytosis from and early endosome to a late endosome to a lysosome, the environment becomes more and more acidic?

Answer 33

T

Question 34

What recruits clathrin for deformation of the cell membrane during endocytosis?

Answer 34

adaptin 2. Dynamin is then responsible for the final cleavage of the vesicle from the membrane.

Question 35

Caveolae mediate the endocytosis of which substances? What cells are they commonly found in?

Answer 35

fatty acids and lipids; epithelial cells

Question 36

What happens to lumenal proteins that fuse with the cell membrane? Transmembrane proteins?

Answer 36

Lumenal- released to cell exterior

Transmembrane- become transmembrane cell membrane proteins

Question 37

The terminal modification of proteins moving through the golgi have what terminal modification?

Answer 37

Sialic acid

Question 38

Receptor Mediated Endocytosis of Membrane Enveloped Virus

Answer 38

Hemagglutinin on the surface of the membrane of the virus binds to non-specific receptors on the cell membrane through sialic acid. Inside acidic endosomes, the acidic pH causes fusion of the viral membrane to the endosome membrane, causing release of the genome of the virus into the cell.