Vesicular Transport (ch 13)

Question 1

What are the 3 intracellular vesicular transport pathways?

Answer 1

1. Biosynthetic secretory pathway
2. Endocytic pathway
3. Retrieval pathway

Question 2

What are the 3 major characteristics of vesicular traffic?

Answer 2

Vesicular traffic is:

1. Organized
2. Balanced
3. Selective

Question 3

What is the structure of phosphatidylinositol (PI)?

Answer 3

2 fatty acid chains bound to glycerol which is in turn attached to a phosphate head group.

Question 4

What is the structure of a phosphoinositide (PIP)? What differentiates PI(3)P from PI(4)P?

Answer 4

A 6-member ring connected to a glycerol with 2 fatty acid tails by a phosphate group. 3 and 4 denote different locations of phosphorylation on the ring.

Question 5

On the phosphate head group of a phosphoinositide, which carbons do not allow phosphorylation?

Answer 5

Carbons 2 and 6 cannot be phosphorylated, only 3, 4, and 5. Carbon 1 is attached to the glycerol and thus can’t be phosphorylated either.

Question 6

Where are phosphoinositides and phosphotidylinositol found on the plasma membrane?

Answer 6

Always on the cytosolic side.

Question 7

What percentage of membrane lipids are phosphotidylinositol or phosphoinositides?

Answer 7

~10% of membrane lipids.

Question 8

What are the 3 coat proteins we went over in lecture?

Answer 8

1. Clathrin
2. COP1
3. COP2

Question 9

What 2 functions does a coat protein perform?

Answer 9

1. Shape/mold the vesicle

2. Concentrate specific proteins in the vesicle

Question 10

Why would we want to change the phosphorylation status of phosphoinositides (PIPs)?

Answer 10

To change the recognition pattern of other proteins and bind selectively.

Question 11

What is the structure of clathrin?

Answer 11

A three-legged “triskelion” structure with 3 light chains and 3 heavy chains.

Question 12

How can clathrin associate with a membrane to form a vesicle?

Answer 12

Requires an adaptor protein which is specific to each membrane and facilitates binding.

Question 13

During vesicular transport, what chaperone protein acts as an ATPase and is responsible for removing the clathrin coat from a vesicle?

Answer 13

Hsp70 chaperone.

Question 14

During vesicular transport, what protein in responsible for budding and separation of a vesicle from a membrane?

Answer 14

Dynamin (+associated proteins).

Question 15

Where do clathrin-coated vesicles travel to?

Answer 15

From the plasma membrane to the early endosome then the late endosome.

Question 16

Where do COP1-coated vesicles travel to?

Answer 16

From the golgi to the endoplasmic reticulum, OR the early endosome to the plasma membrane.

Question 17

Where do COP2-coated vesicles travel to?

Answer 17

From the endoplasmic reticulum to the golgi, OR the golgi to secretory vesicle/late endosome/plasma membrane.

Question 18

What is the function of a phosphoinositide (PIP)?

Answer 18

To be recognized by adaptor proteins during early vesicle formation.

Question 19

What happens to phosphoinositides (PIPs) when they are bound by an adaptor protein? What causes this?

Answer 19

They are depleted by PIP phosphatase.

Question 20

How does a BAR domain dimer facilitate vesicle formation?

Answer 20

It helps to deform the membrane via electrostatic interactions.

Question 21

What are the 2 coat-recruitment GTPases?

Answer 21

1. Arf1 (adenosine diphosphate ribosylation factor 1) protein
2. Sar1 (secretion-associated RAS-related) protein

Question 22

Which coat protein is Sar1 (GTPase) associated with?

Answer 22

COP2 coat assembly.

Question 23

How are the coat-recruitment GTPases activated? How are they de-activated?

Answer 23

They are activated by GEF phosphorylation and deactivated by GAP dephosphorylation.

Question 24

Which coat protein is Arf1 (GTPase) associated with?

Answer 24

COP1 and Clathrin coat assembly.

Question 25

What happens to the alpha-helix binding domain of Arf1 and Sar1 when GTP is bound?

Answer 25

The binding domain is exposed with GTP and hidden with GDP.

Question 26

What 3 steps can outline vesicle delivery to a membrane?

Answer 26

1. Tethering 2. Docking 3. Fusion

Question 27

What purpose does the Rab effector protein serve during vesicle "tethering"? What protein does it bind to?

Answer 27

It binds Rab-GTP on the surface of the vesicle and begins pulling the vesicle toward the membrane.

Question 28

What purpose do the t-SNARE proteins serve during vesicle "docking"? What protein do these bind to?

Answer 28

They bind to v-SNARE on the surface of the vesicle and squeeze out intervening water, locking the vesicle into place on the membrane.

Question 29

How does a "docked" vesicle fuse with a membrane and deliver its cargo? What protein is important for this?

Answer 29

Rab-GDP dissociation inhibitor removes Rab-GDP from the vesicle membrane, allowing the vesicle to fuse with the membrane.

Question 30

What happens to the v-SNARE once the vesicle has fused with the target membrane? How is it recycled?

Answer 30

It exists as a complex with t-SNARE. It is dissociated from the complex by NSF which is driven by ATP.

Question 31

Under what circumstance would we want to pause vesicle transport just before vesicular fusion with the target membrane?

Answer 31

When we need to be able to deliver substrate immediately and on demand. Ex: in the synaptic cleft of the neuromuscular junction.

Question 32

What differentiates homotypic and heterotypic membrane fusion?

Answer 32

Homotypic: both membranes are of the same type Heterotypic: both membranes are of different types.

Question 33

Are all proteins in the endoplasmic reticulum packaged into vesicles? Please elaborate.

Answer 33

No. Resident ER proteins and chaperone proteins remain in the ER.

Question 34

Why is it necessary to have different protein coats like COP1 and COP2?

Answer 34

For target specificity.

Question 35

How does the body retrieve proteins that were not supposed to leave the endoplasmic reticulum?

Answer 35

KDEL receptors which bind ER resident proteins are transported to the Golgi, picking up proteins. Once bound, they travel back to the ER.

Question 36

To make ER resident protein retrieval possible, what coat protein must KDEL receptors bind to?

Answer 36

Affinity for COP1 coat proteins which can transport them back to the endoplasmic reticulum.

Question 37

Why must the affinity of KDEL receptors for ER resident proteins be different in the Golgi vs the endoplasmic reticulum?

Answer 37

Don't want KDEL binding to ER resident proteins in the ER and impeding their function. Only bind in Golgi.

Question 38

How many stacks comprise the Golgi apparatus in animals? What group of organisms possesses the largest Golgi?

Answer 38

Animals have 6 stacks, but some protists can have more than 60!

Question 39

Which Golgi face is closest to the Endoplasmic Reticulum?

Answer 39

The Cis face.

Question 40

What is the function of the cis Golgi network?

Answer 40

To phosphorylate oligosaccharides on lysosomal proteins.

Question 41

What is the function of the cis cisterna of the Golgi?

Answer 41

To remove Man.

Question 42

What is the function of the medial cisterna of the Golgi?

Answer 42

To remove Man and to add GlcNAc.

Question 43

What is the function of the trans cisterna of the Golgi?

Answer 43

To add Gal and NANA.

Question 44

What is the function of the trans Golgi network?

Answer 44

To sulfate tyrosines and carbohydrates.

Question 45

To what 3 locations are vesicles usually transported after leaving the trans Golgi network?

Answer 45

1. Lysosome 2. Plasma membrane 3. Secretory vesicle

Question 46

How many Golgi apparati do animal cells have? What about plant cells?

Answer 46

Animals: one Plants: lots

Question 47

How can we observe the local protein specificity of the Golgi apparatus?

Answer 47

By labeling the stacks with gold.

Question 48

Describe the cisternal maturation model of Golgi function.

Answer 48

Vesicles are delivered to the cis stack, which then moves through the golgi apparatus until being split into vesicles when it reaches the trans face. Vesicular transport in reverse.

Question 49

Describe the vesicle transport model of Golgi function.

Answer 49

The Golgi stacks are static, and vesicles move between stacks as they progress from one Golgi face to the other (bidirectional)

Question 50

In what manner are sugars added to a proteoglycan? What organelle accomplishes this?

Answer 50

The Golgi apparatus adds these sugars one at a time.

Question 51

Where do complex oligosaccharides get modified?

Answer 51

Precursor oligosaccharides are added to proteins in the ER, but must then be moved to the Golgi for complex modifications.

Question 52

In the Golgi, what enzymes facilitate complex oligosaccharide modification?

Answer 52

Glycosyl transferases.

Question 53

What are the 4 main purposes of glycosylation?

Answer 53

1. Determine protein folding 2. Protect protein from enzymes 3. Regulate cell-surface signalling 4. Protect protein from pathogens

Question 54

What characteristic must lysosomal membrane proteins have?

Answer 54

Must be highly glycosylated to prevent breakdown by acid hydrolases.

Question 55

What is the plant cell equivalent to a animal cell lysosomes?

Answer 55

A large, central vacuole.

Question 56

What is the purpose of a lysosome in an animal cell? What enzyme helps accomplish this?

Answer 56

To break down cellular components for recycling. Acid hydrolases catalyze breakdown.

Question 57

What prevents acid hydrolases from being active in the cytosol and indiscriminately breaking down cellular components?

Answer 57

They are inactive before post-translational modification in the lysosomes.

Question 58

How is the low pH inside lysosomes maintained?

Answer 58

Through the action of V-type ATPases which use ATP to pump H+ ions into the lysosome.

Question 59

What 4 pathways mentioned in class can deliver substrate to lysosomes?

Answer 59

1. Phagocytosis 2. Endocytosis 3. Macropinocytosis 4. Autophagy

Question 60

Describe endocytosis.

Answer 60

Form vesicle at the plasma membrane, engulfing nearby extracellular substrate.

Question 61

Describe pinocytosis. At what rate does this process proceed?

Answer 61

Membrane extends to "scoop" in extracellular fluid and substrate. ~2500/cell/minute.

Question 62

Is macropinocytosis spontaneous or non-spontaneous? Please elaborate.

Answer 62

Non-spontaneous. Requires signal activation at receptor.

Question 63

Is fluid-phase endocytosis spontaneous or non-spontaneous?

Answer 63

Spontaneous.

Question 64

What 2 methods exist for sorting membrane proteins in a polarized epithelial cell?

Answer 64

1. Direct sorting in the trans Golgi network | 2. Indirect sorting via early endosomes

Question 65

What are the 2 main functions of phagocytosis?

Answer 65

1. Feeding (esp. Protists) | 2. Immune response (engulf shit)

Question 66

Is phagocytosis spontaneous or non-spontaneous? Please elaborate.

Answer 66

Non-spontaneous. Must be induced by Rho-GTPases.

Question 67

What 3 main steps outline phagocytosis?

Answer 67

1. Rho-GTPases signals PI kinase 2. PI kinase accumulates PI(4,5)P₂ 3. Accumulation signals actin polymerization

Question 68

How is the signal which activates phagocytosis deactivated?

Answer 68

PI(4,5)P₂ is converted to PI(3,4,5)P₃, initiating depolymerization of actin.

Question 69

Describe exocytosis.

Answer 69

Transport out of the cell via vesicles.

Question 70

In what 3 ways can protein sorting occur in the trans Golgi network? Which of these occurs only in secretory cells?

Answer 70

1. Signal-mediated diversion to lysosomes 2. Constitutive secretory pathway 3. Signal-mediated diversion to vesicles (for secretion) SECRETORY CELLS ONLY

Question 71

What might be the advantages of proteolytic processing (post-translational modification)?

Answer 71

To ensure that proteins remain inactive until they reach their destination and to increase cargo concentration.

Question 72

What 6 steps outline secretion at synaptic vesicles?

Answer 72

1. Components delivered to synaptic membrane 2. Endocytosis of components for new vesicle 3. Delivery of vesicle to endosome 4. New vesicle buds from endosome 5. Neurotransmitter loaded into vesicle 6. Exocytosis in response to action potential

Question 73

What 5 steps outline the special vesicle fusion which occurs with synaptic vesicles? What ion triggers fusion?

Answer 73

1. Docking 2. Priming 1 3. Priming 2 4. Fusion pore opening (triggered by Ca2+) 5. Fusion complete

Question 74

What are the 3 main characteristics of a lysosomal protein?

Answer 74

1. Cotranslationally moved to ER 2. N-terminal signal directs to ER 3. Additional signal directs to lysosome

Question 75

What is the pH in the cytosol? What about in the lysosome?

Answer 75

Cytosol: 7.2 Lysosome: 5.0

Question 76

What protein blocks SNARE from completing vesicle fusion in the synapse? What signal triggers the removal of this protein?

Answer 76

Complexin blocks SNARE bundles, but is removed when intracellular [calcium] increases.

Question 77

What are Arf1 (adenosine diphosphate ribosylation factor 1) protein and Sar1 (secretion-associated RAS-related) protein?

Answer 77

Coat-recruitment GTPases.