Liu 2

Question 1

What are the 3 types of coated vesicles?

Answer 1

1) clathrin
2) COPI
3) COPII

Question 2

Where is each type of coated vesicle used?

Answer 2

1) clathrin: from golgi or plasma membrane, also used by secretory vesicle
2) COPI: golgi retrieval process
3) COPII: ER

Question 3

What is assembly of clathrin? What are adaptins?

Answer 3

• 3 large and 3 small light chains to make triskelion

- adaptin recognize diff transmembrane receptors and therefore determine specificity for receptor

Question 4

Describe vesicle formation with adaptin and clathrin

Answer 4

1) Clathrin binds to adaptin which is bound to transmembrane receptor (specific)
2) Vesicle begins to pinch off, needs dynamin (GTPase) to do it
3) Clathrin and adaptin are shortly dissociated after vesicle formed

Question 5

COPI and COPII vesicle formation?

Answer 5

1) use of GTPase ARF (COPI), SarI (COPII) -> important for priming membrane
2) Normally inactive and attached to GDP (floating with hydrophobic tail), when activated by GEF, it is bound to membrane with GTP
3) GTPases also responsibly for hydrolysis of GTP to cause dissociation of coat proteins

Question 6

What is the role of t-SNARE, v-SNARE, and Rabs?

Answer 6

1) v-SNARE on vesicle and t-SNARE on target, they complement
2) They find each other and dock the vesicle to correct target membrane.
3) Interaction is monitored by Rab (GTPase), if correct, hydrolysis of GTP causes locking of the membranes (in combination with ATPase NSF will cause fusing)

Question 7

What is involved in membrane fusion? SNARE dissociation?

Answer 7

SNARE and NSF (ATPase)

-hydrolysis of ATP by NSF needed to prie SNARE apart

Question 8

What else is capable of fusion similar to SNARE?

Answer 8

Viruses can be received by chemokine receptor and use fusion proteins to enter the membrane

Question 9

How do COPI and COPII work to maintain ER

Answer 9

COPI is needed to retrieve lipids, resident proteins, and receptor from golgi back to the ER

COPII transfers from ER to golgi (vesicular tubular cluster) -> retrograde transport

Question 10

Describe resident ER protein retrieval

Answer 10

1) Need KDEL sequence (lys-asp-glu-leu) for ER resident protein (2 sequences then, ER signal and KDEL)
2) KDEL recognizes KDEL receptor and binds in the golgi because of acidic pH
3) brought back by COPI to ER and dissociates in the neutral pH

Question 11

What are the two pathways from trans Golgi network?

Answer 11

1) constitutive secretory pathway (unregulated by COPI)
2) regulated secretory pathway - uses clathrin vesicles. Secretory vesicle is not released unless a signal received (hormone, neurotransmitter, or digestive enzymes)

*remember the vesicle that mediates retrieval is clathrin-coated

Question 12

What are the 3 pathways for trans Golgi network?

Answer 12

1) lysosomal hydrolases (clathrin)
2) secretory vesicle; signal-mediated (clathrin)
3) constitutive (COPI)

Question 13

How do synaptic vesicles work?

Answer 13

1) Synaptic vessicle constitutively released to membrane
2) Endocytosed and is loaded with neutransmittters
3) Neutrotransmitters secreted by exocytosis in response to action potential

Question 14

What proteins needed in synaptic and presynaptic transport?

Answer 14

1) v-SNARE (synaptobrevin) and t-SNARE (syntaxin), and Rab3

Question 15

What cells can undergo phagocytosis? Pinocytosis?

Answer 15

specialized cells (ie. macrophages and neutrophils)

pinocytosis can be unregulated (nonselective) or receptor mediated (very selective)
-use clathrin pits

Question 16

How is LDL taken up by receptor mediated cytosis?

Answer 16

1) LDL taken up by coated pit (receptors are localized in coated pits), vesicle forms regardless of binding of LDL (clustering of receptors stimulate vesicle formation)
2) receptor can be recycled and LDL can be broken to cholesterol by lysosome

Question 17

How is coated pit related to familial hypercholesterolemia (FH)?

Answer 17

• 4 AA chain in C-terminal
• one is tyrosine
• recognition site for adaptin
• LDL receptors does not concentrate in clathrin pit

Question 18

3 outcomes of endocytosis?

Answer 18

1) transcytosis (one membrane to other) (IgG taken up by newborn from mother)
2) degradation (first activation of receptor and dimerization using monoubiquitin marker. Formation of multivesicular bodies and then degradation by lysosome)
3) recycling (ie. LDL and transferrin)

Question 19

Early endosome vs late?

Answer 19

early is made up many vesicles coming together. As some components are broken off to be recycled, it becomes a late endosome and fuse with each other or other preexisting late endosomes.

Question 20

pH of lysosome? Content of lysosome?

Answer 20

pH inside is 5.0, outside is 7.2

contains acid hydrolases (ie. proteases, lipases, etc.)

Question 21

How are things degraded by lysosome?

Answer 21

Endosome to lysosome or endosome fusing with phagosome or autophagosome before fusing with lysosome.

Question 22

How are lysosomal enzymes tagged with mannose 6-phosphate?

Answer 22

1) signal patch with hydrophobic residues recognize and bind to GlcNAc phosphotransferase (N-acetylglucoasminephosphotransferase) KEY ENZYME
2) transfer GlcNAc-P to mannose
3) lysosomal hydrolase with GlcNAc-P attached is released
4) GlcNAc phosphodiesterase uncovers mannose 6-P

Question 23

How is lysosomal hydrolase transported to early endosome?

Answer 23

1) with mannose-6-phosphate, binds to M6P receptor (transmembrane protein) at pH 7 in trans golgi network
2) shipped to early endosome and dissociates at the more acidic pH (6)

Question 24

Disease associated with faulty targeting of lysosomal enzymes?

Answer 24

• inclusion-cell (I-cell) disease
• lack of GlcNAc-phosphotransferase
• lysosomal enzyme not phosphorylated, cannot be recognized by M6P
• secreted by default, buildup of oligosacch, lipid, etc.