Lecture 9: protein sorting to organelles (II)

Question 1

Define secretory pathway

Answer 1

• outward pathway using transport vesicles.

- proteins transported from ER, through Golgi, to lysosome, PM or extracellular env.

Question 2

Name two branches of secretory pathway

Answer 2

1) lysosomal

2) exocytic (aka exocytosis)

Question 3

membrane-bound compartments of the endomembrane system communicates with _________.

Answer 3

transport vesicles.

Question 4

To perform correct function, each transport vesicle must:

Answer 4

1- carry appropriate protein.

2- fuse with appropriate target membrane.

Question 5

Characteristic of vesicles from ER to Golgi

Answer 5

1) exclude ER resident proteins.
2) carry appropriate proteins.
3) fuse only with Golgi membrane.

Question 6

Characteristics of vesicles from trans Golgi to lysosome

Answer 6

1- exclude Golgi residents and proteins destined for PM.
2- carry appropriate proteins.
3- fuse only with lysosomal membrane.

Question 7

Are the processing of vesicles selective?

Answer 7

Yes, from packaging, budding and fusion.

Question 8

Newly synthesized proteins destined to reside in organelles of the endomembrane system are

Answer 8

• co-translationally imported to the rough ER as unfolded, monomeric, an unmodified polypeptides.
• folding, assembly into multimeric complexes and covalent modification occur only in rough ER

Question 9

Name five modifications in which newly synthesized proteins in the ER lumen undergo

Answer 9

• happens before they reach their destinations.
  1) formation of disulfide bonds.
  2) addition and processing of cabs.
  3) specific proteolytic cleavage.
  4) proper folding.
  5) assembly into multimeric proteins.

Question 10

Why are disulfide bonds only formed in the ER lumen?

Answer 10

• cytosol is a reducing environment, high level of GSH (glutathione), ER has an oxidative environment, low level GSH.
• only found in secretory proteins in extracellular env.

Question 11

What are the function of disulfide bonds in the ER?

Answer 11

• sulfhydryl group are sensitive to pH change and proteases. Intrachain disulfide bonds are resistant to pH change and proteases and help stabilize the structure of proteins in plasma membrane, since extracellular env has a) no pH control, b) proteases are not confined within lysosome (like in a cell).

Question 12

Most plasma membrane and secretory proteins that initially enter the ER lumen control one or more carbohydrate chains (T/F).

Answer 12

True, covalent bond of short carb chains convert these proteins (glycosylases) into glycoproteins in ER (glycosylation), but not in cytosol (no glycosylases).

Question 13

Type of glycoproteins

Answer 13

glucose, galactose, mannose.

Question 14

Glycosylases are

Answer 14

glycosylating enzymes otherwise known as oligosaccharide transferases.

Question 15

Describe N-linked glycosylation

Answer 15

• occurs in ER lumen.
• polypeptide chain enters, then glycosylated by covalent bond of oligosaccharides from dolichol (special membrane lipid) to asparagines.

Question 16

Function of attachment of oligosaccharide to protein

Answer 16

1- protect from degradation (resistant to proteases).
2- prevent premature folding.
3- serve as transport signal, by packing into appropriate vesicle.

Question 17

Steps in folding of proteins it multimeric protein complexes

Answer 17

Step 1:
-protein (hemagglutinin) glycosylation.
Step 2:
- binding to Calnexin and Calreticulin.
- form 3 disulfide bonds.
Step 3:
- release ribosome.
- trimeric protein created.

Question 18

Mechanisms controlling protein that exit from ER

Answer 18

1) ER retention signal of ER-resident.
- KDEL receptor binds to ER retention signal, retain AND retrieve ER residents containing specific KDEL sequence that have escaped to the cis-Golgi.

2) quality control in ER.
- chaperone actively retain proteins (monomeric, multimeric) in ER lumen until they are properly folded and properly assemble.
- if chaperone unable to do so they export them to the cytosol for degradation.

3) coat proteins on cytosolic surface of transport vesicle.
- vesicles have distinct protein coats and after bussing coat is completely gone to enable vesicle to interact with target membrane.

Question 19

Name two functions of protein coating

Answer 19

1- shapes membrane into bud.

2- helps capture associated membrane-bound and soluble cargo proteins into vesicles.

Question 20

Steps in protein coating

Answer 20

Step 1: GTP-binding proteins recruit on cytosolic surface of donor membrane, regulates the rate of vesicle formation.

Step 2: Coat and adapters (for membrane protein and soluble protein) recruit.

Step 3:

• Specific sequences for membrane and soluble proteins recognized by adapters.
• adapters select which proteins enter.

Step 4: coat subunit proteins polymerize, helping vesicle pinch off donor organelle.

Step 5: GTP-binding proteins hydrolyze and pinch off the vesicle.

Question 21

Types of coated vesicles and organelle destination

Answer 21

I: COPI: Golgi to ER.
II: COPII: ER to Golgi.
III: Clathrin: Golgi to lysosome.

Question 22

Common features of fusion of transport vesicle

Answer 22

• fusion occurs after depolymerization of coats.
• must recognize correct target membrane.
• vesicle and membrane must fuse.

Question 23

Name conserved set of proteins involved in fusion of vesicle with target membrane

Answer 23

• NSF= N-ethylmaleimide sensitive factor (ATPase).
• SNAPs= soluble NSF attachment proteins.
• v-SNARE= SNAP receptor on transport vesicle.
• t-SNARE: SNAP receptor on target membrane.
• Rab protein= family of GTP-binding proteins.

Question 24

Steps in fusion of transport vesicle and target membrane

Answer 24

Step 1: docking. v-SNAR and t-SNARE bind, Rab facilitate complex formation of the two.

Step 2: fusion. GTP hydrolysis use two membranes and NSF/SNAP complex recruited from cytosol.

Step 3: disassembly of SNARE complex. energy from ATP hydrolyses disassembles complex and are reutilized.