Lecture 11 and 12

Question 1

What proteins are glycosylated?

Answer 1

Most soluble and transmembrane proteins in the ER

Question 2

Two types of glycosylation

Answer 2

O-linked glycosylation (10%)
N-linked glycosylation (90%)

Question 3

Where is the N-linked oligosaccharide precursor formed and what is it linked to?

Answer 3

The N-linked oligosaccharide precursor is preformed in the ER and it is linked to target protein in the ER.

Question 4

Sugars involved in glycosylation

Answer 4

N-acetylglucosamine, mannose and glucose

Question 5

What happens in the ER lumen?

Answer 5

An oligosaccharyl transferase transfers an N-linked oligosaccharide precursor to an Asn on a protein being synthesized. - proteins are only glycosylated on the er lumen side

Question 6

sequence of amino acids for glycosylation of asparagine

Answer 6

asn-X-ser or asn-x-thr
where x is any amino acid except proline

Question 7

What happens after transfer of N linked oligosaccharide to the protein?

Answer 7

1. 3 glucose removes - 1 removed by glucosidse 1 and 2 removed by glucosidase 2
2. 1 mannose removed by ER mannosidase
3. glycosylated protein is transported via vesicles to golgi

Question 8

Cisternae in golgi

Answer 8

cis, medial, trans

Question 9

How is N linked oligosaccharides processed in golgi?

Answer 9

mannose removes, n-acetylglucosamine added, galactose added, N-acetylneuraminic acid (sialic acid, NANA) added

Question 10

Why is glycosylation done?

Answer 10

1. tag to mark state of protein folding
2. protect proteins on cell surface from proteases
3. some glycosylated proteins have a role in cell adhesion
4. allows proteins to form correct 3d structure

Question 11

How can glycosylation be a tag to mark the state of protein folding?

Answer 11

1. Glucose trimming will take place
2. protein + precursor oligosaccharide structure bind to calnexin
3. glucosidase 2 comes in
4. 2 options: protein bound w N-linked oligosaccharide can leave ER or the incompletely folded one binds glycosyl transferase.
5. UDP-glucose comes in to transfer glucose the oligo structure.

process then repeats

Question 12

What do protein coats do?

Answer 12

1. select cargo for vesicle
2. give curvature for vesicle
3. promote vesicle budding

Question 13

COP II

Answer 13

ER to golgi

Question 14

COP I

Answer 14

golgi to ER and within golgi

Question 15

clathrin

Answer 15

trans golgi network to late endosome
both ways between early endosome and plasma membrane

Question 16

retromer

Answer 16

early to late endosome

Question 17

General steps in coat assembly and vesicle formation

Answer 17

1. GEF at the site of membrane budding - recruits GTPase which is GTP bound
2. GTP-bound GTPase recruits coat proteins
3. Vesicle bud formation, cargo selected
4. vesicle buds off
5. vesicle uncoating - COP1 and COP2 coated vesicles involve GAPs, clathrin coated-diff mechanism

Question 18

ARF GTPase

Answer 18

recruited for COP1 and clathrin-coated vesicles

Question 19

2 layers of vesicle coats

Answer 19

1. inner layer - binds to membrane and selects cargo - it has Sec23/24, Sar 1 GTP, and selected membrane proteins
2. Outer layer - associates with inner layer to promote polymerization of the coat- sometimes selects cargo - it has Sec13/31

Question 20

Sar1 GTP

Answer 20

1. amphipathic alpha-helix exposed (interacts with membrane)
   2.recuits coat protein subunits

Question 20

Sar1 GTPase

Answer 20

recruited for COP 2

Question 21

What must coat proteins select?

Answer 21

cargo (tranmembrane proteins), transmembrane cargo recepts, SNARES

Question 22

Uncoating of Cop1

Answer 22

1. gamma COp (inner subunit) binds to ARF-GAP
2. GTP hydrolysis (Arf-GTP to Arf-GDP)
3. Arf GDP detaches from membrane and the coat is released.

Question 22

COP1 - inner and uter layers

Answer 22

inner - 4 subunits
outer - 3 subunits

Question 23

COP 2 coated vesicles

Answer 23

inner 2 units (sec 23/24) outer 4 subunits (sec 13/14)

Question 24

uncoating of cop2 coated vesicles

Answer 24

1. Sec 23 has GAP activity which stimulated by Sec13/31. 2. GTP hydrolysis of (Sar1 GTP to Sar 1 GDP) 3. Sar1 GDP detached from membrane and coat os released.

Question 25

Layers of clathrin coated vesicles

Answer 25

inner - different adaptor complexes outer-clathrin (6subunits)

Question 26

uncoating of clathrin coated vesicles

Answer 26

requires Hsp70 and auxillin

Question 27

what does pinching off of clathrin coated vesicles require

Answer 27

dynamin (has GTPase activity)

Question 27

In clathrin molecule what polymerizes to for a curved lattice?

Answer 27

triskelions - they have light and heavy chains

Question 27

What is specificty of vesicle fusion determined by?

Answer 27

1. Proteins for docking and tethering the vesicle to target membrane - Rab GTPases and Rab effectors 2. Proteins for catalyzing vesicle fusion with the target membrane - SNAREs

Question 28

Mediation of vesicle docking

Answer 28

1. Rab-GTP binds Rab effector 2. Rab GTP + Rab effector will dock and tehter vesicle, they bring v SNARes and t SNAREs together - the helical domains coil around each other

Question 29

v SNARE

Answer 29

vesicle membrane SNARE

Question 30

t SNARE

Answer 30

target membrane SNARE

Question 31

what happens when v SNAREs and t SNAREs bring membrane close together

Answer 31

displace water and prmote membrane fusion