ER/Golgi transport

Question 1

Golgi order

Answer 1

1. Cis-network
2. Cis
3. Medial
4. Trans
5. Trans-network (TGN)

Question 2

Possible destination from the Trans-Golgi-Network (TGN)

Answer 2

—> late endosome —> lysosome

—> constitutive secretion

—> regulated secretion

(Retrograde) to trans-Golgi and beyond

Question 3

As the golgi cisternae move to their new location…the mature through ?

Answer 3

Retrograde movement of enzymes needed for an earlier compartment

Question 4

Anterograde movement =

Retrograde movement =

Answer 4

1. Cisternal maturation

2. Retrograde vesicles

Question 5

Three different types of vesicles

Answer 5

1. COPII (anterograde)
2. COPI (retrograde)
3. Clathrin/adapter protein (TGN —> endosome/lysosome & plasma membrane —> endosome)

Question 6

GTPase for COPII vesicles

Answer 6

Sar1

Question 7

GTPase for COPI vesicles

Answer 7

ARF

Question 8

GTPase for all non-COPII vesicles

Answer 8

ARF

= COPI + all clathrin vesicles

Question 9

Trans-golgi —> endosome

Adapter protein?

Answer 9

Clathrin + AP1 complexes (or GGA)

Question 10

Plasma membrane —> endosome

Adapter proteins?

Answer 10

Clathrin + AP2 complexes

Question 11

Golgi —> lysosome, melanosome, or platelet vesicles

Adapter proteins?

Answer 11

AP3 complexes

Question 12

How are integral proteins carried in COPII vesicles from ER —> cis-Golgi

Soluble proteins?

Answer 12

Integral = inserted in the vesicle membrane

Soluble = bind to specific ER membrane-bound receptors

Question 13

What does the COPII coat protein bind to?

Answer 13

The cytosolic domains of the integral membrane cargo

The receptor for the soluble cargo

The cytosolic domains of the ER membrane…curved nature gives the vesicle a round shape

Question 14

What happens once the formation of COPII vesicle is complete and pinches off the ER?

Answer 14

The vesicle starts to de-coat (lose the COPII proteins)

Exposes the v-SNAREs proteins in the vesicle membrane

Question 15

Role of the v-SNAREs

ER —> cis-golgi

Answer 15

Bind to t-SNAREs (target SNAREs)

Mediating vesicle fusion

At this point…

The integral cargo is now integral to the cis-membrane

The soluble cargo is bound to its receptor, bound to the cis-membrane

Question 16

COPI transport

Answer 16

Same mechanism as COPII

Just in reverse direction and different coat protein

Still uses v and t-SNAREs

Question 17

GTPase function in vesicle transport

Answer 17

• binds GTP and hydrolyzes the GTP —> GDP

COPI = ARF

COPII = Sar1

Two important functions

1. Vesicle formation
2. De-coating

Question 18

GTP-bound form of GTPase (Sar1 or ARF)

Answer 18

N-tail of protein extends into the vesicle membrane (hydrophobic interactions)

Can interact with coat proteins (COPI/II)

Therefore…Sar1 or ARF aids in

1. The binding of the coat proteins
2. Recruitment of cargo (indirectly)
3. Inherent curvature of vesicle

Question 19

GTP hydrolysis by GTPase

Answer 19

Promotes conformational change of GTPase

Retracts N-tail out of membrane

Lessens the coat proteins association with the vesicle membrane

Mediates coat dissassmebly

Question 20

Processing of N-linked oligosaccharide complex

Cis golgi vs. trans golgi

Answer 20

Cis = high mannose glycosylation

Trans = complex glycosylation

**proteins can be hybrids of both

Question 21

High mannose modifications can be enable proteins to be targeted by ?

Answer 21

Lysosomes

Question 22

High mannose gylcosylation

Answer 22

(Man)8(GlcNAc)2 —> (Man)5(GlcNAc)2

**lose 3 mannose

Question 23

In TGN,

To get proper cargo into the correct type of transport vesicle…

Answer 23

The adapter protein (AP) binds a cytosolic domain sorting signal on the cargo (or cargo receptors)

The AP also interactes with the coat protein (usually Clathrin)

Question 24

What transport from the TGN would not have clathrin as the coat protein?

Answer 24

Retrograde from the TGN —> trans-golgi

COPI

Question 25

Cargo signal for TGN —> lysosome transport

Answer 25

Mannose-6-phosphate (M6P)

Question 26

I-Cell Disease General/symptoms

Answer 26

General theme: mis-targeting of proteins from the TGN Symptoms: 1yr old coarse facial features, craniofacial abnormalities, psychomotor retardadtion, stunted growth

Question 27

M6P modification sorting signal | TGN —> lysosome transport

Answer 27

Occurs on one of the mannose sugars on the N-linked oligosaccharyl high mannose complex

Question 28

TGN —> lysosome transport Steps

Answer 28

1. M6P lysosomal proteins packaged into AP3 containing vesicles (M6P receptor) 2. De-coats 2A. Clathrin and AP3 are recycled back 3. Uncoated vesicle fuses with late endosome/lysosome 4. Low pH in lysosome, acid hydrolase (M6P protein) now active 4A. M6P receptor is recycled back to TGN 5. Or to the plasma membrane...in case a hydrolase is accidently released out of cell

Question 29

Mechanism of I-cell Disease

Answer 29

Lysosomal proteins are not tagged with M6P Therefore...all of lysosomal proteins are do not go to lysosomes...but instead are constitutively secreted out of the cell Now lysosomes cannot break down substrates

Question 30

How the M6P cargo signal is created in the cis-golgi

Answer 30

UDP-GlcNAc (substrate) used to add GluNAc-phosphate to one of the mannose sugars Enzyme #1 = GlcNAc phosphotransferase Then GlcNAc is removed to leave the M6P sorting signal Enzyme #2 = phosphodiesterase

Question 31

What enzyme is blocked by I-Cell Disease

Answer 31

GlcNAc phosphotransferase Prevents any M6P from being made Causes a build up of multiple types of inclusion bodies within the lysosome (mainly carbs and lipids)

Question 32

what is I-cell disease referred to as ?

Answer 32

Mucolipidosis (ML-II) Characteristics of mucopolysaccharidoses and sphingolipidoses (both types of LSDs)

Question 33

ML-I

Answer 33

Sialidase deficiency

Question 34

ML-III

Answer 34

Pseudo-Hurler Polydystrophy Less severe case of I-cell disease (same enzyme)

Question 35

What is the root cause of most LSD? And why are ML-II/III different

Answer 35

Single defective hydrolase...so get a buildup of a single class of molecule ML-II/III have a problem in the targeting part of a lysosomal formation...so many types of molecules build up

Question 36

MPS-I

Answer 36

Name = Hurler-Scheie syndrome Enzyme = alpha-L iduronidase

Question 37

MPS-II

Answer 37

Hunter syndrome Iduronate-2-sulphatase

Question 38

MPS-III

Answer 38

Sanfilippo syndrome Heparan-N-sulphatase Alpha-N-acetlyglucoaminidase AcetlyCoA:N-acetyltransferase N-acetylglucosamine 6 sulphatase

Question 39

Gaucher disease

Answer 39

Beta-glucosidase

Question 40

Gangliosidosis type I

Answer 40

Tay Sachs disease Beta-hexosaminidase A

Question 41

Gangliosidosis type II

Answer 41

Sandhoff disease Beta-hexoaminidase A and B

Question 42

Lysosome pH range

Answer 42

5.0-6.0

Question 43

Cytosolic pH

Answer 43

7.2-7.4

Question 44

Endocytosis

Answer 44

Plasma membrane invaginates to form small endocytic vesicles (endosomes)