Mod2 - The Endomembrane System

Question 1

Name two examples of molecular chaperones that assist proteins with correct folding once inside the ER (and state what each one does)

Answer 1

BiP is an ATPase which binds exposed hydrophobic residues
Calnexin binds N-glycosylated proteins

Question 2

Name the three common types of protein modification in the ER

Answer 2

1. Signal sequence cleavage
2. Disulphide bond formation (oxidation)
3. Glycosylation

Question 3

How are disulphide bonds formed chemically (and which enzyme catalyses this)?

Answer 3

Oxidation of cysteine side chains (protein disulphide isomerase)

Question 4

Where can disulphide bonds be formed in the cell?

Answer 4

ONLY in ER Lumen (NOT in cytosol - needs oxidising environment)

Question 5

What occurs in N-linked glycosylation (and which enzyme catalyses this)?

Answer 5

Oligosaccharide added to amino group of Asparagine (Oligosaccharyl Transferase (OST)

Question 6

4 functions of N-linked Glycosylation?

Answer 6

1. Assists protein folding (e.g., allows to bind to chaperones)
2. Can be modified for Mannose-6-phosphate (lysosome signal)
3. Acts as a ligand for cell-cell recognition
4. Plays a role in glycocalyx formation

Question 7

What is the role of the Scramblase enzyme?

Answer 7

Transfers phospholipids between the two leaflets of the membrane non-selectively (to ensure equal growth of each)

Question 8

What is the role of the Flippase enzyme?

Answer 8

Flips phospholipids from the outer to the inner leaflet (in the Golgi specifically - asymmetric membrane)

Question 9

What is the role of coats in vesicle budding?

Answer 9

Deform the membrane into a bud, and capture cargo to be included in the vesicle

Question 10

What is the role of Dynamin?

Answer 10

Helps the vesicle bud to pinch off from the donor compartment (forms as a ring around the neck of the bud; it is a GTPase and hydrolysis causes a conformational change allowing it to pinch off the vesicle)

Question 11

Where do each of the three types of vesicle coat proteins mediate transport?

Answer 11

Clathrin = endocytic pathway (plasma membrane -> Golgi)
COPI = Various stages of Golgi trafficking
COPII = Form at ER, deliver to Golgi

Question 12

What is the role of Adaptins?

Answer 12

Adaptins help Clathrin to attach to the membrane, forming a clathrin-coated pit on the surface (also bind cargo receptors, which recognise sorting signals, and recruit them into the vesicle)

Question 13

What two proteins interact (initially) in vesicle-membrane recognition?

Answer 13

Rab (on the vesicle) and tethers (on the membrane) -> specific pairs

Question 14

Which proteins interact to dock the vesicle to the membrane and catalyse fusion?

Answer 14

SNAREs (vSNAREs with complementary tSNAREs)

Question 15

Describe the structure of the ER retention signal sequence

Answer 15

KDEL (LysAspGluLeu) - at the C-terminus; recruited into COPI-coated vesicles which return the protein to the ER

Question 16

What is the role of the Mannose-6-Phosphate sorting signal?

Answer 16

Addition of M6P to N-linked glycans of some glycoproteins results in sorting to LYSOSOME

Question 17

What is the role of an unusually short transmembrane domain in terms of sorting?

Answer 17

Proteins are retained in the Golgi

Question 18

State the main difference between the Vesicular Transport model and the Cisternal Maturation model of Golgi function

Answer 18

Vesicular Transport: Cisternae viewed as static compartments containing specific enzymes; vesicles bud from one to the next
Cisternal Maturation: Each cisterna matures as it migrates outward through the stack; Golgi resident enzymes are transported back to less mature cisternae via vesicles

Question 19

What is the difference between constitutive and regulated secretion?

Answer 19

Constitutive: constant stream of transport vesicles bud from the trans Golgi and fuse with the plasma membrane
Regulated: proteins are sorted into secretory vessels and stored until a particular signal is received (only in specialised secretory cells)

Question 20

Where are glycolipids found in the plasma membrane and why?

Answer 20

ONLY in the outer leaflet, because their orientation is fixed once they are packaged in vesicles, and there are no flippases which can flip them to the inner leaflet

Question 21

What does the integral region of an integral protein usually consist of?

Answer 21

Around 20 hydrophobic amino acids which form an alpha-helix that crosses the bilayer

Question 22

Why does the plasma membrane surface area remain constant despite the fusion of vesicles?

Answer 22

Endocytosis is balanced by exocytosis

Question 23

Describe the process of phagocytosis

Answer 23

The cell extends pseudopods to engulf the microbe, forming a phagosome, which fuses with a lysosome to destroy the microbe

Question 24

Describe pinocytosis

Answer 24

Non-selective uptake of fluid and macromolecules, mediated by Clathrin-coated vesicles - small areas of plasma membrane and extracellular fluid are internalised

Question 25

What is the benefit of receptor-mediated endocytosis?

Answer 25

The use of cell-surface receptors to capture cargo increases uptake efficiency up to 1000x

Question 26

Why do most cargo (e.g., LDL) dissociate from their receptor once it reaches the endosome?

Answer 26

The endosome has an acidic pH

Question 27

What is the main role of endosomes?

Answer 27

They are the main sorting station in the endocytic pathway (equivalent to the trans Golgi network in the secretory pathway)

Question 28

How is the acidic pH maintained in lysosomes?

Answer 28

An ATP-dependent proton pump

Question 29

How does autophagy occur?

Answer 29

Damned organelles are engulfed by a Double Membrane that forms in the cytosol, forms an autophagosome, and fuses with a lysosome