Module 2 - The endomembrane system

Question 1

How are proteins refolded after entering the ER?

Answer 1

Chaperone proteins fold the proteins again

BiP binds to hydrophobic residues
Calnexin binds N-glycosylated proteins

Question 2

How can proteins be modified after entering the ER?

Answer 2

Signal sequence clevage
Disulfide bridge formation
N-terminal glycosylation

Question 3

What is the function of most proteins entering the ER?

Answer 3

Some will function in the ER but most will be moved around for use in other subcellular locations or secreted out of the cell

Question 4

Vesicular transport with the ER: how does it work?

Answer 4

The membrane buds around the cargo and the vesicle moves to the target location before fusing with the target location

The formation that the bud has (ie any proteins sticking out of the vesicle) will remain after fusing with the target (proteins that were facing the cytosol will still face the cytosol after fusing)

Question 5

The secretory pathway

Answer 5

Allows material moving outwards (ER-Golgi-Plasma membrane-endo/lysosome)

Question 6

The endocytic pathway

Answer 6

Allows material to move inwards (plasma membranes-endosomes-lysosomes)

Question 7

What are protein coats?

Answer 7

Coats that are formed by proteins

Adaptin binds to the cargo receptor and clathrin binds to this which forms a protein coat.

This coat helps pull the vesicle off and dynamic (GTPase) pinches the bud off

Question 8

COPI, COPII, and Clathrin: what movements do they facilitate?

Answer 8

COPII -> ER -> Golgi and rest of cell
COPI -> Golgi to ER
Clathrin -> Outer cell -> inner cell organelles

Question 9

Rab proteins: what do they do?

Answer 9

Bind to the vesicle and interact with a tethering protein on the target membrane and, along with t-snares which bind with v-snares, they pull the vesicle to the target membrane and fusion occurs

Question 10

V-snares: what do they do?

Answer 10

Bind to the vesicle and interact with a t-snare on the target membrane and, along with Rab proteins which bind with tethering proteins, they pull the vesicle to the target membrane and fusion occurs

Question 11

Fusion between vesicles and target membrane: how close do they have to be for fusion to occur, is it energetically favourable, and is another signal needed?

Answer 11

Bilayers must be brought within 1.5nm for lipids to mix (fuse)

Water must be displaced => energetically unfavourable

Fusion may require an additional signal

Question 12

KDEL sequences: what are they and what do they do?

Answer 12

Sequence (LysAspGluLeu) at the C-terminus of soluble proteins recognised by KDEL receptor in Golgi

Recruited into COPI vesicles that return the protein to the ER

Question 13

What is the signalling sequence for the Golgi apparatus?

Answer 13

18 amino acids (unusually short)

Question 14

What is the signalling sequence for the lysosomes?

Answer 14

Addition of Mannose-6-Phosphate to N-linked glycans of some glycoprotein

Question 15

What is the Golgi apparatus and what does it do?

Answer 15

Sacs of cisternae located near the nucleus

Control the movement of proteins from the ER (cis) into the Golgi body and then out of the Golgi to other locations (trans)

Question 16

Two models explaining protein movement through the Golgi

Answer 16

Vesicular transport
Cisternal maturation

Question 17

Vesicular transport: what is the idea behind it, how does the theory work, and what suggests this theory is accurate?

Answer 17

Golgi cisternae viewed as static compartments containing specific enzymes.

Vesicles bud from one cisterna and fuse with the next transporting cargo through each cisterna in turn

Experimental evidence shows cargo molecules present in small transport vesicles that are ≈100 nm in diameter

Question 18

Cisternal maturation: what is the idea behind it, how does the theory work, and what suggests this theory is accurate?

Answer 18

Each Golgi cisterna matures as it migrates outward through the stack

Golgi resident enzymes carried forward with the cisterna are returned to an earlier compartment in vesicles

Explains the transport of large cargo like procollagen (300 nm long) that are too big for typical vesicles of 100 nm in diameter

Question 19

Protein modifications in the Golgi apparatus

Answer 19

O-linked oligosaccharides are added to some –OH side chains of Serine (S) and Threonine (T) residues in proteins as they transit the Golgi.

N-linked oligosaccharides added to proteins in the ER can be trimmed and rebuilt at the Golgi

N-linked oligosaccharides on lysosomal proteins modified with Mannose-6-Phosphate

Question 20

Constitutive secretion of transport vesicles: what is it, what does it do, and how is it initiated?

Answer 20

Involves vesicles being sent to the membrane which supplies it with membrane proteins and lipids for growth

Allows protein secretion – cell surface, extracellular matrix or fluid

No signal required – ‘default’ pathway

Question 21

Regulated secretion of transport vesicles: what is it, what does it do, and how is it initiated?

Answer 21

Proteins are sorted into vesicles until a signal is received

Occurs with specialised secretory cells

Question 22

What are the three secretory mutations?

Answer 22

Secretory mutant A - Mutation at rER which prevents proteins from moving to Golgi
Secretory mutant B - Mutation at Glogi which prevents proteins from moving to membrane
Secretory mutant C - Mutation at the membrane which prevents proteins from being secreted

Question 23

The three forms of endocytosis

Answer 23

Phagocytosis - Using the membrane to form a phagosome to break down engulfed material
Pinocytosis - Non-selective uptake of material mediated by clathrin-coated vesicles
Receptor-mediated - Receptors bind to molecules before forming a vesicle with a clathrin coat which moves to the endosome for intracellular sorting

Question 24

Endosomes: what are they and what type of molecules are sorted?

Answer 24

Intracellular sorting organelles composed of tubules and vesicles (with a low pH which causes the release of the receptors from the molecules) which are vital in the endocytic pathway

Cholesterol (LDL), Essential metabolites (VB12, iron), Signalling molecules (Epidermal growth factor (EGF)), and Virus particles (Influenza, HIV, and coronavirus)

Question 25

What happens to the receptors after dissociating from the bound molecules?

Answer 25

• Can be recycled (LDL receptors)
• Can be degraded (EGF receptors)
• Transcytosis (movement to a different domain of the plasma membrane)

Question 26

Lysosomes: what are they, what is the optimum pH, and how is it maintained?

Answer 26

Contains 40 hydrolytic enzymes (inc proteases, nucleases, glycosidase, etc) which break down macromolecules before pumping the products into the cytosol

Optimum pH = 5

pH maintained by an ATP-dependent proton pump

Question 27

Sorting enzymes to lysosomes

Answer 27

Lysosomal enzymes made at the ER, moved through the Golgi and modified with Mannose-6-phosphate, and transported to the lysosome after interacting with M-6-P receptors in the trans-Golgi network.

Question 28

Autophagy: what is it?

Answer 28

Self-consuming within the cell by forming an autophagosome which transports damaged organelles/obsolete parts of the cell to the lysosome