Protein Targeting

Question 1

What is proteolytic cleavage?

Answer 1

Breaking of peptide bonds to remove part of a protein

Question 2

What is chemical modification of a protein?

Answer 2

Addition of functional groups to the amino acid residues of a protein

Question 3

On which ribosomes are proteins for lysosomes synthesised?

Answer 3

Ribosomes associated with the rough ER

Question 4

What is required for protein sorting? (4)

Answer 4

A signal intrinsic to the protein
A receptor that recognises to the signal and directs it to the membrane
Translocation machinery (e.g. Proteins/cell structures to move and allow sorting)
Energy to transfer the protein

Question 5

What is a peroxisome and its function?

Answer 5

Small spherical membrane enclosed organelle

Contains enzymes for oxidation reactions, e.g. Catalase to break down hydrogen peroxide and the breakdown of very long fatty acids

Question 6

What signal do proteins targeted to peroxisomes possess? Where on the protein is this located?

Answer 6

Serine-Lysine-Leucine
(PTS)

On c terminus of protein

Question 7

Are proteins targeted to peroxisomes usually folded or unfolded before import?

Answer 7

Folded

Question 8

What happens to a protein destined for peroxisome import in the cytoplasm?

Answer 8

Pex5 receptor recognises PTS and binds to cargo protein
13 Pex proteins form a transport channel across the peroxisomal membrane
The transport channel binds to the Pex5-cargo complex - allowing entry into peroxisome

Question 9

Is energy required during targeting of proteins to peroxisomes?

Answer 9

Yes, hydrolysis of ATP for recycling of Pex5

Question 10

What signal is found in proteins targeted to the ER? Where on the protein is this found? When and by which protein is the signal recognised?

Answer 10

Signal sequence
N terminus
During translation - SRP

Question 11

After binding of the SRP how do proteins get imported into the ER?

Answer 11

On the ER membrane there is a translocon (channel) associated with a docking protein and SRP receptor.
SRP receptor binds to SRP/Protein complex
Hydrolysis of GTP - translocon opens
Protein synthesised through ER membrane into lumen
Signal sequence cleaved by signal peptidase
Folded in ER lumen

Question 12

Is energy required when targeting proteins to the ER?

Answer 12

Yes - hydrolysis of GTP by SRP - opens channel

Question 13

Is the signal sequence cleaved or retained in protein targeting to the ER? Is it folded or unfolded during transfer?

Answer 13

Cleaved - signal peptidase

During transfer = unfolded

Question 14

What is the signal for proteins that are retained in the ER? Where on the protein is it found?

Answer 14

KDEL Sequence
Lys-Asp-Glu-Leu

C terminus

Question 15

How is a protein destined to be retained in the ER, moved out and returned to the ER?

Answer 15

COP2 transports the KDEL protein towards the golgi
KDEL receptors bind to the KDEL sequence in cis golgi
COP1 transports the KDEL sequence back towards the ER

Question 16

Is a protein destined to be retained in the ER folded or unfolded during transfer? Does it use energy?

Answer 16

Folded

No energy required - pH dependent

Question 17

What is the signal for proteins that are targeted to the lysosome?

Answer 17

Addition of mannose-6-phosphate in the cis golgi through N linked glycosylation

Question 18

What happens to the lysosomal protein after Mannose-6-phosphate has been added to it?

Answer 18

Travels through the golgi, binds with M6P receptors in trans golgi
Receptor dependent transport takes place, vesicle moves towards lysosome membrane
There is dissociation of the receptor from the molecule at the acidic pH of a lysosome
Phosphate is cleaved of by phosphatase

Question 19

Is energy required for targeting of lysosomal proteins? Are the proteins folded or unfolded during transfer?

Answer 19

Yes

Folded in a vesicle

Question 20

What is the signal found in proteins that are targeted to the mitochondria? What is a feature of this sequence? Where on the protein is it found?

Answer 20

MTS
N terminus
Amphipathic

Question 21

Are proteins for the mitochondria folded or unfolded during transfer?

Answer 21

Held (partially) unfolded by chaperone proteins

Question 22

How is a mitochondrial protein moved to the mitochondria from the cytoplasm?

Answer 22

MTS binds to an import protein on the outer mitochondrial membrane
Protein is fed through the TOM protein on the outer mt membrane
Protein is fed through the TIM protein on the inner mt membrane
MTS cleaved by matrix peptidase
Protein folds in matrix

Question 23

Is energy required in the targeting of proteins to the mitochondria?

Answer 23

Yes - hydrolysis of ATP for association of chaperone proteins

Question 24

What is the signal found on proteins targeted for the nucleus? What composition does it have? Where is it found?

Answer 24

NLS
Basic amino acids
Can be found anywhere in protein

Question 25

What are two examples of basic amino acids found in the NLS?

Answer 25

Lysine, arginine

Question 26

What happens in the targeting of a protein to the nucleus?

Answer 26

Importin binds to the NLS of cargo protein, moves protein into the nucleus Ran-GTP binds to importin in the nucleus Displacement of the protein in the nucleus Ran-GTP/Importin complex recycled to the cytoplasm

Question 27

Does the targeting of proteins to the nucleus require energy?

Answer 27

Yes - hydrolysis of GTP

Question 28

Is the NLS cleaved or retained? Why?

Answer 28

Retained, to allow protein to re-enter the nucleus after it breaks down during cell division

Question 29

On which ribosomes are membrane proteins synthesised?

Answer 29

Ribosomes attached to the ER

Question 30

What is the difference between regulated and constitutive secretion? Give an example of each.

Answer 30

Constitutive = constant secretion (e.g collagen from fibroblasts) Regulated = at specific times, controlled (e.g. Endocrine, exocrine, neurocrine cells)

Question 31

What do the 'pre' and 'pro' parts of a protein mean? | E.g. Preproalbumin

Answer 31

Pre = refers to part of the protein (signal sequence) that is cleaved during processing Pro = referees to partially mature protein

Question 32

Which receptor recognises the signal sequence? What is the composition of this receptor? What organelle does the receptor recognise?

Answer 32

SRP 6 proteins and a short piece of RNA Ribosome

Question 33

The signal sequence is found on cells destined for the ______

Answer 33

ER

Question 34

For secretory proteins that need to enter the ER, how does translation begin? How is the protein produced?

Answer 34

Translation begins at a 'free' ribosome Signal sequence is is produced and is recognised by SRP, binds Translation stops SRP binds to ribosome This complex binds to the SRP receptor on the ER membrane Translation begins GTP hydrolysis SRP falls off Protein is made through the ER membrane into the lumen + folds

Question 35

What do membrane proteins contain in their sequence that affects translation at ER ribosomes?

Answer 35

Stop transfer sequences

Question 36

What is the function of stop transfer sequences in membrane proteins?

Answer 36

They halt the transfer of the peptide across the ER membrane Acts as an anchor to hold the protein in position

Question 37

What are some functions of the ER?

Answer 37

Specific proteolytic cleavage Glycosylation Formation of disulphides bonds Hydroxylation of selected lysine and proline residues etc.

Question 38

Where does n linked glycosylation take place? What is it?

Answer 38

In the ER Sugars are added onto an asparagine side chain (NH group)

Question 39

What is glycosylation important for in proteins?

Answer 39

Proper folding of proteins Stability of proteins Allows interactions with other molecules

Question 40

Which enzyme in the ER ensures the correct disulphide bonds are formed?

Answer 40

Protein Disulphide Isomerase (PDI)

Question 41

Misfolding of proteins in the ER can be corrected by which proteins?

Answer 41

ER chaperone proteins

Question 42

If protein misfolding cannot be resolved by chaperone proteins in the ER, what happens to the protein? (2)

Answer 42

Retained in the ER | Returned to the cytosol for degradation

Question 43

What modifications take place in the golgi?

Answer 43

Glycosylation Sulfation Removal of mannose

Question 44

Where does O linked glycosylation occur?

Answer 44

Golgi

Question 45

What is o linked glycosylation? In which molecule does o linked glycosylation play an important role?

Answer 45

Attachment of sugar to hydroxyl groups Proteoglycans