Lecture 8

Question 1

What proteins have to start in the ER?

Answer 1

Membrane bound

Question 2

Where do proteins translated at ribosomes tethered to the ER membrane go?

Answer 2

Straight into the lumen

Question 3

How can different vesicle fractions be isolated?

Answer 3

Density gradient centrifugation

Question 4

How is density gradient centrifugation carried out (steps)?

Answer 4

1) Rough and smooth ER cell are homogenized into rough and smooth microsomes
2) Put in test tube and mix with a gradient of increasing sucrose concentration
3) Centrifuge
4) Smooth microsomes have a low density and stop sedimenting at low sucrose concentration and rough microsomes have a high density and stop sedimenting at high sucrose concentration

Question 5

What is post-translational transport?

Answer 5

Import of proteins into nucleus, peroxisomes, and mitochondria after translation on free ribosomes in cytosol

Question 6

What is co-translational transport?

Answer 6

Import of proteins into ER occurs while still attached to ribosomes

Question 7

What types of proteins are synthesized into the ER?

Answer 7

Transmembrane proteins and soluble proteins

Question 8

What happens to transmembrane proteins?

Answer 8

• Partially translocated across and embedded into membrane (when hydrophobic amino acids are reached)
• Some proteins have a single transmembrane domain, others have many
• Some function in the ER and others are delivered to other membrane-associated organelles or plasma membranes

Question 9

What happens to soluble proteins?

Answer 9

• Fully translocated across membrane and are released into the ER lumen
• Either secreted from the cell or stay in the lumen of an organelle

Question 10

What do all proteins destined for the ER have?

Answer 10

An ER signal sequence (leader peptide) at 5’ where translation starts

Question 11

How is the ER signal peptide recognized?

Answer 11

The signal-recognition particle holds together a multisubunit complex (6 proteins) + RNA (ribonucleoprotein complex) which binds the SRP receptor in the ER membrane and large ribosomal subunit and then recognizes the ER signal that contains more than 8 nonpolar amino acids as an alpha-helix

Question 12

What does the signal-recognition particle (SRP) do?

Answer 12

1) Binds to the large ribosomal subunit and binds the signal peptide as it emerges from ribosome
2) Second site on SRP blocks translation from continuing (safety mechanism, until SRP binds to SRP receptor)
3) SRP-ribosome complex binds to SRP receptor in membrane of ER
4) SRP-ribosome-receptor complex interacts with the protein translocator (Sec61) embedded in the ER membrane

Question 13

What can errors in proper ER processing result in?

Answer 13

• Cystic fibrosis (CF)
• Retinitis pigmentosa (RP)

Question 14

What is cystic fibrosis?

Answer 14

• CFTR protein is a transmembrane protein mutated in cystic fibrosis (deltaF508 mutation)
• In CF patients, CFTR deltaF508 protein is made in the ER and functional but retained due to the mutation so it’s eventually targeted for degradation

Question 15

What is retinitis pigmentosa (RP)?

Answer 15

• Most common inherited form of retinal degradation due to loss of specialized rod photoreceptor neurons in the eye
• Gene for opsin is mutated (P23H)
• In RP patients, opsinP23H is misfolded in the ER and makes it sensitive to protease degradation