Intracellular Compartments and Protein Sorting

Question 1

What percentage of the cell volume is cytosol?

Answer 1

About half. The other half are organelles (nucleus takes up 10%)

Question 2

What organelles make up the exocytic network?

Answer 2

Endoplasmic reticulum

Golgi apparatus

Secretory vesicles

Question 3

What organelles make up the endocytic network?

Answer 3

Plasma membrane

Endosomes

Lysosomes

Question 4

How are proteins ‘tagged’ with a destination to travel to following synthesis?

Answer 4

Sorting signals address the protein to enter/exit cellular organelles

This sequence can be linear at end of protein or formed during protein folding (patch)

Question 5

What property is common in ER signal peptides?

Answer 5

A hydrophobic center

Question 6

What property is common in mitochondrial signal peptides?

Answer 6

Amphipathic alpha helices

Have positively charged edge on side of alpha helix

Question 7

A signal peptide is leucine rich. Where is the protein most likely going?

Answer 7

Leucine rich signal peptides signal export from the nucleus

Question 8

Describe the nuclear membrane

Answer 8

A double membrane that is perforated with about 4000 nuclear pores

The perinuclear space (between membranes) is continuous with the endoplasmic reticulum

Question 9

Describe the structure of a nuclear pore

Answer 9

Nuclear pores are octamers with fibrils extending into the cytosolic and nuclear sides of the pores

On the nuclear side, the fibrils join into an 8-fiber basket that restricts the passage of large particles (>60 kDa)

Question 10

How are porteins larger than 60 kDa moved in/out of the nucleus?

Answer 10

Nuclear localization signals (NLS) or Nuclear export signals (NES) trigger active energy dependent gated transport through the nuclear membrane

Question 11

Why is [Ran-GTP] high in the nucleus, but low in the cytosol?

Answer 11

RanGEF is a nuclear protein that exchanges GDP bound to Ran for GTP to form Ran-GTP

This gradient drives cargo transport across nuclear pores

Question 12

Describe the steps for nuclear export.

Answer 12

RanGTP promotes cargo with NES to bind to an export receptor (exportin) which binds to the NPC fibrils and travels through nuclear pores

RanGAP hydrolyzes the RanGTP to dissociate the cargo

The exportin enters back into the nucleus to repeat the cycle

Question 13

Describe the steps for nuclear import

Answer 13

Cytosolic cargo with NLS binds to an importin which interacts with the NPC to be imported into the cell

In the nucleus, RanGTP binds the complex and stimulates dissociation

Importin-RandGTP exits back into cytoplasm

Question 14

How is the nuclear uptake of NF-kB regulated?

Answer 14

NFkB is housed in the cytoplasm where it is bound to I-kB and inactivated.

Following signal transduction, I-kB is phosphorylated and dissociates from NFkB revealing an NLS

Question 15

True or false: Nuclear import signals can be reused during repeated rounds of mitosis.

Answer 15

True.

Although the nucleus breaks down and reforms in each cell cycle, the NLS on proteins stay put and direct the proteins into the correct place during division

Question 16

Describe the structure of the mitochondrion.

Answer 16

Has two membranes.

The inner membrane is very folded (large surface area) but the outer membrane is not.

There are four compartments: matrix, inner membrane, inter-membrane space, outer membrane

Question 17

True or false: Mitochondrial proteins are coded for from mitochondrial DNA

Answer 17

False. Most mitochondrial proteins are nuclear gene products

Question 18

Describe protein transport into the mitochondrion

Answer 18

Post-translational

Signal sequence binds to TOM (outer membrane) receptors and TIM (inner membrane) receptors lined up

Unfolded protein passes through aqueous pores in the membrane

Question 19

What is required for proteins to be inserted into the mitochondrial matrix?

Answer 19

Cytoplasmic heat shock protein (hsp70) bind the unfolded cargo protein, push protein through pore

Electric potential across inner membrane is dissipated to electrophorese the unfolded protein into the matrix space

Chaperones (hsp70) in the matrix bind unfolded protein and pull through the pore

Question 20

What are the two types of mitochondrial chaperones that may assist in protein folding?

Answer 20

Hsp70: binds to unfolded protein domains and protects them during folding process

Hsp60: “cage” in which unfolded protein is protected while it finds its way through the folding process

Both are ATP-dependent

Question 21

What is the general function of peroxisomes?

Answer 21

Peroxisomes oxidize things like fatty acid chains, cholesterol (into bile acids), metabolic intermediates (acetyl CoA production)

Detoxify poisons (alcohol)

Question 22

How are peroxisomes formed?

Answer 22

They bud off of the endoplasmic reticulum and can expand and divide by fission

Question 23

True or false: Proteins are introduced into peroxisomes during translation.

Answer 23

FALSE

Proteins are introduced post-translationally into peroxisomes

Question 24

What processes occur within the endoplasmic reticulum?

Answer 24

Membrane and secreted proteins are produced

Lipids are produced

Calcium is stored

Lipid soluble drugs/toxins are detoxified

Question 25

When are most proteins transported into the ER?

Answer 25

As they are tranlated (“cotranslational”)

Signal peptides are produced at cytoplasmic ribosomes, which cause ribosomes to localize to the ER so that the protein can be pushed through a pore into the ER lumen

Question 26

What does a signal recognition particle do?

Answer 26

They guide ribosomes to the ER

SRP bind to signal peptides and arrest translational elongation until the SRP is bound to an SRP receptor on the ER membrane near the Sec61 translocation channel

Question 27

How are free, soluble polypeptides synthesized into the ER lumen

Answer 27

The signal sequence directs translation to the ER surface and the protein is synthesized directly into the ER lumen

Signal peptidase cleaves the signal peptide upon completion of translation leaving a free, soluble protein in the ER

Question 28

How are membrane proteins produced?

Answer 28

Stop-transfer signals are regions are charged residues flanking hydrophobic stretches that will end up being the transmembrane spans

After a stop-transfer, the ribosome will finish translation cytosolically

Sec61 will be pried open to release the protein into the ER membrane

Question 29

How are proteins N-glycosylated?

Answer 29

Asparagine residues in the sequence N-X-S/T can be glycosylated in the lumen of the ER

Dolichol lipids in the membrane are substrates for buildup of big sugars that can be transferred onto N by oligosaccharyltransferase

Question 30

What amino acid residues other than arginine can be glycosylated?

Answer 30

In O-glycosylation, the hydroxy groups of Serine or Threonine are glycosylated

Question 31

Describe the unfolded protein response

Answer 31

ER can sometimes be overwhelmed with unfolded proteins

Aggregation occurs, which can be recognized by sensors that dimerize into ribonucleases

The sensors trigger splicing of mRNA that encodes a gene regulator protein that drives transcription of ER chaperone genes

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