Block C Lecture 4: Protein Targeting to the Nucleus and Mitochondria

Question 1

Despite mitochondria containing their own DNA, ribosomes and other components needed for protein synthesis, where are most mitochondrial proteins encoded and imported from?

Answer 1

Encoded in the nucleus and imported into the mitochondria from the cytosol

(Slide 4)

Question 2

What must each protein have to enable it to reach a specific sub-component of the mitochondrion?

Answer 2

They must contain targeting signals

(Slide 4)

Question 3

How are new mitochondria formed?

Answer 3

By growth and fission of existing mitochondria

(Slide 5)

Question 4

What does the growth and fission of mitochondria require?

Answer 4

The import of new proteins

(Slide 5)

Question 5

In what state are proteins translocated into the mitochondria?

Answer 5

An unfolded state

(Slide 5)

Question 6

Does mitochondrial protein translocation occur pre or post-translationally?

Answer 6

Post-translationally

(Slide 5)

Question 7

What do signal sequences which specify entry into the mitochondrial matrix consist of?

Answer 7

An N-terminus amphipathic helix with positive charges on one side and hydrophobic / uncharged residues on the opposite side

(Slide 5)

Question 8

Why are the signal sequences of proteins which have transmembrane domains not as well characterised as other signal sequences?

Answer 8

As they aren’t cleaved

(Slide 5)

Question 9

What are present on mitochondrial membranes which act as protein translocators?

Answer 9

Protein complexes

(Slide 6)

Question 10

What are the protein complexes which transfer proteins across the mitochondrial outer and inner membranes called?

Answer 10

TOM complex - for protein transfer across the outer membrane

TIM complex - for protein transfer across the inner membrane

(Slide 6)

Question 11

What are the steps of mitochondrial protein transfer across the mitochondrial membrane?

Answer 11

1. Mitochondrial precursor proteins remain unfolded after being synthesised in the cytosol via interactions with chaperones of the Hsp70 family, which prevent aggregation or folding
2. Import receptors in the TOM complex bind to the signal sequence

3.Interacting proteins (Hsp70) are stripped off as the polypeptide is threaded through the translocation channel

4. The signal sequence is then recognised by the TIM complex and threaded into the matrix
5. Mitochondrial Hsp70 hydrolyses ATP, generating a conformational change that acts like a ratchet to pull the protein through the TIM channel
6. Signal sequence is then cleaved

(Slide 7)

Question 12

What does the release of HSP70 from the translocating polypeptide in the cytosol require?

Answer 12

ATP hydrolysis

(Slide 8)

Question 13

Other than the hydrolysis of ATP by mitochondrial Hsp70, what else helps move the protein into the the mitochondrial matrix?

Answer 13

The electrochemical gradient (H+ gradient) across the inner membrane attracts the positively-charged signal sequence into the matrix

(Slide 8)

Question 14

What is the nuclear envelope?

Answer 14

A double-layered membrane that surrounds the nucleus of a cell

(Slide 11)

Question 15

What is the outer membrane of the nuclear envelope continuous with?

Answer 15

The ER membrane

(Slide 11)

Question 16

What does transport occur through between the cytosol and the nucleus?

Answer 16

Aqueous nuclear pores

(Slide 11)

Question 17

What is a nuclear pore complex (NPC)?

Answer 17

A large, multi-protein structure embedded in the nuclear envelope, functioning as a selective gateway for the transport of molecules between the nucleus and the cytoplasm

(Slide 12)

Question 18

How many nuclear pore complexes (NPCs) does the nuclear envelope of a typical mammalian cell have?

Answer 18

4,000-6,000

(Slide 12)

Question 19

How do proteins diffuse through the nuclear pore complex according to size?

Answer 19

Proteins with a Da or less than 5000 can diffuse freely whereas proteins with a Da of more than 60,000 can’t

(Slide 12)

Question 20

Can a nuclear pore complex (NPC) transport in both directions at the same time?

Answer 20

Yes

(Slide 12)

Question 21

What are nuclear localisation signals?

Answer 21

Short amino acid sequences that tag proteins for transport into the nucleus of a cell

(Slide 13)

Question 22

What do nuclear localisation signals mediate?

Answer 22

Active transport processes

(Slide 13)

Question 23

What do nuclear localisation signals (NLS) typically contain?

Answer 23

1 or more stretches of positively charged amino acids (such as lysine or arginine) exposed on the surface of a protein

(Slide 13)

Question 24

What are nuclear localisation signals recognised by?

Answer 24

Protein import receptors called importins

(Slide 13)

Question 25

What do importins do?

Answer 25

They bind to NPCs and mediate transport of proteins through the pore and into the nucleus (Slide 13)

Question 26

What are the steps of protein transport into the nucleus?

Answer 26

1. Nuclear localisation signal on a protein binds to importin 2. The protein-importin complex enters the nucleus and binds to Ran-GTP. 3. The protein dissociates 4. The importin-Ran-GTP complex moves to the cytoplasm 5. GTP is hydrolysed into GDP, which causes importin to dissociate (Slide 14)

Question 27

Where are the Ran-GTP and Ran-GDP proteins localised?

Answer 27

Ran-GTP - nucleus Ran-GDP - cytosol (Slide 14)

Question 28

What 2 proteins regulate the nucleotide-bound state of Ran-GTP/GDP, what do these proteins do, and where are they localised?

Answer 28

Ran-GAP - A GTPase activating protein which converts GTP into GDP which is localised to the cytosol Ran-GEF - A guanine nucleotide exchange factor which exchanges GDP for GTP, and is localised in the nucleus (Slide 15)

Question 29

Why is protein movement through nuclear pore complexes energy-dependent?

Answer 29

As it it involves GTP hydrolysis (Slide 15)

Question 30

What is a nuclear export signal?

Answer 30

A short peptide sequence in a protein that directs it to be exported from the nucleus to the cytoplasm (Slide 16)

Question 31

What do nuclear export signals usually consist of?

Answer 31

4 hydrophobic amino acids, usually arranged LxxxLxxLxL, with L being a hydrophobic amino acid (usually leucine) and X being any amino acid (Slide 16)

Question 32

What are the steps of the transport of a protein from the nucleus to the cytoplasm?

Answer 32

1. Exportin binds to Ran-GTP in the nucleus 2. This changes the conformation of exportin, allowing it to bind to an nuclear export signal 3. The Exportin-Ran-GTP-Protein complex moves through the nuclear pore complex 4. The conversion of Ran-GTP to Ran-GDP results in the release of exportin and the cargo protein (Slide 16)

Question 33

What does it mean to enhance protein localisation?

Answer 33

To increase the concentration or presence of a protein in a specific cellular compartment (Slide 17)

Question 34

How can protein phosphorylation enhance protein localisation?

Answer 34

By changing the affinity of nuclear localisation signals for importin or nuclear export signals for exportin, it can reduce affinity, leading to the retention of the protein in the component, or increase affinity leading to increased transport (Slide 17)

Question 35

What is an example of a way in which nuclear import / export of proteins can be regulated?

Answer 35

Via signalling pathways which can mask or unmask nuclear localisation signals, such as nuclear factor of T cells (NFAT) transcription factors (Slide 18)

Question 36

What does SRY stand for?

Answer 36

Sex-determining region of the Y chromosome (Slide 19)

Question 37

What does the SRY gene encode?

Answer 37

A transcription factor which is important for proper development of genitalia in males (Slide 19)

Question 38

What do mutations in the SRY gene cause?

Answer 38

Male-to-female sex reversal which is known as Swyer syndrome. It results in an XY female which has external female genitalia but lacks both ovaries and testes (Slide 19)