Protein targeting

Question 1

What are the 2 main outcomes for a protein when it has begun to be synthesised via translation in the cytoplasm?

Answer 1

1. Synthesis of the protein occurs completely in the cytoplasm and the protein is released into the cytosol - it can then be transported to other organelles
2. Protein can go through an ER signal sequence which directs it to the surface of the endoplasmic reticulum where its synthesis resumes - protein can remain in ER or be transported to other organelles of secretory pathway e.g. lysosome

Question 2

What is the key aspect of protein targeting?

Answer 2

• Signal sequences which allow a protein to be transported to specific organelles e.g. the ER, mitochondria or the nucleus

Question 3

Describe the nature, the signal location within the protein and whether the signal sequence is removed from the protein once it enters its target organelle for the signal sequences that target proteins to the ER, mitochndria, peroxisome and the nucleus.

Answer 3

Question 4

Describe the structure of the nuclear envelope

Answer 4

• Nucleus has inner and outer membrane
• Between the 2 membranes is the perinuclear space
• Nuclear laminar is on the inside of the inner membrane
• Contains lots of nuclear pores which allow nuclear content to be transported out of the nucleus
• Nuclear envelope is continuous with the ER

Question 5

Describe the structure of a nuclear pore complex

Answer 5

• Contains about 30 different proteins
• However, there are multiple copies of each protein so the total no. of protein molecules in each complex ranges from 500-1000
• Cytosolic side of the complex has cytosolic fibril
• Nuclear side of the complex has a structure called a nuclear basket

Question 6

How is nuclear import dependent on the size of the molecule?

Answer 6

• Molecules that are smaller than 60kDa are able to freely diffuse through the nuclear pore complex into the nucleus
• Molecules that aer larger than 60kDa have to be transported through the nuclear pore complex into the nucleus via active transport

Question 7

Explain the mechanism of nuclear import of a protein targeted to the nucleus

Answer 7

1. A complex is formed between the cargo protein and the nuclear localisation signal (NLS)
2. Importin α and β then associate with the cargo protein to form a trimeric complex
3. This trimeric complex is then able to interact with the proteins of the nuclear pore complex allowing it to be imported into the nucleus
4. Once in the lumen of the nucleus the trimeric complex interacts with a protein called Ran-GTP
5. This intercation causes the cargo protein with the NLS to dissociate from the rest of the complex which allows it to move freely within the nucleus
6. The Ran-GTP importin-β complex and importin α are both transported out of the nucleus
7. Once in the cytoplasm Ran hydrolyses its GTP into GDP - facilitated by Ran GAP
8. This causes Ran to undergo a conformational change meaning it can no longer bind to importin β
9. Ran-GDP is imported back into the nucleus where it switches back into Ran-GTP - facilitated by GEF

Question 8

Why does the interaction between Ran-GTP and the trimeric complex release the cargo protein from the complex?

Answer 8

• Because Ran-GTP has a high affinity to importin-β and so is able to bind to it
• This binding then displaces importin-α from the complex leaving only the cargonprotein and the NLS

Question 9

GTPases such as Ran require other proteins in order to hydrolyse GTP and to unbind GDP, what are the names of the proteins that perform these 2 functions?

Answer 9

• GAP (GTPase activating protein) - Help GTPases to hydrolyse GTP more efficiently
• GEF (GTPase exchange factor) - Help GTPases dissociate from GDP nad re-associate with GTP

Question 10

Are mitochondrial proteins synthesised using nuclear DNA or mitochondrial DNA?

Answer 10

• Most mitochondrial proteins are synthesised using nuclear DNA and are imported into the mitochondria
• However, some mitochondrial proteins are synthesised using mitochondrial DNA

Question 11

Briefly describe the structure of the mitochondria

Answer 11

• Double membrane structure - outer membrane and inner membrane
• Inner membrane highly inverluted forming structures called cristae
• Innermost space of the mitochondria is aclled the matrix
• Space between the 2 membarnes is called the intermmebrane space

Question 12

Explain the mechanism that allows a protein targeted for the matrix of the mitochondria to be imported into the matrix

Answer 12

1. The mitochondrial signal sequence on the protein binds to a receptor present within the TOM (transport outer membrane) complex
2. The channel component of the TOM complex then allows the protein to move through the outer membrane into the intermembrane space
3. Once in the intermmebrane space the protein interacts with a receptor present withtin the inner membrane called the TIM23 (transport inner membrane 23) complex
4. TIM23 allows the protein to be transported across the inner membrane into the matrix
5. Once in the matrix the mitochondrial signal sequence is cleaved off the protein by a signal peptidase

Question 13

The process of transoprt of a protein into the matrix of the mitochondria requires free energy. Explain the sources of free energy throughout the process

Answer 13

• When travelling through cytosol to reach mitochondria the protein associates with the ATPase Hsp70
• In order for the protein to asociate with the TOM complex Hsp70 must dissociate from the protein and this process requires free energy (produced via hydrolysis of ATP)
• Second source of free energy is provided by the electrochemical gradient between the intermembrane space and the matrix
• The electron transport chain generates this electrochemical gradient by pumping protons from the matrix into the intermebrane space
• This electrochemical gradient allows for the protein to move through the TIM23 complex into the matrix
• As the protein emerges from the TIM23 complex it associates with mitochondrial Hsp70
• As the protein travels through the matrix mitochondrial Hsp70 goes through cycles of association and dissociation
• Dissociation requires hydolysis of ATP which is the 3rd source of free energy
• This association requires

Question 14

Explain the first mechanism of protein transport into the inner mitochondrial membrane

Answer 14

1. The mitochondrial signal sequence on the protein binds to a receptor present within the TOM (transport outer membrane) complex
2. The channel component of the TOM complex then allows the protein to move through the outer membrane into the intermembrane space
3. The protein interacts with a receptor present withtin the inner membrane called the TIM23 (transport inner membrane 23) complex
4. As soon as the signal sequence emerges from the TIM23 complex it is cleaved off
5. Immediately upstream from the signal sequence is a highly hydrophobic region which acts as a transmembrane domain for the protein
6. This hydrophobic region allows the protein to dissociate laterally from the TIM23 complex meaning the protein remains anchored in the inner membrane

Question 15

Explain the second mechanism of protein transport into the inner mitochondrial membrane

Answer 15

1. Protein passes through TOM complex and then through TIM23 complex to reach the matrix (signal peptide cleaved off as it passes through TIM23)
2. Once in the matrix the protein intercats with the OXA complex within the inner membrane via its second signal sequence
3. OXA complex mediates the proteins insertion into the inner membrane so that it becomes ancjored within said membrane

Question 16

Explain the third mechanism of protein transport into the inner mitochondrial membrane

Answer 16

1. Protein travels through TOM complex to enter intermmebrane space
2. As it moves through TOM complex intermembrane space chaperones bind to the protein
3. Once in the intermmebrane space these intermembrane space chaperones guide the protein to the TIM22 complex within the inner membrane
4. The TIM22 complex uses the electrochemical gradient between the intermembrane space and the matrix to arrange the protein so that certain parts of it face the intermembrane space and certain parts face the matrix
5. This creates a protein within the inner membrane that contains multiple transmembrane domains

Question 17

Explain the mechanism of protein transport into the intermembrane space of the mitochondria

Answer 17

1. Protein is inserted into inner membrane via hydrophobic domain
2. Once inserted the protein is cleaved at the hydrophobic domain so while the hydrophobic domain remains in the inner membrane the protein moves freeley within the intermembrane space

Question 18

What organelles are involved in the secretory pathway?

Answer 18

• Endoplasmic reticulum (rough)
• Golgi apparatus
• Lysosomes
• Secretory granules
• Plasma membrane

Question 19

Explain how the secretory pathway was discovered

Answer 19

• Used slices of hamster pancreas - used because they needed a tissue good at secretion
• Performed a pulse chase experiment
• The tissue slices were incubated with a radioactive amino acid, 3H leucine for 3 mins
  
  • This allowed for the 3H leucine enough time to be incorporated into any new proteins synthesised during the 3 mins but not enough time for proteins to move in the cell
• The tissue slices were washed - to remove any unincorporated 3H leucine
• Then non-radioactive leucine was added after either 7, 37, 117 mins
• After each time period, the tissue slices were fixed then underwent audioradiography
• Finally, percipitate produced was viewed using electron microscopy
• The experiment concluded that the radioactivity from the 3H leucine, and therefore the proteins, were found first at the ER, then golgi appartus and then secretory granules which collectively became known as the secretory pathway

Question 20

Explain the process of Co-Translational Protein Targeting to the ER

Answer 20

1. At the start of of translation the ribosome scans the mRNA until it locates the translation initiation codon (AUG)
2. Then the ribosome begins synthesis of the protein with the N terminus being synthesised first - signal peptide is produced firs as it’s located at N terminus
3. Once the signal peptide is synthesised it causes translation to stop
4. The SRP (Signal recognition particle) then binds to the ribosome-mRNA-protein complex and causes it to bind to a complex of proteins on the ER
5. The SRP binds to the SRP receptor and the ribosome binds to the peptide translocation complex which causes translation to resume
6. This causes the protein to move through the peptide translocation complex into the lumen of the ER as its synthesised
7. Once the ribsome is bound to the peptide translocation complex the SRP dissociates from the SRP receptor which requires hydrolysis of GTP - SRP is then recycled to cytosol to find another signal sequence
8. A signal peptidase then cleaves off the signal sequence and eventually the protein is fully synthesised

Question 21

What are the different types of membrane protein?

Answer 21

• Type I - N terminus in ER lumen, C terminus in cytoplasm
• Type II - N terminus in cytoplasm, C terminus in ER lumen
• Signal anchor type II - Transmembrane domain at N terminal acts as anchor
• C-terminal anchor - Transmembrane domain at C terminal acts as anchor
• Polytopic - Protein with multiple transmembrane domain

Question 22

Explain the process of ER targeting of tail-anchored (C-terminal anchored) membrane proteins

Answer 22

1. While the protein is being synthesised by the ribosome a complex of a Get3 dimer, Get5 and Get5 binds to the protein
2. Once synthesis of the protein is complete the complex takes the protein to the ER membrane
3. Get4 and Get 5 dissociate from the complex
4. Get3 binds to the Get1-Get2 hetero-oligomer bringing the protein with it
5. Then intergrase integrates the protein into the membrane of the ER