Zaidi lecture 2 9/19

Question 1

Define translocation

Answer 1

the act of proteins crossing inner and outer mitochondrial membranes

Question 2

What directs the proteins to the right compartments in mitochondria?

Answer 2

signal sequences

Question 3

Define an amphiphilic alpha helix

Answer 3

the reaction between positively charged residues on one end of the protein and the uncharged hydrophobic residues on the other

Question 4

What do the receptor proteins look for when scanning the proteins?

Answer 4

The configuration of the amphiphilic alpha helix

Question 5

Define a protein translator

Answer 5

a multisubunit complex that mediates translocations

Question 6

Where is the SAM complex located?

Answer 6

On the outer mitochondrial membrane

Question 7

Where is the OXA complex located?

Answer 7

On the inner mitochondrial membrane

Question 8

Define TOM

Answer 8

translocate of the OUTER membrane

required for the import of all nuclear encoded proteins by inserting them into the outer membrane

Question 9

Define TIM

Answer 9

Translocate of the inner membrane

Question 10

What are the two TIM complexes?

Answer 10

TIM22 and TIM23

Question 11

What is TIM23 in charge of transporting?

Answer 11

soluble proteins into the matrix and helps insert these proteins into the inner membrane

Question 12

What does TIM22 do?

Answer 12

mediates the insertion of a specific proteins

Question 13

What are the two components of the TIM and TOM complexes?

Answer 13

receptors for the mitochondrial proteins and translocation channels

Question 14

What does the SAM complex do? And what does it stand for?

Answer 14

Sorting and Assembling machinery; inserts and folds the beta barrel proteins into the outer membrane

Question 15

What does the OXA complex do?

Answer 15

inserts proteins that are synthesized in the mitochondria

Question 16

Describe the process of protein import into the mitochondrial matrix.

Answer 16

1. proteins are unfolded via interactions with chaperone proteins (HSP70)
2. Receptors on TOM bind to the signal sequence that is present on the protein
3. unfolded protein is pulled through the translocator and the chaperone proteins are stripped off (via ATP)
4. TOM transports it through to the inner membrane space
5. The protein binds to the TIM complex and transportation begins(via membrane potential)
6. Mitochondrial HSP70 is bound to the TIM23 on the matrix side and pulls the protein through
7. HSP70 released protein in an ATP dependent step
8. signal sequence is cleaved by the signal peptidase
9. HSP60 helps to fold the protein using ATP

Question 17

What is the role of HSP60 inside the mitochondrial matrix?

Answer 17

folds the protein with the help fo ATP

Question 18

Describe the process of a protein being inserted into the outer mitochondrial matrix

Answer 18

1. Passes through the TOM complex into the inter-membrane space
2. binds to chaperone proteins
3. binds to the SAM complex
4. SAM refolds and inters into the outer membrane

Question 19

What are the 5 different ways that a protein can get imported from the cytosol into the IMM and the inter membrane space

Answer 19

1. Nuclear encoded proteins can become a part of the inner membrane via TOM and TIM23
2. A mitochondrial encoded protein can be inserted into the inner membrane via TOM, TIM, and OXA
3. The protein can be released in the inter membrane space
4. Protein reduced the SH can be transported via the TOM complex and into the inter membrane space, where it is reduced by mia 40 and interacts with the electron transport chain
5. protein is looped through the TOM complex and accompanied by chaperones where if travels to the TIM 22 complex and is incorporated into the inner membrane

Question 20

Describe the steps required for a nuclear encoded protein to be transported to the inner membrane and incorporated into the inner mitochondrial membranous layer

Answer 20

1. The N terminal initiates the the import to the inner membrane space via TOM
2. the hydrophobic sequence that is present behind the signal sequence “gets stuck” in the inner membrane
3. signal sequence is cleaved
4. protein is pulled through the TOM complex and remains as an inner membrane protein

Question 21

Describe the steps required for a mitochondrial encoded protein to be transported to the inner membrane and incorporated as an inner mitochondrial membrane protein.

Answer 21

1. Signal sequence is recognized by TOM and transported through to the inner mitochondrial space
2. The signal sequence travels through the TIM and is cleaved (There is a second signal sequence behind the one that was cleaved)
3. Entire protein is passed through to the mitochondrial matrix
4. The second sequence is recognized by the OXA complex, where it is inserted into the inner membrane

Question 22

Describe the steps required for protein transport that will be released into the inner membranous space.

Answer 22

1. signal sequence recognized by TOM and transported into the inner membrane space
2. signal sequence is recognized by TIM and the signal sequence is cleaved
3. the hydrophobic portion of the protein is stuck in the inner membrane layer like glue
4. A protease comes by and cleaves the protein above the hydrophobic portion, and the protein is released into the inner membrane

Question 23

Describe the steps required for a reduced protein to be imported.

Answer 23

1. reduced protein travels through the TOM complex
2. Protein is oxidized by Mia 40 in the inner membrane space
3. disulfide bonds form between the newly oxidized SH groups (folds over on itself into a half moon-ish shape)
4. Mia 40 is reduced via the ETC

Question 24

Describe the steps required for the translocation of a protein that has an internal protein sequence.

Answer 24

1. the internal sequence is recognized by tOM and the protein is “looped” through the TOM (meaning it folds up and the U part of the protein goes through the sequence)
2. The entire protein is transported to the inner membrane where it is accompanied by chaperone proteins
3. The sequence is recognized by TIM22
4. protein is inserted into the inner membrane

Question 25

What is the endoplasmic reticulum, and what are its functions?

Answer 25

network of branching tubules with a continuous nuclear membrane plays a role in lipid and protein synthesis

Question 26

Describe co-translational translocation

Answer 26

1. ER signal sequence emerges from the ribosome, the protein is directed by the ribosome to the pore on the ER while translation is occurring 2. entire ribosome binds to the ER and the protein is eventually translocated once it is made

Question 27

Describe post-translational translocation

Answer 27

The protein is made by the free ribosome outside of the ER | 2. The protein is then taken into the ER

Question 28

What are the 2 components that help to guide the signal sequence to the ER?

Answer 28

Signal recognition particle (SRP) | SRP receptor

Question 29

What is at the center of an ER signal sequence?

Answer 29

8 or more non-polar amino acids

Question 30

Describe the basic structure of an SRP

Answer 30

rod shaped with a large hydrophobic pocket that is liked with methionines There is a signal sequence binding site and then the particle is able to hinge up and bind to the ribosome at the elongation factor binding site or whatever machinery is present

Question 31

Describe the steps necessary to get a protein into the ER from when it is being translated to the insertion into the inner mitochondrial membrane.

Answer 31

1. The signal sequence of a growing polypeptide emerges from the ribosome 2. conformation chance in SRP and binding site is exposed 3. (in the cytosol) the SRP binds to the signal polypeptide and pauses translation 4. SRP receptor in the rough ER membrane binds to the SRP and the complex is taken to a protein translocator 5. signal sequence is recognized by the pore and it opens allowing the protein and the signal sequence in 6. . The SRP/SRP receptor complex leaves the ribosome and the protein at the translocator 7. signal sequence is recognized and translation continues to occur completing the protein 8. SRP is recycled

Question 32

Describe the translocator in the ER membrane

Answer 32

- has a water filled pore - core is made of the sec61 complex - 3 subunits with the largest at the central pore - pore is gated by a short helix that opens and closes to protect the ER

Question 33

Describe the reaction of the pore in the ER during a translocation

Answer 33

1. Protein is brought to the pore with the SRP and SRP receptor 2. pore recognizes the signal sequence and opens, allowing the SRP complex to leave. (start transfer signal) 3. signal sequence is bound in the middle and the remaining portion of the protein is near the outside of the translocator 4. signal peptidase cleaves the signal and escapes from a lateral opening in the pore 5. Protein is released into the lumen

Question 34

Describe the translocation of a transmembrane protein in the ER

Answer 34

1. N terminal sequence initiates 2. pore is opening and the protein is brought into the translocator 3. hydrophobic region in the polypeptide stops the transfer process and acts like glue in the inner membrane layer (STOP TRANSFER SIGNAL) 4. signal sequence (start-transfer signal) is cleaved by a signal peptidase 5. lateral gated allows the start transfer peptide to be removed and to integrate the stop transfer signal into the bilayer

Question 35

Describe what happens with a polypeptide that needs to be translocated but has an internal signal sequence

Answer 35

The signal sequence is noticed by the translocator and the protein is looped through and incorporated into the membrane EITHER the C or the Terminus enters (determined by the amino acids before and after the signal sequence

Question 36

What happens if there are more positively charged amino acids prior to the sequence?

Answer 36

The C terminus is inserted into the lumen (CHECK ME ON THIS-in book)

Question 37

What happens if there are more positively charged amino acid after the sequence?

Answer 37

The N terminus is interred into the lumen (CHECK ME ON THIS-in book)

Question 38

Describe the translocation of a protein that has two TM domains

Answer 38

The start and stop transfer sequences are hydrophobic and act like glue in the membrane and stick and the protein remained on the inside and outside of the ER, with the N and C termini in the cytosol

Question 39

Describe the translocation of a membrane protein with multiple domains

Answer 39

Same principle as the 2 TM domains, but it is looped through the membrane N group is on the inside of the ER and the COOH is on the outside in the cytosol with several loops of the protein present in the membrane