Lecture 14

Question 1

Where are most mitochondrial proteins encoded, where are they synthesized, and how do they reach their final destination?

Answer 1

encoded in nuclear DNA

synthesized on ribosomes

Transported into the mitochondria

(Some mito proteins are made in the mito, though)

Question 2

What is the movement of proteins to their final destination and what directs this process?

Answer 2

translocation

signal sequences on the proteins

Question 3

Describe the structure of signal sequences on proteins destined for the mitochondria

Answer 3

postively charged on one end and uncharged hydrophobic on the other end

this forms a amphiphilic alpha helix

Question 4

what is the signifigance of amphiphilic alpha helixes on signal sequences?

Answer 4

receptor proteins recognize this configuration (not necessarily the specific sequence) and then multi-subunit protein complexes called protein translocators mediate the translocation

Question 5

Describe the location and action of the following mitochondrial translocators
TOM
TIM

Answer 5

TOM: present in the outer membrane of the mito. imports all nuclear encoded proteins and inserts them into the membrane if they need it

TIM: TIM22 and TIM23
TIM22: mediates the insertion of a specific subclass of proteins (ATP, ADP, and Pi)

TIM23: transports soluble proteins into the matrix and helps insert membrane proteins into the inner membrane.

Question 6

What does SAM stand for, and what does it do?

Answer 6

Sorting and Assembly Machinery

it translocates and inserts/folds beta barrel proteins into the outer membrane of the mito.

Question 7

Describe the function of OXA complexes when it comes to mitochondrial proteins.

Answer 7

they mediate insertion of proteins synthesized in mitochondria.

Question 8

What is the main purpose of chaperone proteins, such as cytosolic Hsp 70, and what happens to them after the protein binds to the receptors of a TOM complex?

Answer 8

they inhabit the cytosol and interact with proteins that are being translocated to prevent folding

chaperone proteins are stripped off as the signal sequence binds to TOM receptors and the rest of the translocated protein enters the mito

Question 9

What does peptidase do to proteins that are being translocated into the mitochondria?

Answer 9

it cleaves off signal sequences once the protein reaches it’s final destination.

this prevents any further translocation from occurring

Question 10

Describe the energy sources used for the activities of TOM and TIM complexes

Answer 10

ATP is used by TOM when dissociating the Hsp70 from the protein

Translocation through the TIM complex does not require energy bc it uses the electrochemical H+ gradient that exists across the inner membrane of the mito.

mitochondrial Hsp70 exists on the matrix side of the TIM23 complex and uses ATP to drive it’s “motor” movement that pulls the precursor protein into the matrix space.

Question 11

explain how mitochondrial Hsp70 releases the precursor protein after it’s ratcheting action and describe how this happens and what role Hsp 60 plays.

Answer 11

Hsp70 releases the precursor protein in an ATP-dependent step

Hsp60 (in the mito. matrix) assists with the folding of the precursor protein in another ATP-dependent step.

Question 12

Describe the path of a protein that is inserted into the outer mito. membrane by the SAM complex.

Answer 12

The protein passes through the TOM complex, binds to chaperone proteins, and then binds to the SAM complex.

SAM then inserts and folds
the protein into the outer membrane (porins are an example of beta barrel proteins that SAM incorporates into the outer membrane)

Question 13

Describe the nuclear encoded pathway that incorporates an intrermembranous protein into the inner membrane of the mitochondria.

Answer 13

the protein’s N-terminal sequence (used by TOM) binds to TIM23, is cleaved and that unmasks a hydrophobic sequence.

this hydrophobic sequence is released into the inner membrane, and anchors the rest of the protein there

Question 14

Describe the mito encoded pathway that incorporates an intermembranous protein into the inner membrane of the mitochondria.

Answer 14

It is incorporated into the inner membrane by the OXA-dependent pathway, by passing from the mito matrix, into the inter-membranous space until it’s “anchoring” portion stays in the inner membrane

Question 15

Describe the pathway that an intermembrane space protein is translocated through.

Answer 15

These proteins are anchored by their hydrophobic sequences via either the nuclear encoded pathway or the mito encoded pathway.

Then protease cleaves the protein from the hydrophobic sequence anchored in the inner membrane and the rest of the protein freely occupies the intermembrane space.

Question 16

provide the full name of Mia40 and it’s role in modifying proteins that have been translocated to the inner membrane space.

Answer 16

Mia40: mitochondrial intermembrane space assembly

Mia40 proteins oxidize some proteins as they are imported through the TOM. Mia40 adds disulfide bonds to the protein to create an intermediate. Mia40 reduced and then reoxidized by the Electron transport chain to prepare for another round of import.

Question 17

Describe what TIM22 is specialized for

Answer 17

the insertion of multipass inner membrane proteins into the inner membrane

Once through the TOM, mulitpass inner membrane proteins bind to inner membrane space chaperones that handoff to the TIM22 complex.

Question 18

What 2 components guide ER signal sequences to the ER membrane?

Answer 18

SRP (signal recognition particles):

SRP receptors

Question 19

Describe SRP’s

Answer 19

made of 6 different polypeptides bound to a single small RNA molecule

it is rod shaped with a large hydrophobic pocket lined by methionines (this is where the hydrophobic signal sequence go)

Question 20

Where are all transmembrane proteins produced?

Answer 20

the ER

this includes the transmembrane proteins for the ER itself

Question 21

Describe where SRP’s and SRP receptors are located

Answer 21

SRP’s are found in the cytosol “hugging” the ribosome that is synthesizing a protein

SRP receptors are within the membrane of the ER

Question 22

During cotranslation describe the interaction of SRP and the ribosome that is synthesizing the protein. How does this affect synthesis?

Answer 22

SRP wraps around the large ribosomal subunit and the binding of the SRP to the ER signal sequence on the protein transiently stops translation

SRP then binds to the SRP receptor in the ER membrane, and translation resumes

this allows the protein to be translated through a translocator and into the ER lumen

Question 23

Describe ER translocators in terms of their location and structure

Answer 23

found in the ER membrane

have a water filled pore that is gated by a short helix that may open and close the translocator as needed (plug fills closed translocators)

its core is made of Sec61 complex

Question 24

During the translocation of a soluble protein into the ER, how does this transmembrane movement begin, ensure specificity, and dispose of the ER start transfer signal sequence?

Answer 24

a start transfer signal on the protein interacts with a specific site on the core of the translocator to open it.

dual recognition ensures specificity (start transfer signal and interaction with the lipid core of the ER membrane)

Signal peptidase cleaves off the start transfer signal into the ER membrane (leaving just the mature soluble protein in the ER lumen)

Question 25

Define the role that start and stop transfer signal sequences play in determining the topology.

Answer 25

the N terminal start transfer signal begins translocation and then the stop/start signals after that determine the topology (location in the ER that the protein will end at) of the protein

Question 26

What region of a polypeptide stops the transfer process before the entire polypeptide is translocated, in intermembranous proteins?

Answer 26

the hydrophobic region

Question 27

______ helps remove _____ in order to stop the translocation process and integrate the protein into the ER bilayer.

Answer 27

Lateral Gating

Cleaved start-transfer peptide sequence

Question 28

Where are the 2 different types of Hsp70 located? what are their respective functions?

Answer 28

Cystosolic Hsp 70: binds to and stabilizes unfolded proteins prior to their translocation across the mitochondrial membrane (requires ATP to remove these from the protein so that it may be completely translocated)

Mitochondrial Hsp 70: Acts like a motor (using ATP) to pull the protein into the mitochondrial matrix (assists with translocation into the matrix)