Lecture 14 Flashcards
Where are most mitochondrial proteins encoded, where are they synthesized, and how do they reach their final destination?
encoded in nuclear DNA
synthesized on ribosomes
Transported into the mitochondria
(Some mito proteins are made in the mito, though)
What is the movement of proteins to their final destination and what directs this process?
translocation
signal sequences on the proteins
Describe the structure of signal sequences on proteins destined for the mitochondria
postively charged on one end and uncharged hydrophobic on the other end
this forms a amphiphilic alpha helix
what is the signifigance of amphiphilic alpha helixes on signal sequences?
receptor proteins recognize this configuration (not necessarily the specific sequence) and then multi-subunit protein complexes called protein translocators mediate the translocation
Describe the location and action of the following mitochondrial translocators
TOM
TIM
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.
What does SAM stand for, and what does it do?
Sorting and Assembly Machinery
it translocates and inserts/folds beta barrel proteins into the outer membrane of the mito.
Describe the function of OXA complexes when it comes to mitochondrial proteins.
they mediate insertion of proteins synthesized in mitochondria.
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?
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
What does peptidase do to proteins that are being translocated into the mitochondria?
it cleaves off signal sequences once the protein reaches it’s final destination.
this prevents any further translocation from occurring
Describe the energy sources used for the activities of TOM and TIM complexes
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.
explain how mitochondrial Hsp70 releases the precursor protein after it’s ratcheting action and describe how this happens and what role Hsp 60 plays.
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.
Describe the path of a protein that is inserted into the outer mito. membrane by the SAM complex.
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)
Describe the nuclear encoded pathway that incorporates an intrermembranous protein into the inner membrane of the mitochondria.
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
Describe the mito encoded pathway that incorporates an intermembranous protein into the inner membrane of the mitochondria.
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
Describe the pathway that an intermembrane space protein is translocated through.
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.
provide the full name of Mia40 and it’s role in modifying proteins that have been translocated to the inner membrane space.
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.
Describe what TIM22 is specialized for
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.
What 2 components guide ER signal sequences to the ER membrane?
SRP (signal recognition particles):
SRP receptors
Describe SRP’s
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)
Where are all transmembrane proteins produced?
the ER
this includes the transmembrane proteins for the ER itself
Describe where SRP’s and SRP receptors are located
SRP’s are found in the cytosol “hugging” the ribosome that is synthesizing a protein
SRP receptors are within the membrane of the ER
During cotranslation describe the interaction of SRP and the ribosome that is synthesizing the protein. How does this affect synthesis?
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
Describe ER translocators in terms of their location and structure
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
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?
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)
