Protein trafficking: ER and mito Flashcards
Describe the basic principles of protein sorting in the cell.
- information to target protein to a particular organelle is encoded in the amino acid sequence of the protein
- each organelle carries a set of receptors that bind to specific signal sequences (e.g. SRP receptor in ER)
- unidirectional transfer is often achieved through coupling transfer to an energetically favorable process such as ATP hydrolysis
Why would evolution stop without mito?
Glycolysis harnesses only 10% of the power of ATP
What does it tell us about the origin of mito that they are surrounded by two membranes?
That they came from invagination of bacteria
Matrix vs intermembrane space of mito
Matrix is at very center, intermembrane space is between the two membranes
Where are mito proteins synthesized?
Fully synthesized in cytoplasm before being translocated into mito
Describe signal sequence for proteins destined to the mito.
proteins destined for matrix space have Nterminal signal sequence:
- amphipathic alpha helix with hydrophobic residues clustered on the opposite side
- alternating positive, nonpolar, and hydrophobic residues to get hydrophobic residues all clustered on the same side in secondary structure
- the structure gets the protein translocated, not the primary sequence
List the principle protein translocators needed to import proteins into mito.
each is a multiprotien complex:
- TOM (translocator of the outer mito membrane)
- TIM23
- TIM22
- SAM
- OXA
What is the role of the TOM complex in transport of proteins to mito?
Used for translocation of all proteins encoded by the nucleus. Consists of receptor and translocation channel. It is on the outer mito membrane and recognizes the unfolded protein’s signal sequence structure. Transports proteins from the cytosol to the intermembrane space through translocation channel, as the Hsp70 chaperones that kept the protein unfolded are shed.
Describe the TIM23 complex.
Spans inner to outer membrane right near TOM. Further transports proteins into the matrix space after TOM moves them into intermembrane space (requires ATP). After signal sequence is cleaved in matrix by signal peptidase, the protein folds properly.
Also helps to insert some inner membrane proteins.
Describe the TIM22 complex.
Needed for the insertion of a different subset of inner membrane proteins
Describe the SAM complex.
Helps to fold proteins properly within the outer mito membrane
Describe the OXA complex.
involved in inserting proteins synthesized within the matrix into the inner membrane of the mito.
What experiments can we do to know mitochondrial membrane transport takes place?
- incubate labelled protein with mito and centrifuge. If the protein is transported into the mito, they will fractionate together in the centrifuge tube.
- Add protease to degrade signal sequence if it has not been imported into the mito. (removal of sequence can be monitored using gel – if removed, it will move further)
- If signal is not removed, we know it has been imported. Can confirm by adding detergent to disrupt membrane of mito and then add protease.
How can we use varying temperatures to study import of proteins into the mito?
- expose mito to proteins on ice (nothing will happen, too cold, or will occur slowly. Add protease, and might only find signal sequence inside mito, showing that it is imported N-term first)
- if you warm up the reaction and allow it to proceed, we will find the whole protein inside the mitochondria even after protease is found because the mito protects the protein from degradation
- can then add detergent to break down mito membrane and recover protein
What steps of mito protein import require energy?
- release of unfolded proteins in the cytosol from Hsp70 chaperones after binding to TOM complex requires ATP
- translocation of protein through TIM23 channel relies on proton gradient (higher in intermembrane space than matrix)
- mitochondrial Hsp70 helps pull unfolded protein into matrix space and requires ATP hydrolysis to be released from chaperone