How to build organelles Flashcards
Do the mitochondria encode their own proteins?
Yes however only a handful of the ~300 proteins found in the mitochondria
How can we study mitochondrial import?
Many of the original experiments were done in yeast as it is a good model EUK system. It lives happily on glucose - ferments and does not need the mitochondria to do this. However to grow on ethanol/glycerol you need mitochondria function.
Discovered mutations which would only grow on glucose and not ethanol/glycerol and these mutants had impaired mitochondria - either the import of the proteins or the enzymes themselves.
Can test by knocking something out and then reintroducing it on a plasmid
Describe the matrix targeting signals (MITO)
Typically N-terminal
Amphipathic helix - charged on one side and hydrophobic on the other
From comparing the cDNAs - not a specific sequence
Discovered in 1980s when cloning and sequencing DNA - found extra sequences on the cDNA which were not present in the protein
Can take it off and put it one different proteins e.g. GFP and visualize where it goes in the cell
Targeting sequences can also have a transmembrane domain sequence if the destination is IM/OM
Internal transmembrane domains with no N-terminal seq are for member of the metabolite carrier family for the IM
How did they test the theory of matrix targeting signals? (MITO)
Created a synthetic targeting sequence made of only 3 aa only - tested with a yeast mutant which lacked a subunit of cytochrome oxidase so could only grow on glucose not ethanol/glycerol. Tested different sequence combinations of the targeting sequence which showed the primary sequence was not important rather if it could form an amphipathic helix
What chemical can be used to stop unfolding of DHFR and how can this be useful? (MITO)
MTX - anticancer drug which binds tightly to DHFR and prevents unfolding. It can start to be imported however it cans stuck and this allows cross-linking to see what machinery it uses for import
How was TOM 70 identified?
By manipulating ATP and membrane potential in vitro. AAC accumulated on the surface and was cross-linked to TOM 70
How was GIP identified?
Adding a tag to one component allow the pull-down of interacting partners
Draw the TOM complex
See diagram
TOM 20, 22, 6, 40, 7, 5 and 70
How to proteins cross the outer mitochondria membrane?
TOM complex
What are the components of the general import pore (GIP)
TOM 5, 6, 7, 22, 40
Which TOM forms the central pore in the TOM complex?
TOM 40
What is the initial receptor in the import of N-terminal amphipathic helix targeting signals? (MITO)
TOM 20
Describe TOM 20
Initial receptor for amp. helix signals
Have solved the XRC structure of TOM 20 soluble domain in complex ith amp. targeting signal - showed the cytosolic domain provides a groove lined by hydrophobic residues which interacts with the hydrophobic side of the amp. helix (hydrophobic interactions) and the positive side of the amp. helix extends out into the soluble solution phase - shows why a specific sequence is not necessary
Describe TOM 70
Other initial receptor for internal TM targeting signals e.g. metabolite carrier family which have internal hydrophobic repeats.
TOM 70 is abundant in the membrane.
Also receptor for proteins using MSF chaperone and cytosolic hsp70 - Precursor-MSF complex binds TOM 70 and ATP hydrolysis occurs which releases MSF and get the transfer of precursor to TOM 20/22.
Binds multiple copies of TOM 70
Describe the GIP and what each protein does
GIP is made up of TOM 5, 6, 7, 22 and 40
- TOM 22 - receives the proteins recognized by TOM 20/70 and acts as the organizer of the GIP (involved in the assembly of the whole complex). It spans the membrane and has a large cytosolic domain which interacts with charged pre-sequences. Has a IMS domain where it provides a binding site for the pre-sequence as has a negative charged domain which interacts with the positive part of the helix.
- TOM 5 - acessory receptor which helps in the trasnfer of proteins into the TOM 40 pore and receptor for some IMS such as the tiny TIMs
- TOM 6 -assembly of TOM 22 with TOM 40
- TOM 7 - translocase disassembly
- TOM 40 - import chanell - oligomeric complex
How was TOM 40 visualized?
Single particle EM His-tagged one component and it pulled the whole complex through TOM 22 sits between 2x TOM 40 Salt bridges may help interactions Did cross-linking of the TOM complex See diagram
Explain the binding site or acid chain hypothesis (MITO)
The TOM proteins and some TIM proteins provide a sequential set of acidic and hydrophobic binding sites for the positively charged amp. helix pre-sequence
Allows both sides of the helix to interact in an energetically favorable way
Specificity arises from multiple low-affinity interactions (don’t want too high affinity as needs to be transient to allow movement along the chain)
Describe the insertion of beta-barrel proteins in the OM of mitochondria
OM proteins which carry an amp. helix followed by a stop-transfer enter through the TOM complex but recognized and segregated into the OM using MIM 1/2.
Beta-barrel proteins also enter via the TOM complex but when they enter and come through the other side they are bound by small TIMs (chaperones)
The SAM machinery inserts them - in yeast it is made up of 4x components ( mdm 10, SAM 50, SAM 37 and SAM 35)
See diagram
Is the machinery for inserting beta-barrels homologous to the machinery in bacteria OM? (MITO)
Yes - mitochondria are descents of free-living PRO cells and were engulfed by a EUK cell and over evoution the genes have moved into the nucleus.
SAM 50 is homologous to Omp85 and both have POTRA domains
Tiny TIM chaperones are homologous to Skp/SurA
What is SAM 50 homologous too in bacteria? (MITO)
Omp85 - both have POTRA domains and insert OM beta-barrels
What are the tiny TIMs homologous to in bacteria?
Skp/SurA
How can we study mitochondria import in vivo?
Use yeast mutants which can only grow on glucose no ethanol/glycerol - resp defective
Easy to transform gene in yeast so can look for the ones which grow again - lots of components were identified this way
Some protein knock outs are lethal and therefore they couldn’t be identified this way e.g. TOM 40 therefore had to use thermosensitive mutants which are unable to grow at high temperatures - lots of missense mutants don’t kill the cell but make it less stable
Describe mitochondrial reconstituted import assays
In vitro
Easy to isolate mitochondria by differential centrifugation
Use labeled translated proteins and mix with isolated mitochondria with energy and ATP. Split into two and add protease to one. Reisolate and carry out SDS-PAGE/Western blot/Blue-native PAGE for complexes. Can see where the precursor and mature proteins are.
Can manipulate the conditions e.g. collapse membrane potential (no longer see import), study the import of TOM 40 as it needs SAM so K.O. SAM and see the import of TOM 40 is blocked.
See diagram.
All proteins use TOM complex to enter the mitochondria - True or False?
True
All proteins use the same TIM machinery - True or false?
False
Which proteins use TIM 22 complex?
Proteins withOUT a pre-sequence e.g. MCF
Which proteins use TIM 23 complex?
Protein with a pre-sequence e.g. matrix proteins. Translocated via TIM23 and processed by matrix processing protease (MPP)
Which proteins use TIM23-SORT?
Proteins which carry a matrix targeting signal followed by a stop-transfer signal and they get integrated by a variant of TIM23 called TIM23-SORT
What are the components of the TIM 23 complex?
TIM 17/23 - 2x central components, essential for cell viability (need to use temperature sensitive mutants), cation selective voltage-gated channel which responds to the pre-sequence. TIM 17 regulates the opening of the TIM 23 pore.
TIM 50 - Binds the pre-sequence of protein emerging from TOM complex. Closes the TIM 23 channel in the absence of the pre-protein
TIM 21 - links TOM and TIM complexes via the interaction with TOM 22 IMS domain. Disassociates TIM 23 from PAM complex. Role of intermediate in contacting interacting partners of TIM 23. Helps guide protein into the TIM complex.
Gating of the channel is important for maintaining the membrane potential of the IM
Which protein regulates the opening of the TIM 23 pore?
TIM 17
Which protein closes the TIM 23 channel?
TIM 50
Which TOM does TIM 21 contact?
TOM 22 IMS domain