Compartments and Protein Sorting (ch 12) Flashcards
At what stage of protein translation does the signal recognition particle (SRP) recognize the emerging protein?
The SRP goes partially into the ribosome tunnel to scan the protein before it even emerges.
What are the 4 distinct intracellular compartment families?
- Nucleus + cytosol
- All secretory+endocytic organelles
- Mitochondria
- Plastids
What are the 3 distinct protein transport pathways?
- Gated Transport (cytosol>nucleus)
- Transmembrane transport (>ER)
- Vesicular transport (secretory transport)
What is the purpose of a protein signal sequence?
An “address” for proteins. Can be hydrophilic/phobic, polar/non-polar, -ve/+ve charge. Just something recognizable.
In folded protein, signal sequence is on ______. In unfolded protein, signal sequence is on ______.
Folded: protein surface
Unfolded: N-terminus
Does most protein transport occur while the protein is folded or unfolded?
Unfolded.
What end of the protein enters the ER initially? What about if the protein has to re-enter the ER after leaving?
Enter initially: N-terminus
Re-enter: C-terminus
Where would you expect to find shorter proteins? What about longer ones?
Shorter: in cytosol, lost signal sequence
Longer: in transit to other locations
What is transported into the nucleus through the nuclear pore complexes?
Only mRNA and transcription factors.
At what rate are molecules transported into and out of the nucleus?
~500 molecules per second!
How does the nuclear membrane bend around the nuclear pore complexes?
With the help of membrane ring proteins and nuclear basket proteins which distort the membrane.
How many different proteins make up a nuclear pore complex?
~30 different proteins.
How are large molecules excluded from the nucleus by the nuclear pore complexes?
Because of the channel nucleoporins which project into the opening and filter away large molecules but allow small ones to pass.
What is the function of nuclear import receptors?
To enable the movement of cargo proteins into the nucleus when directly bound to the receptor or indirectly through an adaptor protein.
Does changing one amino acid on a nuclear localization signal affect the import of a protein?
It can. Sometimes resulting in aggregation and no transport.
How do nuclear import receptors carry cargo through the nuclear pore complex?
By attaching to FG repeats on nucleoporins and “hopping” through the channel.
How is a monomeric GTPase activated? By what?
Guanine exchange factor (GEF) replaces GDP on inactive GTPase with GTP to activate.
How is a monomeric GTPase inactivated? By what?
GTPase activating protein (GAP) removes a phosphate from GTPase-bound GTP, converting it to GDP and inactivating the GTPase.
What is the name for the monomeric GTPase involved in transport across the nuclear pore?
Ran-GTP and Ran-GDP depending on localization and activation status.
What mechanism imposes transport directionality through the nuclear pore?
The activation/inactivation of Ran-GDP/GTP.
How is Ran-GDP activated as it enters the nucleus? By what?
Ran-GEF replaces Ran-bound GDP with GTP, activating Ran.
How is Ran-GTP deactivated as it exits the nucleus? By what?
Ran-GAP converts Ran-bound GTP to GDP, deactivating Ran.
How does Ran-GTP assist nuclear import receptors to transport proteins into the nucleus?
Ran-GTP replaces cargo protein by binding to nuclear import protein, releasing the cargo into the nucleus. The import protein with bound Ran-GTP then exits the nucleus.
How does Ran-GTP assist nuclear export receptors to transport proteins out of the nucleus?
Binds to export receptor in nucleus, causing it to pick up the cargo protein and take it out of the nucleus. Ran-GDP and cargo both release once in the cytosol.
How does mRNA export differ from Ran-driven protein export through the nuclear pores?
mRNA export is driven by ATP rather than Ran-GTP.
How is transport through the nuclear pore complex regulated?
By limiting access to transport machinery and turning import/export signals on/off via phosphorylation.
What is SREBP? What about SCAP?
SREBP: sterol response element binding protein
SCAP: SREBP cleavage activating protein
What 2 proteins are involved in feedback regulation of cholesterol synthesis via the nuclear pore complex?
- SREBP
2. SCAP
Do cargo proteins always just pass smoothly through the nuclear pore complex?
No. Many cargo proteins circulate in the pore for a while before passing through or even being rejected.
Is Ran absolutely required for transport through the nuclear pore complex? What effect does it have?
No. But it makes import much more effective (50% vs 0.01%).
Are proteins transported into the nucleus folded or unfolded? Where would the nuclear localization signal be present?
Proteins are transported in their folded conformation with the nuclear localization signal exposed.
What 2 spaces exist in the mitochondria?
- Intermembrane space
2. Matrix space
What 3 spaces exist in the chloroplast?
- Intermembrane space
- Stroma (matrix space)
- Thylakoid space
Can mitochondria be generated by the cell? How are they replicated?
No. They must arise from mitochondrial DNA contained within existing mitochondria.
How many types of proteins are provided to a new mitochondria by the nucleus? How many are provided by another mitochondria?
~1000 from the nucleus, 13 from mitochondrial DNA.
Where are mitochondrial precursor proteins synthesized? When are they translocated into the mitochondria?
Fully synthesized in the cytosol. Translocated into the mitochondria post-translationally.
What is an example of a signal sequence for import and localization to a mitochondrial subcompartment?
Cytochrome oxidase subunit IV.
What is the function of a TOM complex in the mitochondria membrane?
Translocase on outer mitochondrial membrane, identifies linear proteins.
What is the function of a SAM complex in the mitochondria membrane?
Sorting and assembly machine, folds linear proteins imported by TOM into β-barrels.
What is the function of a TIM22 complex in the mitochondria membrane?
Translocase on inner mitochondrial membrane, inserts proteins into inner membrane.
What is the function of a TIM23 complex in the mitochondria membrane?
Translocase on inner mitochondrial membrane, moves proteins into matrix space.
What is the function of an OXA complex in the mitochondria membrane?
Cytochrome oxidase activity, inserts proteins into inner membrane.
In what conformation are proteins transported into the mitochondria? What assists this?
Unfolded and bound to chaperones to prevent folding.
What mechanism drives protein import into the mitochondria?
Active transport from ATP hydrolysis or membrane potential.
What other mitochondrial process assists the import of protons by creating an electrochemical gradient?
Oxidative phosphorylation (produces ATP).
Do all eukaryotes have mitochondria?
Nope!
In bacteria, the SAM complex in the mitochondria is replaced by the ___ complex on the outer bacterial membrane.
BAM complex.
What must occur following import before a mitochondrial protein can be activated?
Cleavage of the signal sequence.
What is the purpose of a “stop-transfer” sequence in a mitochondrial protein?
Allows protein to stop partway through the inner (or outer) membrane because of hydrophobic interactions.
How does protein translocation into chloroplast differ from translocation into mitochondria?
TOM complex replaced by TOC complex, TIM complex replaced by TIC complex.
What is Mia40? What is its role in the mitochondria?
Mitochondrial intermembrane space assemby. Folds proteins once imported into the intermembrane space.
What are the 3 main functions of the endoplasmic reticulum?
- Lipid biosynthesis
- Protein biosynthesis of transmembrane proteins
- Ca2+ storage
How can the ER be isolated? What else occurs during this process?
Centrifugation will break up the ER into rough and smooth microsomes which can be separated by density in a tube with a sucrose gradient.
What 2 main ways can proteins be translocated?
- Co-translationally
2. Post-translationally
How does translocation of proteins occur in the ER? Why?
Co-translationally. Proteins are not fully made in the cytosol and must be finished in rough ER ribosomes.
Why do proteins being translocated into the ER not need chaperone proteins?
Folding is not possible because translocation is done co-translationally and identification occurs before the protein is exposed out of the ribosome channel.
Describe the structure of a signal recognition particle in the ER.
6 proteins connected by an SRP RNA molecule.
What 4 steps outline the function of a signal recognition particle in the ER?
- Recognition (of new protein + binding)
- Targeting (of membrane protein translocator)
- Release (of ribosome onto protein translocator)
- Recycling (of SRP to next ribosome)
What 2 ER proteins can recognize an ER signal sequence?
- Signal recognition particle
2. Protein translocator
In what 2 ways can a protein translocator in the ER open?
- Pore for hydrophilic portions
2. Opens laterally for hydrophobic portions
On a hydrophobicity plot, how can we tell the number of transmembrane domains in a transmembrane protein?
By counting the number of peaks.
Are proteins targeted for translocation always recognized by their N-terminus sequence?
No. Some proteins are recognized by their C-terminus, in which case the normal membrane localization is reversed and the C-terminus can be anchored.
What 2 events constitute the “dual recognition” of proteins by the ER?
- SRP recognizes protein in the cytosol
2. Protein translocator recognizes protein during binding
Why is “dual recognition” of proteins by the ER important?
It ensures that inappropriate proteins do not enter the ER lumen.
Where in the cell are signal recognition particles (SRPs) mainly found?
In and around the rough endoplasmic reticulum (ER).
If a protein is engineered to have two different localization signals, which determines its destination?
The one closest to the N-terminus or which will be read first.
Where is a protein sent if it has no localization signal or ER retention signal?
To the cytosol, where it will eventually be picked up and degraded.
What are some examples which support the theory that mitochondria and chloroplasts are engulfed prokaryotes?
- Neither can be made from scratch by the cell
- Distinct transmembrane transport methods
- Have their own ribosomes
- Have their own DNA
Transmembrane proteins have start-transfer and stop-transfer sequences. In a recombinant experiment, you change the order of those sequences. What happens to the integration of that recombinant protein into the membrane?
It integrates normally in the same way as the wild-type protein.
Excitatory neurotransmitters ___ channels which results in a ___ charge on the cytosolic side of the cell. Inhibitory neurotransmitters ___ channels which results in a ___ charge on the cytosolic side of the cell.
Opens sodium. Positive. Opens potassium of chlorine. Negative.
In what case are proteins transported from the ER to the cytosol via the retrotranslocation pathway?
When a protein has failed to fold properly in the ER it is moved to the cytosol and degraded via a proteosome.
