Topic 4 - Protein Sorting Flashcards
1
Q
What is the SRP and what is its functions?
A
- the signal recognition particle
- will sneak into the ribosome tunnel, scanning the substrate (protein) translation
- if it recognizes the correct protein it will bind to it and position itself at the entrance
- it forms a stable complex with the protein before transporting it to its target site or the ER membraine
2
Q
Explain in short the evolution of organelles.
A
An aerobic bacterium was engulfed and lived symbiotically as a protomitochondrion
- basically a prokaryote with its surrounding membrane
- membrane formed around the host cells DNA (nucleus)
3
Q
What is one key feature of mitochondria?
A
They lack any vesicle budding (compared to the Golgi Apparatus)
4
Q
What are the 4 distinct intracellular compartment families?
A
- Nucleus and cytosol
- any organelles (this consists of the secretory and endocytic pathway - ER, golgi, lysosomes)
- mitochondria
- plastids (chloroplast)
5
Q
What are the three distinct protein transport ways?
A
- Gated transport (cytosol and nucleus - nuclear pore complex)
- Transmembrane transport
- Vesicular transport (ER to the golgi)
6
Q
What does monoterpene and sesquiterpene synthases refer to? - the end terminus signal sequences?
A
- refers the protein signal sequence at the end of the expressed sequence which determines where the protein will end up in the cell
- if there is no protein signal sequence, protein is bound to the cytosol
- the presence of an end terminus signal means the protein is bound for a specific organelle
7
Q
What sort of protein transport is used between the cytosol and nucleus
A
- gated transport
8
Q
How do nuclear pores work, and what is transported?
A
- they function bidirectionally and are highly selective
- mRNA transported out, proteins transported in
9
Q
What is an NPC, and what are the key features?
A
Nuclear Pore Complex
- tethered to the nuclear envelope & regulates membrane curvature
- channel nucleoporins creating a large unstructured regions for a messy tangle - preventing macromolecules
10
Q
NPC and diffusion?
A
- small molecules may enter the nucleus via diffusion
- while large molecules will require an active transporter through nuclear pore receptors (DNA and RNA polymerases)
11
Q
How do nuclear localization signals work?
A
- a cargo protein has a nuclear localization signal (NLS) which will bind the nuclear import receptor
12
Q
What does the cargo protein require besides the nuclear localization signal?
A
- it requires positively charged AA to bind the nuclear import receptor
- an adapter protein may be required as well
13
Q
Does it matter if you only change ONE amino acid in the nuclear localization signal?
A
YES - there may be deleterious effects to the cell, that affects functionality
14
Q
How do you regulate monomeric GTPases?
A
- an active monomeric GTPase will be bound to GTP (the GEF will remove the GDP on the inactive monomeric GTPase and add a GTP activating it)
- an INactive monomeric GTPase will be bound to GDP (the active monomeric GTPase with GTP bound will be removed and replaced with a GDP to inactivate the monomeric GTPase by GAP)
15
Q
What is a monomeric GTPase?
A
- an enzyme that undergoes conformational change when either bound to GDP or GTP to be inactive or active respectively
- functions like a light switch
- GEF activates by binding GTP
- GAP inactivates by binding GDP
16
Q
What monomeric GTPase is involved with the the transport across the nuclear pore?
A
Ran-GTPase
17
Q
How is the nuclear pore complex involved in regulation? (2 ways)
A
- it controls transport by controlling the access of transport machinery
1) keeps gene regulatory proteins in cytosol until needed
2) turns export/import signals on/off through phosphorylation
18
Q
Give an example on how the NPC is involved in gene regulation
A
- in high [Ca2+] in a cell: a regulatory protein is phosphorylated but a protein regulator phosphatase, removes the phosphates from the regulatory protein, then calcineurin binds the nuclear export signal allowing the nuclear import signal to activate and allow the gene regulatory protein to enter the nucleus and activate gene transcription
- compared to low [Ca2+] in the cell the regulatory protein is phosphorylated and the nuclear export signal is activated removing it from the nucleus
19
Q
Explain how SCAP and SREBP are involved in cholesterol feedback regulation and the NPC?
A
- SREBP: sterol response element binding protein
- SCAP: SREBP cleavage activation protein
- these two are bound on the ER membrane, high cholesterol these two will be inactive, low cholesterol will cause these to proteins to form a vesicle bound for the golgi where two proteases cleave the sterol response element which can be transported through the NPC to direct the biosynthesis production of sterol enzymes
20
Q
What effect does Ran have one getting cargo proteins through the NPC?
A
- cargos can be transported through the NPC, however with much greater effect with the monomeric Ran-GTPase
21
Q
What is going on with NPC selectivity?
A
- cargos may be rejected and returned to the cytoplasm through an accumulation of multiple reverse sub-steps and a final irreversible exit step of cargo proteins not bound to Ran or that are too large
22
Q
Where does energy harvesting electron transport chain occur in mitochondria and chloroplasts?
A
- cristae
- thylakoid space/membrane
23
Q
What are some interesting points about mitochondria and chloroplasts?
A
- new formation of each occurs through growth and fission (they can replicate themselves)
- the proteins they require are encoded in the nucleus, synthesized in the cytosol and transported into the mitochondria or chloroplasts for its intended purpose via protein translocation
24
Q
Explain translocation into the mitochondria
A
- mitochondrial precursors are a) fully synthesized in the cytosol
b) post-translational translocation to mitochondria - thus a signal sequence for the import and localization to the specific mitochondrial compartment is required
25
What does the TIM22, OXA, and SAM complexes have in common?
- each of these channel complexes will embed the proteins bound for the mitochondria in the: inner membrane, inner membrane, outer membrane respectively
- each of these proteins will utilize a second protein localization signal sequence after the first protein localization sequence is cleaved off
26
In which membrane are TOM and TIM23 Complexes embedded in? what do they have in common?
- TOM is embedded in the outer mitochondrial membrane with a receptor domain and a channel domain
- TIM23 is embedded in the inner mitochondrial membrane which aligns itself with TOM and also has a channel domain but instead has an anchor domain
27
What drives the active transport of the translocation of proteins into the mitochondria matrix?
1) ATP hydrolysis - at each of the channel proteins (especially the TOM complex and TIM23 Complex)
2) The proton motif force between the inter membrane space through the inner membrane to the matrix (inter membrane space is typically positively charged due high [H+] - thus having a positively charged N-terminus signal to pull the protein to the negative matrix side - thanks to the generation of the electrochemical proton gradient)
28
What sort of protein is typically transported in mitochondria
unfolded proteins, where HS70 chaperone proteins are required to fold the mature proteins after the N-terminus signal sequence is cleaved
29
Compare the insertion of transmembrane proteins between mitochondria and bacteria
- mitochondria can utilize the TOM complex to transport the protein to the inter membrane space and then the SAM complex to insertion of beta-barrels into the outer membrane OR use a TIM22 complex to insert a transmembrane protein in the inner membrane (key is the signal peptide)
- a bacterium will use a Sec transporter and periplasmic chaperones before using the BAM complext to insert the PORIN into the outer bacterial membrane
30
What is the function of Mia
- mitochondrial inter membrane space assembly
- through the reduction of Mia40 it will cause the oxidation of the imported protein and formation sulphide bridges and sulfhydryl bonds
31
Compare the translocation into mitochondria and chloroplasts
- the translocator complexes used are in order: TOC complex and TIC complex
- the thylakoid precursor protein has two signals: the chloroplast signal sequence and the thylakoid signal sequence
- the transport into the stroma is GTP-dependent and where the cleavage of the first signal occurs in the stroma
- now there are 4 routes possible for the translocation into they thylakoid space
32
What is the function of the endoplasmic reticulum?
- lipid synthesis
- protein biosynthesis of transmembrane proteins
- site for intracellular Ca2+ storage
33
What is the SRTP and the SRP receptor?
- signal recognition particles
- this particle will assess the assess the hydrophobic signal sequence on a growing peptide in a ribosome
- if there is a match the particle will bind this sequence and wait for the protein to complete synthesis before transporting it to its to the ER membrane via an SRP receptor
34
What happens after the SRP binds the signal peptide and the SRP receptor?
a protein translocator will begins translocation of the still translating protein either:
a) as a water soluble protein in the ER lumen
b) embedded in the ER membrane due to transfer sequences
35
The ER signal sequence on the newly translated protein will have completed these two steps?
- interaction with the SRP
| - protein translocator
36
The ER translocator is gated in two directions?
- will function either as a pore for hydrophilic protein portions
- OR opens laterally for hydrophobic portions
37
What do ER resident proteins have?
ER retention signal
38
How would you observe the translocation domains of a transmembrane domain?
- a hydrophobicity plot will be able to identify the hydrophobic and hydrophilic domains
39
How do form a SNARE protein?
an active ATP bound Get3 ATPase will bind the signal recognition particle at the END of the protein, the hydrolysis of the ATP causes the protein tail to be anchored in the membrane
40
What is the role of glycosylation?
- physical properties include: folding, trafficking, packing, stabilisation, protease protection, quaternary structure and organization of water structure
