How to Build Intracellular Organelles Flashcards
1
Q
What is the import targeting signal for the mitochondria?
A
N-terminal pre-sequence, amphipathic helical structure. No specific sequence, a variety that direct the protein to specific areas. All but simple outer membrane protein sequences are cleaved.
2
Q
What is the import machinery in the mitochondria?
A
TOM translocase and TIMs.
3
Q
What are the initial receptors?
A
TOM20 for those with an amphipathic signal and TOM70 for hydrophobic proteins (which have multiple internal signals or use chaperones).
4
Q
What forms the pore?
A
TOM40 and TOM5, 6 and 7.
5
Q
Where does TOM20/70 dock?
A
TOM22.
6
Q
What is the role of TOM5?
A
Assisting in the transfer of pre-protein to TOM40 pore, and the rector for some inter membrane space proteins.
7
Q
What is the role of TOM6?
A
Aids in the assembly of the complex (TOM22/40).
8
Q
What is the role of TOM7?
A
For translocase complex dissociation and dissembly.
9
Q
Describe TOM40?
A
Forms the import channel, an oligomeric complex with three very clear areas of low electron density (pretzel or monkey-face).
10
Q
Describe details of the pore formed?
A
Formed using TOM40, 22, 7 and 6, diameter is 2.1 nm.
11
Q
What does the active protein complex have?
A
Three TOM40, three TOM22 transmembrane domains all with a TOM5, 6 and 7.
12
Q
What is the driving force?
A
A set of sequential acidic and hydrophobic interactions.
13
Q
How does specificity arise?
A
From multiple low affinity interactions.
14
Q
What is the SAM complex required for?
A
The assembly of the TOM translocase by inserting the multi-membrane spanning TOM40 beta-barrel protein.
15
Q
Describe the SAM complex?
A
A beta-barrel protein, destined for the outer membrane.
16
Q
What methods are available to study import?
A
In vivo: use yeast mutants.
In vitro: use reconstituted assays.
17
Q
What does TIM stand for?
A
Translocon of the inner membrane.
18
Q
What are the 2 fates of proteins with a pre-sequence?
A
Complete translocation by TIM23 and matrix processing centre or becoming inner membrane proteins due to a stir transfer signal.
19
Q
What is the role of TIM23?
A
Essential for cell viability, forms a cation selected voltage gated channel in the membrane with TIM17.
20
Q
What is the role of TIM17?
A
Also essential for cell viability, forms the channel with TIM23 and used for regulating pore opening.
21
Q
What is the role of TIM22?
A
For proteins of the metabolise carrier family, proteins without a pre-sequence.
22
Q
What TIM extends a drain into the inter membrane same and binds pre-proteins and/or other translocase subunits?
A
TIM54.
23
Q
Which TIM may regulate assembly of the complex?
A
TIM18.
24
Q
What is TIM50 similar to and why?
A
TIM54, as it also has a domain that sticks out into the inter membrane space.
25
What is the role of TIM50?
Binding the pre sequence of proteins and TOM22 to aid in the closure of the channel in absence of a pre-sequence.
26
Which TIM linked the TOM and TIM complex?
TIM21.
27
What does PAM stand for?
Pre-sequence associated motor complex.
28
When and why does mtHSP70 bind?
Once the protein leaves the TIM23 channel TIM44 and mtHSP70 binds which then allows of rebinding to TIM44 and aids in hydrolysis of ATP to energies the transport into the matrix.
29
What is mtHSP70 nucleotide exchange factor?
Mgel.
30
What is PAM18?
A stimulator of ATPase activity of HSP70, binds to TIM17.
31
What controls the activity of PAM18?
PAM16.
32
What organises the PAM-TIM complex?
PAM17.
33
Describe the tiny TIMs?
8, 9, 10, 12, 13.
Similar in structure, contain a conserved twin CX3C motif (2 disulphide bonds folded between the cysteine of two motifs).
34
What do the tiny TIMs form?
A 70 kDa hexamer complex, consisting of 3 TIM9 and 3 TIM10 or 3 TIM8 and 3 TIM13.
35
What is the difference between TIM9/10 and TIM8/13?
Both jellyfish structure but one (?) has longer tentacles.
36
Describe what happened to the tiny TIMs?
They are imported into the inter membrane space via the TOM complex, Mia 4 and Erv 1 catalyse the folding of them by oxidising the disulphide on the tiny TIM and reducing the disulphiding on Erv 1.
37
What are the other possible import mechanisms in mitochondria?
C-terminal pre-seqeunce, Oxa pathway
38
How is TIM energised?
Combination of trapping model, pulling model and regulating channel opening.
39
What is delta si?
The charge difference across the membrane, essential for import of all proteins that cross or insert into the inner membrane.
40
Draw the TOM/TIM translocase system?
Look at pic.
41
How is cholorplast import similar to mitochondrial?
Also has an N-terminal targeting sequence, can be bipartite,
TOC and TIC (as with TOM and TIM), ATP is also required (as is GTP but mitochondria don't require this), proteins are translocated unfolded.
42
How can you study chloroplast import?
Use recombinant proteins mixed with isolated chloroplasts and GTP/ATP.
43
What are the energy requirements for chloroplasts and how do these differ from mitochondria?
ATP (in the dark) and GTP are required but no membrane potential. In mitochondria ATP and a membrane potential is required.
44
What is the cytosol-guidance complex?
May and Soll., 2000
Transit sequences of chloroplast-destined precursor proteins are phosphorylated on a serine or threonine residue. The amino acid motif around the phosphorylation site is related to the phosphopeptide binding motif for 14-3-3 proteins and a HSP70. 14-3-3 subunit recognises the phosphorylated transit peptide.
45
What does the cytosol-guidance complex do?
Docks at the Toc Complex. Precursor de-phosphorylation precedes import.
46
How important is the cytosol-guidance complex?
Not essential but highly stimulatory of most precursors in vivo. Importance in vivo in uncertain.
47
How can you identify components of the import machinery in chloroplasts?
Use translocation intermediates by either limiting ATP concentrations or use purified outer enveloped vesicles (as inner vesicles are missing, translocation cannot be completed).
48
What was Jarvis, P (2008) steps of protein import into chloroplasts?
1. Energy independent reversible binding
2. Insertion into the outer membrane (low ATP + GTP)
3. Translocation at high ATP.
49
How can you identify components of the translocation machinery in chloroplasts?
1. Cross-linking of the translocation intermediate to adjacent proteins followed by solubilisation and SDS PAGE.
2. Solubilisation of a protein complex under native conditions and isolation of proteins associated with the translocation intermediate.
50
What is the role of TOC159?
A major membrane-bound receptor for imported proteins, contains motifs that bind to ATP and GTP. N terminal domain is negatively charged (acidic) and is likely to be binding the positively charged transit peptide. Antibodies against block early intermediate formation.
51
What TOC has sequence homology to TOC159 and binds GTP?
TOC34.
52
Describe TOC75?
A 16 transmembrane beta-barrel (homologous to TOM40). Forms a voltage-gated cation selected channel which acts as a crosslink to import intermediates. Essential for cell viability.
53
Which TOC stabilises associations with the other TOCs and interacts with early import intermediates?
TOC64.
54
Which TOC is proposed to be the binding site for guidance complex?
TOC64.
55
What is the role of TIC110?
Possibly to recruit two stroll chaperones in order to drive translocation and folding of incoming proteins.
56
Which TIC is associated with precursor proteins and has an N-termianl protein anchor?
TIC 22.
57
What is the role of TIC20?
Cross-links to precursor proteins, hydrophobic and membrane embedded, could form a channel.
58
Who describes TIC22/20?
Chen et al., (2002)
59
Describe the model for import?
1. Precursor protein synthesised which interacts with HSP90 or guidance complex
2. Delivered to Toc machinery by Toc64 for HSP 70 interacting proteins or by Toc34 for guidance complex protein interacting proteins
3. Toc 75 and motor protein 159 and Toc 20 for non-photosynthetic
4. In space is the HSP 70 isoform thought to aid in transit
5. Delivered to Tic machinery (not sure what it is formed of 110 or 20, or combination), chaperones act as the motor protein to pull though the polypeptide
6. Processing peptidase removes the transit peptide on the stroll side.
60
How is import into the chloroplast potentially regaled?
By redox; photosynthesis produces reducing power which you would expect to feedback to monitor how well photosynthesis is going.
61
How is import into the outer membrane of chloroplasts different to the inner membrane?
AKR2, targets signal and chaperones two outer membrane proteins OEP7 and 64 (involved in Toc machinery). Requires Toc 75 but not 159/34. Probably more than one mechanism.
62
How specific are chloroplast transit peptides?
Generally quite specific, although examples of proteins that can be targeted by both chloroplasts and mitochondria.
63
How does plant TOM22 differ from mammalian or fungal TOM22?
Plant lacks the acidic pre-sequence binding domain and is much shorter.
64
How does plant TOM20 differ from mammalian or fungal TOM20?
Plant Tom20 is also more divergent in sequence and is anchored to the OM at the C rather than the N terminus.
65
Describe thylakoid target signals?
Thylakoids have a bipartite target signal (as in chloroplasts and mitochondria). A stromal transit peptide and a thylakoid transit peptide. Similar to the bacterial peptide sequence which is used in mitochondria.
66
How can you study protein import into thylakoids?
Isolate chloroplast and take a precursor protein either recombinant or fluorescent. Allow for it to be passed through TOC/TIC into the stoma and then it is processed by the stromal processing peptide and the stromal intermediate made can take multiple pathways to the thylakoid membrane.
67
What did studying the energetics of import into isolated chloroplasts show?
Proteins fall into two groups, one requiring the protein motive force and one not.
1. Import of OE23/17 was severely inhibited my ionophores like Nigericin and Valinomycine that dissipate the pmf. They accumulated as stromal intermediates and could not cross the thylakoid membrane.
2. Plastocyanin and LHCP were not effected by the ionophores.
68
Name the pathways of import?
ATP dependent OE23 and plastocyanin, PMF dependent OE23 and 17, GTP dependent light harvesting complex protein (LHCP), spontaneous Cfo2.
69
Give the steps of the sec pathway of protein export in bacteria?
1. Synthesis of pre-protein in the cytosol and chaperones bind (eg. Secb)
2. Then past to secA an ATPase
3. SecA allows passing through the membrane via a proteinacious channel
4. A PMF also stimulates transport.
70
What is TAT pathway also known as?
pH. (Twin arginine translocase).
71
What do proteins that use the TAT pathway have?
A twin arginine preceding the hydrophobic core of the transit peptide.
72
What in E.coli has similarities to the TAT pathway?
In E.coli there is a pathway which also has arginines that are used for protein export to the periplasm which bind cofactors in the chloroplast.
73
How were components of TAT identified?
By genetics: Hcf 106 was identified in maze.
A mutant in the gene was defective in assembly of the photosynthetic apparatus
• E. coli mutant’s defective in export of tat pathway precursors led to identification of Tat A, B, C and E
• Hcf106= TatB
• Tha4= TatA/E
Palmer and Berks,. (2012)
74
Describe the proposed mechanism of the TAT translocase?
1. Substrate with TA motif binds signal peptide
2. The TatBC binds
3. TatA polymerises
4. The passenger domain croses the membrane via TatA
5. Signal peptide is cleaved and TatA is depolymerised
75
What type of pathway do LHCP and other integral membrane proteins have to use for insertion into the thylakoid membrane?
A prokaryotic pathway.
76
What do LHCP have to do to be competent for insertion?
Form a transit complex, containing chloroplasts SRP, GTP and a stroll factor are also required.
77
What type of signal sequence does LHCP have?
Internal
78
What does spontaneous insertion mean?
Without any other proteins, ATP/GTP or membrane potential.
79
Do any proteins insert spontaneously?
- For many years it was believed that some small bacterial proteins like phage coat proteins could insert ‘spontaneously’
- However, it is now known that these proteins all require an E. coli membrane protein yidC for insertion
- For some small integral envelope and thylakoid membrane proteins like CFoII and OEP14 no requirement for proteins or an energy source could be detected.
- There is a yidC homologue in chloroplasts called Alb3 involved in the integration of LHCP but it seems not to be involved in insertion of the ‘spontaneous’ pathway proteins.
- Maybe in chloroplasts there are some that can insert without the need for other proteins.
80
How are peroxisomes structurally different to mitochondria and chloroplasts?
They are bound by a single membrane and they have no DNA (instead they have nuclear encoded proteins).
81
How can you screen for mutant peroxisomes?
Look for those that grow only on glucose, and not on oleic acid.
82
How can you visualise peroxisomes?
Fuse YFP to peroxisome membrane or GFP to metrix protein.
83
Describe the peroxisome targeting sequence?
PTS-1: at the C-terminus, an essential tripeptide, with a small neutral side chain (eg. serine), a basic (eg. lysine) and a hydrophobic (eg. methionine). - PTS-2: toward the N-terminus and have a more complex sequence (a basic then an aa then a space of 5 then an aa).
84
What recognises PTS-1 and 2?
PEX5 and PEX7.
85
What PEX are required for recycling?
1, 4 and 6.
86
What PEX are RING proteins which can act as E3 ubiquitin ligase in recycling?
2, 10 and 12.
87
Which PEX form the channel?
5 and 14.
88
What are PEX13 and 14 for?
Docking.
89
How is peroxisomal protein import energised?
- ATP is needed for ubiquitination by AAA ATPase’s which remove PEX5 from the membrane
- Recycling is necessary for import
- Import is presumably driven by favourable binding energies
- Because peroxisomes import folded proteins peroxisome specific chaperones are not invoked although general chaperones presumably play a role in folding as they do for cytosolic proteins.
- While there are some reports of chaperones associated with peroxisomes their roles are uncertain.
90
What are the types of peroxisome membrane proteins?
Single and multi span.
91
What is PEX3?
A membrane receptor for PEX19
| - Integral peroxisome membrane protein with a cytoslically orientated domain, PEX19 binds in groove.
92
What is PEX19?
- A chaperone/receptor for PMPs
- Predominantly cytosolic
- Binds multiple peroxisomal protein at its C terminal domain
- Binds newly synthesis peroxisomal protein preferentially
- In its absence PMP are degraded or mistargeted
- Binds to sequences that frequently (although not allows) overlap with the membrane protein targeting signal
- Binds PEX3 with its N terminal domain
- Can for ternary complex with bot PEX 3 and PMP
- Globular c terminal domain that binds PMPs
- Flexible N-terminal domain binds PEX3
- Possible c terminal farnesylation for membrane insertion
- C-terminal domain is a globular structure with conserved regions across species. Can bind with um infinity.
93
Which PEX is a peroxisomal membrane protein, clearly implicated in PMP import in mammals?
PEX16.
94
Give a summary of PMP targeting signals?
- Not a simple sequence
- Most compromise of a PEX19 binding sequence and TM domain
- PEX19 recognisees 11mer helical peptides with invariant basic residues and flanked by hydrophobic amino acids e.g. KVLRLLQYLARFLAV
- Some PMPs contain multiple co-operative sequences
- Integral to the protein. Not cleaved.
95
What regulates peroxisome division?
PEX11.
96
What are the three models as to where peroxisomes come from?
1. Derivation from the ER
- Apparent connections in EM- looked joined together
- Cells fractionation studies- ER fractions sometimes contained some peroxisomal enzymes (may have just been contaminated fractions)
2. Autonomous organelles
- Peroxisomes have distinct targeting signals and import machinery
- Most protein are imported post transnationally
- KO of Sec61 has no effect
- Clear evidence for peroxisome division in many species
3. A hybrid model
- Persistent reports of some PMPS in the ER
- Mutants that lack detectable peroxisomes can regain them when the missing gene product is supplied
- Class 2 PMP targeting signals don’t bind PEX19.
97
How can you look at recovery of peroxisomes?
By real-time microscopy.
98
What are the three groups of human diseases associated with peroxisomes?
Group A: mutations in all other PEX genes.
Group B: Mutations in PEX7.
Group C: Mutations in individual peroxisome enzymes.
99
Give a disease in group A?
Zellweger syndrome (ZS): severe neurological and hepatic dysfunction, cranofacial abnormalities, hypotonia and early death.
100
Give a disease in group B?
Rhizomelic Chrondrodysplasia Punctata (RCDP): Growth defects, rhizomelia, cataracts, epiphyseal calcifications and ichthyosis.
101
Give a disease in group C?
X-linked adrenoleukodystrophy: A collection of generally milder (although still severe) disorders.
102
In plants, equivalent mutations in peroxisomes, result in what?
Failure of seeds to germinate unless an alternative carbohydrate source is supplied reduced vegetative growth.
In some cases, embryogenesis. This is because peroxisomes are required for multiple metabolic pathways required a various stages of the plants life cycle.
