Biogenesis of Mitochondria and Chloroplasts II Flashcards
1
Q
Describe the basics of chloroplastic protein import
A
- “transit peptide”
- PT
- unfolded conformation; similar chaperones invoked
- 2x membranes crossed: outer and inner “envelope”
- mediated by translocons: TOC, TIC
- simultaneous 2 “contact sites”
- ATP and GTP-dependent but proton gradient-independent
- SPP (related to MPP) cleaves transpeptide after TOC interaction
- stroma: internal aqueous compartment (analogous to matrix)
2
Q
Where is the proton gradient localised in chloroplasts?
A
the thylakoid
3
Q
Mechanism of chloroplastic protein import
A
- Toc159, 33
- Toc75
- Tic22
- Tic236
- Tic20, 21, 56, 100, 214, 12
- Tic110, 40
4
Q
Toc159, 33
A
- GTPases; controls recognition and pore transfer
- recognise transit peptide
5
Q
Toc75
A
- beta barrel protein
- import pore @ OCM
6
Q
Tic22
A
- IMS chaperone
- aids passage -> TIC
7
Q
Tic236
A
physical link between TOC and TIC
8
Q
Tic20, 21, 56, 100, 214, 12
A
- translocon
9
Q
Tic110, 40
A
- ATP-powered motor (similar to PAM)
10
Q
TOC-TIC supercomplex
A
- algal
- visualised under cryo-EM
- heavily integrated; TOC is fused with TIC
11
Q
Structure
A
- Chloroplastic import description
- Chloroplastic import mechanism
- internal sorting
- thylakoid targeting
- differential targeting
- dual targeting
- genome retention
12
Q
internal sorting occurs
A
between envelope membranes
13
Q
IEM
A
- targeted by stop-transfer or conservative sorting
- no identified carrier protein system
- standard transit peptide identified, but not ubiquitous (which would insinuate a carrier protein system is there)
14
Q
OEM
A
- simple proteins with helical TM domains
- SAM-type mechanism
15
Q
Thylakoid trageting
A
- 4 conservative sorting pathways (2 lumenal, 2 membrane)
1. CpSec
2. CpTat
3. CpSRP
4. spontaneous
16
Q
lumenal clients
A
- bipartite targeting sequences
- N-terminal transit peptide
- bacterial signal peptide (removed in lumen by TPP)
17
Q
Describe CpSec
A
- ATP-dependent
- unfolded proteins
18
Q
Describe CpTat
A
- twin arginine transferase
- H+ gradient powered
- folded proteins
19
Q
Describe CpSRP
A
- polytopic light harvesting proteins
- Alb3 (oxa1 homologue)
20
Q
Describe the spontaneous pathway
A
simple membrane proteins require no assistance
21
Q
Compare presequences to transit peptides
A
- more hydrophobic
- more hydroxylated
- more positive
- less negative
- shorter
- different structures
- different receptors and membranes
- less phosphorylation
- subtle differences in the first 16 N-terminal residues
22
Q
Chloroplast proteins, when expressed in yeast…
A
…are targeted to the mitochondria
23
Q
Describe the differences in the first 16 N-terminal aas between mitochondrial presequences and chloroplastic transit peptides
A
- mTPs: more arginine (+ve charges are a chloroplast avoidance signal)
- cTPs: more Ser, Pro
24
Q
Describe the differences in length between mitochondrial presequences and chloroplastic transit peptides
A
- mTPs: 42-50 residues
- cTPs: ~58 residues (length helps stromal penetration w/o electrophoretic effect)
25
Describe the differences in structure between mitochondrial presequences and chloroplastic transit peptides
- mTPs: amphipathic alpha-helices
- cTPs: less structured (Pro; increased penetration)
26
Describe the differences in receptors between mitochondrial presequences and chloroplastic transit peptides
- Tom20 and absent in planta (replaced by functionally similar, non homologs: Tom20, OM64)
- Tom22 has lost its charged cytosolic binding domain
27
Describe the differences in membrane composition between mitochondrial presequences and chloroplastic transit peptides
- unique lipids
- CE: galactolipids
28
How does mRNA allow differential targeting?
- proteins are synthesised close to their final destination
29
Dual targeting
~400 (5%)
30
Give the 3 functional classes of dual targeted proteins
1. transcription machinery
2. translation machinery
3. antioxidants
31
Describe the options for dual targeting mediation
1) twin targeting sequences
2) ambiguous targeting sequences
32
Describe twin targeting sequences
separate signals exist in tandem in a gene with alternative TSSs / alternative splicing
33
Describe ambiguous targeting sequences
ambiguous characteristics
34
Why do organelles retain a genome at all?
- there are a similar core set of genes retained in the chloroplast genome across all lineages
- this is also true of mitochondria
35
What are the two major theories for organelle genome retention?
1) hydrophobicity hypothesis
2) co-location for redox regulation (CORR)
36
Describe the hydrophobicity hypothesis
- organellar genomes are enchriched in hydrophobic- TM domain genes
- this is a major obstacle in protein transport due to aggregation and mistargeting
37
Challenges to the hydrophobicity hypothesis
- organelles do import hydrophobic proteins (IMM; thylakoids)
- not all organellar genes are hydrophobic (e.g. RbcL)
38
RbcL
- yields are functional protein when nuclear
- this could be co-opted for protein engineering!
39
Describe the CORR hypothesis
- the best way to ensure balanced ETC flow is to co-locate ETC genes within the ETC system; in the organelle
40
Challenges to the CORR hypothesis
- not all organellar genes are redox
- most organellar genes act in multi-protein complices including nuclear genes; nuclear activity is required anyway
41
Other possibilities to explain genome retention?
1) close co-ordination of protein synthesis and assembly into multi-protein complices
2) genetic disparities (codon usage, RNA editing)
3) cytosolic toxicity of gene products
4) "limited transfer window" hypothesis
5) "essential tRNAs hypothesis"
42
Describe the "limited transfer window hypothesis"
- organellar transfer precluded by evolutionary reduction to 1x organelle / cell
- this links to Doolittle's Hypothesis
43
Describe the "essential tRNAs" hypothesis
- impossible to functionally transfer organellar RNA genes to the nucleus; e.g. tRNAGlu is essential in the chloroplast
