Biogenesis of Mitochondria and Chloroplasts I

Question 1

Structure

Answer 1

1. Organellar genomes intro
2. Organelle -> nuclear transfer
3. targeting sequences
4. crossing the organellar membrane
5. pathways for protein import
6. pre-sequence dependent import

Question 2

Organellar biogenesis

Answer 2

• mitochondria and chloroplasts cannot be synthesised de novo
• they are inherited cytoplasmically (often maternally)
• and are propagated via organellar division accompanied by organellar growth (and the associated metabolism)
• the endogenous genome is retained, alongside the necessary transcriptional and translational machineries

Question 3

Describe the organellar genomes

Answer 3

• ~1-3000 proteins
• 90% genes are nuclear-encoded
• insufficient

Question 4

Describe the mitochondrial genome

Answer 4

• humans: 13 genes
• yeast: 8 genes
• plants: 20-50 genes

Question 5

Describe the chloroplastic genome

Answer 5

50-200 genes

Question 6

What percentage of the A. thaliana genome is cyanobacteria?

Answer 6

18

Question 7

Doolittle’s Hypothesis

Answer 7

gene ratchet

Question 8

Organelle -> nucleus transfer

Answer 8

1) organellar lysis
2) DNA uptake
3) genome integration
4) eukaryotic regulatory acquisition (promotor, targeting sequences) by random and rare recombination
5) organellar copy lost (reductive/purifying selection)

Question 9

Measuring chloroplast -> nuclear transfer experimentally

Answer 9

• transform tobacco seed chloroplasts with chimeric gene for kanomycin R + nuclear promotor
• sow thousands of progeny on kanamycin
• 1/16000 seedlings survive

Question 10

Why nuclear transfer?

Answer 10

• host-endosymbiont control
• organellar ETC: mutagenic (ROS; NHEJ)
• avoids Muller’s ratchet
• decreased genome maintenance metabolism
• faster dna replication

Question 11

What is the implication of organellar to nuclear transfer?

Answer 11

proteins are cytosolically translated, and require organellar targeting

Question 12

Targeting sequences

Answer 12

encrypted for subcellular localisation

Question 13

What are the different types of targeting sequence?

Answer 13

1) N-terminal extension
2) C-terminal tag
3) internal sequences

Question 14

N-terminal extensions

Answer 14

usually cleavable

Question 15

internal sequences

Answer 15

form “signal patches”

Question 16

N-terminal mitochondrial presequence

Answer 16

• ~60%
• no conserved aa sequence
• variable length (15-50aas)
• conserved structure (amphipathic alpha-helix, +ve, neutral and hydrophobic facial residues)

Question 17

Internal target sequences in mitochondria

Answer 17

• hydrophobic TM domains
• inner membrane metabolite carrier proteins

Question 18

What are the 3 modes of transport for crossing the organellar membrane?

Answer 18

1) gated transport
2) vesicular transport
3) TM transport

Question 19

describe gated transport

Answer 19

• large, aqueous, selective pore
• cross in folded state

Question 20

describe TM transport

Answer 20

• membrane-spanning protein translocators
• small pores
• cross in unfolded state

Question 21

Describe mitochondrial protein import

Answer 21

• mostly PT
• 2x membranes to cross
• via membrane-spanning translocases
• ATP-dependent Hsp70 and 90 ensure unfolded conformation
• translocation N-terminus first

Question 22

Specificities of mitochondrial protein import

Answer 22

1) Tom20 recognises presequence, delivers to Tom40
2) simultaneous import at “contact sites”

Question 23

Tom40

Answer 23

OMM general import pore

Question 24

“contact sites”

Answer 24

• where the OMM and IMM touch
• observed under EM; gold particles bound to translocating protein
• similarly observed in chloroplasts

Question 25

What drives the import process?

Answer 25

- the binding chain drives presequence through TOM - membrane potential (proton gradient) drives presequence through TOM - MPP cleaves presequence for protein maturity

Question 26

the binding chain

Answer 26

increasing affinity of TOM binding sites to the presequence

Question 27

MPP

Answer 27

mitochondrial processing peptidase

Question 28

Describe the 3 different pathways for IMM transport

Answer 28

1) stop-transfer 2) conservative sorting pathway 3) carrier protein

Question 29

Describe the stop-transfer pathway generalities

Answer 29

- proteins have 1x standard N-terminal presequence and 1x stop-transfer sequence - ATP-independent

Question 30

Describe the stop-transfer pathway - specificities

Answer 30

1) Tom20 translocation 2) endopeptidase cleaves N-terminal presequence 3) stop-transfer signal results in translocation arrest @ Tim23 4) single TM span

Question 31

Describe the conservative sorting pathway - the basics

Answer 31

proteins have 1x standard N-terminal presequence and 1x oxa1 target sequence

Question 32

Oxa1

Answer 32

- oxidase assembly 1 - ATP-dependent insertase - delivers client proteins into IMM - conserved since endosymbiogenesis - related to ydc in bacteria

Question 33

Describe the conservative sorting pathway - the specifics

Answer 33

1) matrix delivery and presequence cleavage 2) oxa1 engages Tim23 machinery 3) results in multiple membrane spans

Question 34

Describe the carrier protein pathway - the generalities

Answer 34

- for multi-spanning proteins involved in metabolite transport - complex - no presequence - hydrophobic TM spanning domains (prone to aggregation)

Question 35

Describe the carrier protein pathway - the specificities

Answer 35

1) Tom70 reception 2) Tom40 import 3) Tim22 insertion machinery

Question 36

Describe presequence-dependent import

Answer 36

- very well conserved in animal mitochondria; also present in yeast - Tom20; - downstream TOM elements; - Tom5, 22; - Tom40; - Tim21; - Tim23, 50 - PAM + Hsp70

Question 37

Tom20

Answer 37

- primary presequence receptor - 3x alpha-helices make hydrophobic surface groove - recognise non-polar presequence helix - visualised under NMR, cryo-EM

Question 38

downstream TOM elements

Answer 38

can interact with charged, basic presequence residues

Question 39

Tom22, 5

Answer 39

- mediate transfer to Tom40 - very high affinity acidic binding site in the IMS; promotes transport

Question 40

TOM complex

Answer 40

- dimeric - close juxtaposition determines client specificity - can undergo oligomerisation to exist as higher order structure

Question 41

Tom40

Answer 41

beta-barrel

Question 42

beta-barrel

Answer 42

multiple anti-parallel beta strands

Question 43

Tim23, 50

Answer 43

- concave cavity - Tim17 homology - spans IMS - recognises presequence on TOM emergence

Question 44

Tim 21

Answer 44

- contacts presequence early - Tim23 regulator

Question 45

PAM

Answer 45

- presequence associated translocase motor - incorporates chaperones

Question 46

List the two models of MtHsp70 + PAM action

Answer 46

1) thermal/Brownian ratchet (Neupert) 2) power stroke (Pfanner) - could be a combination - could be circumstance-dependent

Question 47

Describe the thermal/Brownian ratchet model

Answer 47

- thermal motion moves precursor back and forth in translocon - Hsp70 is passive; prevents backwards movement - ATP cycles chaperone units

Question 48

Describe the power stroke model

Answer 48

- conformational change exerts pulling forces on precursor - repeat cycles of chaperone binding and ATP hydrolysis pulls the protein into the organelle

Question 49

Overview mothcondrial protein import

Answer 49

1) MIM 2) SAM 3) MIA

Question 50

MIM

Answer 50

- mitochondrial import - simple alpha-helical transmembrane spans deliver the protein into the OM

Question 51

SAM

Answer 51

- sorting and assembly machinery (aka TOB) - delivery of complex beta barrel topologies

Question 52

MIA

Answer 52

- mitochondrial inter membrane space assembly - recognises cys-rich targeting sequences