Genesis of mitochondria, plastids, peroxisomes Flashcards
Model systems
Major model systems studied are S. cerevisiae (bakers yeast), Neurospora crass (a fungus), Mammalia and more and more Viridiplanta. As mitochondria of these organisms differ in their protein content, the details of the models discussed in this section are perhaps only valid for taxa of the Opistokonta (“animals” and “fungi”).
Four main types of signals target proteins to Mitochondria
- simple cleavable N-terminal signals (here signal to matrix)
- complex signals (here cleavable matrix signal + cleavable targeting signal to inter membrane space)
- internal signals (here internal signal to inner membrane (carrier-protein type))
- C-terminal signals (here sigmal to outer membrane (tail-anchored membrane proteins))
Matrix-Targeting Sequence
- positive charged amphipatic helix
Different signals may direct to the same compartment - signals for sorting to the inner membrane
Proteins are coded in nucleus (nu) or mitochondria (mt)
a) Cleavable signal to matrix - nu
b) Complex signal - nu
c) Non-cleavable signal to matrix - nu
d) IMS-targeting signal - mt
e) Internal signals (carrier protein type) - nu / mt (Mt-coded possible without charged regions?)
Targeting and quality control I
MT-membrane anchors require special factors
Ubiquilins support the targeting of MT membrane proteins (in particular tail-anchored protein) and are essential for degradation of non-targeted proteins.
Import of beta-barrel proteins
- TOM
- Porin
- SAM
- Tom40
Function of SAM50 during import
1) Closed gate
2) Open lateral gate: beta-signal exchange -> beta-hairpin insertion
3) Release of folded beta-barrel protein
Import of proteins with alpha-helices
-> tail anchored
-> MIM dependent
- Mim1
-> SAM dependent
-TOM
- SAM
Import of small TIMs
1) Precursor recognition
2) Disulphide transfer
3) Release of oxidized precursor
4) Reoxidation of Mia40 by Erv1
tRNA import into mitochondria
- observed in many species; some, like yeast, import only one type, others, like plants, several and kinetoplastida (Leishmania, Trypanosoma) have to import all needed tRNAs (= 24)
- in many cases specific signals in the tRNA exist implicating a selective import
- import mechanisms are different between different organisms and evolved several times
Modification, Folding, Quality control and degradation
- Processing by MPP (soluble in the matrix) and MIPs (anchored in the inner membrane and exposed to the inter membrane space); MIPs are homologous to let B in bacteria
- second processing of several matrix proteins by Oct
- N-terminal processing of single large destabilizing AA by lcp55
- Most mitochondrial proteases are also involved run degradation of damaged proteins.
Modification, Folding, Quality and degradation - example
- Folding by systems homologue to bacterial ones (e.g. DnaK-like systems and mtHSP60 system)
- Degradation by systems homologue to bacterial ones (e.g. mtClpXP, mtLON or FisH-like membrane-bound proteases)
- HtrA-like protease and ATP23 in inter-membrane space
- Mts1p (AAA ATPase) at the cyt side of OM for relocation in cyt (e.g. TA-proteins)
Role of cytoplasm in degradation of mitochondrial proteins
a) MAD (mitochondria Associated Degradation)
- substrate are ubiquitinylated OM proteins
- components: CDC48-Npl4,Udf1 / Doa1 / proteasom
b) Identification of mistargeted tail-anchored proteins
- degradation by AAA Msp1
c) Identification of blocked protein import
mitoCPR (mitochondrial Compromised Protein import Response)
mitoRQC (release of stalled ribosomes engaged in mitochondrial protein import)
mitoTAD (mitochondrial Translocation Associated >Degradation)
mitoTAD
-> Accumulating precursors
-> Burden on proteasome system
-> Proteostatic imbalance
-> Burden on chaperone system
Mitochondrial Stress Response
a) UPRmt
b) mPOS (precursor over accumulation stress)
c) UPRam (unfolded protein response activated by mistargeted proteins)
UPRmt - signalling via TIM/TOM
Transport to the nucleus:
- OXPHOS recovery
- Mitochondrial proteostasis
- ROS detoxification
- Mitochondrial import
Can be inhibited by:
- OXPHOS defects
- ROS
- Mitonuclear protein imbalance
- Unfolded proteins
Could mitochondria evolve before protoeukaryote development?
- common idea is, that only eukaryotic organisms that a developed phagocytosis could give rise for endosymbionts
- however, new findings suggest, that also prokaryotes can harbor endosymbionts
Endosymbionts in prokaryotes
gamma-protepbacterium as endosymbiont in a beta-proteobacterium, which in turn is an endosymbiont in an insect cell
The different plastid types in viridiplantae
Proplastid:
- Etioplast
- Amyloplast
- Chloroplast -> Chromoplast
Targeting of nucleus-encoded proteins to plastids
- OM pathway
- ER-CP pathway
- Uncleaved TP pathway
- TOC-TIC pathways
Major targeting and sorting signals of plastid proteins
A) TARGETING TO THE OUTER MEMBRANE
usually no transit peptide (24 proteins in A.t.)
- tail-anchored proteins have the signal probably located in their flanking region
- some proteins with alpha-helical membrane anchor use TOC75
- beta-barrel TOC75 has a bipartid transit peptide (transit peptide followed by a glycine-rich sequence); is imported via TOC/TIC and processed by an inner-membrane lepB-like protease
- signals in other beta-barrel proteins unknown
B) IMPORT TO INTER MEMBRANE SPACE
very few proteins, transit-peptide like signal
- some use TOC/TIC and stroll processing peptidase
- some use only TOC
C) TARGETING TO INNER MEMBRANE
- with transit peptide via TOC/TIC using either a stroll intermediate (conservative sorting!) employing a cpSEC2 called systems or a stop-transfer mechanism without such intermediate
- without cleavable signal, nut using TOC159 system, pathway unknown
D) IMPORT INTO STROMA
- with cleavable transit peptide, across TOC/TIC system (90% of all nuclear coded proteins)
* transit peptides have very variable structure; (e.g. length from 25 to 150 amino acids)
* are usually processed in the storm by a specific peptidase
* are enriched basic AS, hydrophobic AS and Ser
* show no defined secondary structure in water but may adopt an amphipathic helix in lipid (especially in the presence of monogalactolipids)
- with uncleared signal, not using TOC159 system, pathway unknown (about 100 polypeptides)
- with signal sequence and unknown features via the ER using an unknown pathway (few N-glycosylated proteins)
E) IMPORT INTO THYLAKOIDS
- thylakoid targeting signal - signal (leader) peptide like hydrophobic sequence using different pathways (e.g. I)
- Tat-like signal
* nuclear coded proteins have a composite signal with a transit peptide in front of the thylakoid targeting signal/Tat-like signal
Main types of signals target proteins to plastids
- simple cleavable N-terminal signals (here transit peptide to stroma)
- Complex signals (here cleavable transit-peptide + cleavable targeting signal to thylakoid)
- Internal signals (no special example; any membrane anchor forms an internal signal)
- C-terminal signals (here signal to outer membrane (tail-anchored membrane proteins))
Targeting to chloroplasts
Transit peptide
-> phosphorylation sites (common); depending on substrate addition of phosphate leads to:
a) formation of guidance complexes consisting of 14-3-3 proteins, HSP70 a.o.
b) higher affinity to import receptors
binding of precursors to import receptors needs ATP and GTP
Import into the storma via TIC/TOC
- TOC159-GTP binds precursor; alteration of conformations during GTP/GDP-cycle as driving force for import across oE?
- TOC34-GTP binds precursor and 14-3-3
- ATP (< 100µM?) via imsHSP70?
- for some outer membrane proteins (e.g. TOC75) the second part of a bipartid transit peptide is cleaved by plastid signal peptidase 1 (Plsp1)
Import via TIC ATP-dependent (>3 mM), due to involvement of cpHSP70, HSP90C, and HSP93
- regulation of import capacity depending on the redox-potential (=photosynthetic activity) via Tic32/Tic55/Tic62/FNR or the Ca-level via calmodulin (CAL)
Import into the storma via TIC/TOC
- TOC159-GTP binds precursor; alteration of conformations during GTP/GDP-cycle as driving force for import across oE?
- TOC34-GTP binds precursor and 14-3-3
- ATP (< 100µM?) via imsHSP70?
- for some outer membrane proteins (e.g. TOC75) the second part of a bipartid transit peptide is cleaved by plastid signal peptidase 1 (Plsp1)
Import via TIC ATP-dependent (>3 mM), due to involvement of cpHSP70, HSP90C, and HSP93
- regulation of import capacity depending on the redox-potential (=photosynthetic activity) via Tic32/Tic55/Tic62/FNR or the Ca-level via calmodulin (CAL)
Regulation of the TOC
Toc complex is composed of four Toc75, four (to five) Toc34 and one Toc159 (only half of the Tic c). Precursor proteins are recognized by GTP-charged receptors and might be recognized by either Toc34 or Toc159. This induces dimerization between Toc34 and Toc159. Both, dimerization and precursor recognition activates GTP hydrolysis, initiating the translocation. Recharging of the receptors with GTP is required for the next round of translocation. Phosphorylation of Toc34-GDP inhibits GTP loading and thus inactivates the complex. Dephosphorylation reactivates the complex.
Regulation of the TOC
Toc complex is composed of four Toc75, four (to five) Toc34 and one Toc159 (only half of the Tic c). Precursor proteins are recognized by GTP-charged receptors and might be recognized by either Toc34 or Toc159. This induces dimerization between Toc34 and Toc159. Both, dimerization and precursor recognition activates GTP hydrolysis, initiating the translocation. Recharging of the receptors with GTP is required for the next round of translocation. Phosphorylation of Toc34-GDP inhibits GTP loading and thus inactivates the complex. Dephosphorylation reactivates the complex.
Regulation of the TOC
Toc complex is composed of four Toc75, four (to five) Toc34 and one Toc159 (only half of the Tic c). Precursor proteins are recognized by GTP-charged receptors and might be recognized by either Toc34 or Toc159. This induces dimerization between Toc34 and Toc159. Both, dimerization and precursor recognition activates GTP hydrolysis, initiating the translocation. Recharging of the receptors with GTP is required for the next round of translocation. Phosphorylation of Toc34-GDP inhibits GTP loading and thus inactivates the complex. Dephosphorylation reactivates the complex.
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Vesicular traffic between inner membrane and thylakoid membrane in mature chloroplasts
- Fusion machinery consist of calmodulin and protein phosphatase 1
- vesicles are best visible after inhibition of these components
- proposed function: membrane (lipid) exchange between thylakoid and IM
Mitochondria and plastids - signal specificity and promiscuity
- plastid targeting signals are in average less positively charged than matrix targeting signals
- many (about 100 in A. th.) plant mitochondrial pre sequences, in contrast to pre sequences from non-plant sources, but in accordance with transit peptides, are rich in serine and intermediate in their content of positive charges resulting in a dual targeting to both organelles
Protein import into chloroplast organelles evolved from a bacterial protein-export system
PLANT CELL
Protein -> TOC75 -> TIC236 -> TIC
BACTERIUM
Membrane protein -> SEC -> Tam B -> BamA/TamA
Import into “Microbodies”
- Microbodies - homologous organelles with diverse functions:
-> “true” Peroxisomes, Glyoxisomes (plants), Glycosides (Kinetoplastida), Voronin bodies (Pezizomycotina) - import occurs post transcriptional
- different signals
- factors involved in transport are called peroxins (PEX)
Biogenesis of peroxisomes is strongly regulated
- many organisms show strong proliferation of peroxisomes (e.g. yeasts) starting from early remnants
- in plant seedlings the protein content of peroxisomes may change completely
- change in nutrition may lead to a selective degradation of peroxisomes
Models for peroxisome biogenesis
1) Early (at least Class I type) peroxisomal membrane proteins are targeted directly to pre-peroxisomes
2) Some peroxisomal membrane proteins (at least Class I type) reach preperoxisomes via the ER
3) Preperoxisomes develop to nascent peroxisomes which import proteins, proliferate or develop to mature peroxisomes, which may grow and divide
4) Most peroxisomal membrane proteins reach nascent peroxisomes via the ER
Currently favored models: 2, 3, 4
Typical signals for peroxisomal proteins
MATRIX PROTEINS
- PTS1 - a C-terminal tripeptide (e.g. Luciferase)
- PTS2 - an N-terminal nonapeptide (e.g. Thiolase)
- PTS3 (rare) - an internal peptide
MEMBRANE PROTEINS
- mPTS - 20 aa (e.g. CbPmp47p)
- 40 aa (e.g. PpPex3p)
- 25 aa (e.g. PpPex22p)
- 55 aa (e.g. ScPex15p)
Topogenesis of peroxisomal membrane proteins
Class I peroxisomal membrane proteins (PMPs) contain a peroxisomal membrane-targeting signal, which consist of a PEX19-binding site and a membrane anchor sequence. These PMPs are synthesized on free ribosomes in the cytosol and recognized by PEX19, which is thought to act as a soluble PMP-receptor and chaperone. PEX19 ferries class I PMPs to the peroxisomes, where the membrane association of the PMP-receptor-cargo complex is mediated by PEX3. The mechanism of PMP insertion into the membrane remains to be elucidated. In several organisms this process requires PEX16, which might act as a tethering factor for PEX3.
Class II PMPs are characterized by a PEX19-independent targeting mechanism. Instead, these proteins, which might also include PEX3 and PEX16, are targeted to the ER before their transport to peroxisomes.
Topogenesis of peroxisomal membrane proteins
Class I peroxisomal membrane proteins (PMPs) contain a peroxisomal membrane-targeting signal, which consist of a PEX19-binding site and a membrane anchor sequence. These PMPs are synthesized on free ribosomes in the cytosol and recognized by PEX19, which is thought to act as a soluble PMP-receptor and chaperone. PEX19 ferries class I PMPs to the peroxisomes, where the membrane association of the PMP-receptor-cargo complex is mediated by PEX3. The mechanism of PMP insertion into the membrane remains to be elucidated. In several organisms this process requires PEX16, which might act as a tethering factor for PEX3.
Class II PMPs are characterized by a PEX19-independent targeting mechanism. Instead, these proteins, which might also include PEX3 and PEX16, are targeted to the ER before their transport to peroxisomes.
Working model for import of peroxisomal matrix proteins
- Receptor recycling
- Cargo translocation: Perhaps by transient pore formed by cargo receptor and Pex14
Import substrates can be folded and can be oligomers!
Working model for import of peroxisomal matrix proteins
- Receptor recycling
- Cargo translocation: Perhaps by transient pore formed by cargo receptor and Pex14
Import substrates can be folded and can be oligomers!
The ring ligase may promote Pex5 export working as a bilayer disturbing device
Receptor recycling and degradation use different ring finger domains
The ring ligase may promote Pex5 export working as a bilayer disturbing device
Receptor recycling and degradation use different ring finger domains
The ring ligase may promote Pex5 export working as a bilayer disturbing device
Receptor recycling and degradation use different ring finger domains
Fission in peroxisomes
Pex11-family of proteins elongates peristomes which are then split by a dynamin-related protein (e.g. DLP1).
The peroxisomal QC-system
- as most matrix proteins might get imported folded, a first checkpoint is sometimes the cargo binding during import
- human peroxisomes have refolding capacity, but the molecules employed are unknown; viridiplanta have stress induced sHSP and probably also chaperoning thioredoxins in the peroxisome
- some species (oil. fungi, plants) have a Lon-protease
- some species can direct protein aggregates for degradation by autophagy using specific fission events
- an retrograde transport of damaged matrix proteins is discussed but seems not be a general pathway (one shaky case in plant); membrane proteins may be extruded by an Ub/proteasome dependent pathway in some cases
The peroxisomal QC-system
- as most matrix proteins might get imported folded, a first checkpoint is sometimes the cargo binding during import
- human peroxisomes have refolding capacity, but the molecules employed are unknown; viridiplanta have stress induced sHSP and probably also chaperoning thioredoxins in the peroxisome
- some species (oil. fungi, plants) have a Lon-protease
- some species can direct protein aggregates for degradation by autophagy using specific fission events
- an retrograde transport of damaged matrix proteins is discussed but seems not be a general pathway (one shaky case in plant); membrane proteins may be extruded by an Ub/proteasome dependent pathway in some cases
Peroxisome biogenesis disorders (PBDs)
- Zellweger like diseases: Zellweger Syndrome (ZS); Neonatal adrenoleukodystrophy (NADL); Infantile Refsum disease(IRD); Rhizomelic chondrodysplasia punctata (RCDP)
- Partially or complete loss of the import apparatus
-> Mislocation and therefore impaired function of peroxisomal enzymes
-> Malformation und neurological failures; death of distinct cells - Main problem is the lack of degradation of certain lipids (VLCFA, phytanic acid) and the lack of synthesis of certain lipids (e.g. docosahexaenoic acid, plasmalogens, steroids)
Evolution of the micro bodies
- originated most likely from ER (based on proteome analysis of years and rat)
- during evolution retargeting from nuclear coded proteins of alpha-proteobacterial or eukaryotic origin originally destined to mitochondria into micro bodies
- relationship of ERAD and Pex5 recycling
Dual targeting of proteins
Protein in closely related species may adapt different topologies e.g. AGT1 depending on the type of natural nutrition in peroxisomes, in mitochondria or in both compartments.
Protein topogenesis may depend on environmental signals e.g. enzymes of glyoxylate cycle or catalase A in yeast.
Proteins may generally adopt two different topologies:
- alpha-methylacyl CoA racemase of mammals - Mito and Peroxi
- different types of CytP450, Alzheimer amyloid precursor - ER and Mito
- topology may be regulated in these cases
Dual targeting of proteins
Protein in closely related species may adapt different topologies e.g. AGT1 depending on the type of natural nutrition in peroxisomes, in mitochondria or in both compartments.
Protein topogenesis may depend on environmental signals e.g. enzymes of glyoxylate cycle or catalase A in yeast.
Proteins may generally adopt two different topologies:
- alpha-methylacyl CoA racemase of mammals - Mito and Peroxi
- different types of CytP450, Alzheimer amyloid precursor - ER and Mito
- topology may be regulated in these cases
Alanin:glyoxylat aminotransferase 1 (AGT1)
HERBIVOR
- AGT1 mainly peroxisomal
- mainly to lower the level of oxalate
CARNIVOR
- AGT1 mainly mitochondrial
- mainly for gluconeogenesis
Ala + glyxylat -> AGT -> Gly + pyruvat
Ser + Pyruvate -> hydroxypyruvat + Ala
Presence of a cryptic MTS in human may result in kinetic partitioning
Mistargeting of Alanine:glyxylate aminotransferase (AGT) 1 from peroxisomes to mitochondria results in primary hyperoxaluria type 1 (PH1) due to an excessive production of oxalate out of the unused glyoxylate
Presence of a cryptic MTS in human may result in kinetic partitioning
Mistargeting of Alanine:glyxylate aminotransferase (AGT) 1 from peroxisomes to mitochondria results in primary hyperoxaluria type 1 (PH1) due to an excessive production of oxalate out of the unused glyoxylate
Other pathways for transport of soluble proteins out of the cytoplasm in eukaryotes
- ABC-dependent secretion across the PM - a-factor in yeast using STE6
- atypical secretion across the PM - unknown transmembrane (?) pathway(s) and pathway employing secretary lysosomes (3 …5 % of the secretome)
- special pathway for protein delivery by eukaryotic cellular parasites into their host cells
- import of proteins into lysosomes for degradation (chaperone-mediated autophagy; CMA)
Energy requirements of transmembrane transport
GEP
co-transport does not need energy in addition to synthesis
- post transport in yeast -> 1 ATP per 10 aa
- post transport in bacteria -> 1 ATP per 35 aa (=per proOmpA 7 ATP)
Measured costs per Mol substrate:
spTAT:
8 x 10exp4 protons (equivalent to 10.000 ATP)
GEP
70 aa Substrate, unfolded -> close to detection limit
proOmpA (could fold before transport) -> 1000ATP + PMF or 5000 ATP in the absence of PMF
Energy requirements of transmembrane transport
GEP
co-transport does not need energy in addition to synthesis
- post transport in yeast -> 1 ATP per 10 aa
- post transport in bacteria -> 1 ATP per 35 aa (=per proOmpA 7 ATP)
Measured costs per Mol substrate:
spTAT:
8 x 10exp4 protons (equivalent to 10.000 ATP)
GEP
70 aa Substrate, unfolded -> close to detection limit
proOmpA (could fold before transport) -> 1000ATP + PMF or 5000 ATP in the absence of PMF
