Mitochondria and Chloroplast biogenesis Flashcards
(21 cards)
Mt genomes and variation
Mt 1st described as granules In striated muscle sarcoplasm. Contain outer mem, inner (ETC) mem, IMS, matrix (TCA creating 36ATP per glucose). Typical mt in multicellular euks, many unicellular euks. Some species (protozoan parasites, parasitic worms, some molluscs) have many mt functionalities reduced (ox Pi, carbon/aa/fatty acid metabolism, Fe-S cluster assembly, homeostasis, apoptosis). Functions can vary substantially among euk lineages.
Mt variation: helped by SEM significantly, show variation even in same species/ diff tissues, but morphology based solely on microscopy misleading as mt dynamic. BAT has UCPs-> significant proton leak. Liver mt enzymes for urea synth not present in other tissues. Plant leaf mt have many enzymes for photorespiratory pathway operating only in photo tissues. Yeast w/ no O2-> promt, inactive in respiratory ETC/ATP synth, develop-> functional mt when O2 present.
Genome relic of alphaproteobacterial genome, encode some ETC/ genetic machinery. Majority migrated to nucleus, imported in.
Mt ETC, yeast, inheritance
ETC transfers e-s from NADH+ FADH2 to protein complexes+ mobile carriers-> E conversion into ATP, consuming O2 using large protein complexes I-V, all 5 w/ subunits encoded in mtDNA- Stoichiometry vital. Yeast mt cyt bc1 complex= mem-spanning cyt b+c1, Rieske Fe-S protein. bc1 carried e- ubiquinol-> cyt c soluble protein in IMS. Chloroplast b6f similar, gives e-s to plastocyanin soluble protein in thylakoid lumen. Both complexes have numerous small subunits, several bound to haem.
Mt genomes 1st characterised in yeast: S cerev. Normally grows on fermentable+ non-fermentable (need mt) substrates. Petite mutants arise naturally @ low freq due to mt loss/mutation. In wild type, erythromycin+ some other antibacterials inhibit formation/ respiratory enzymes in mt, inhibiting translation on mt ribosomes-> inhibit growth on non-fermentable substrates, but not on fermentable substrates.
Inheritance: resistant strain x wt crosses->non-Mendelian/ cytoplasmic inheritance. Simple (single) mutations in nuclear genome-> ½ daughters mutant phenotype, but both wt+ mutant mt may be inherited. Molecular technologies-> genetically characterise mutants: phenotypes correlated w/ loss/mutations of specific mt genes. Mapping mutants by recombination in diploid zygotes-> circular linkage map. Restriction mapping+ seq confirmed map+ gave more detail, incl full gene complement
Mt genome variation and splicing
Mt genome variation: mtDNA ID’d in yeast by Schatz 1964. EM showed dsDNA in most mt. Animal mt genomes small, inherited maternally. One of smallest mt genomes in Plasmodium (malaria), most size variation in plants. Generally, small mt genomes of Chlamydomonas+ animals most crowded, larger genomes have more intergenic space. Generally, encode r/tRNAs, ribosomal proteins, ETC+ ATP synth proteins, membrane protein assembly genes in larger genomes. Yeast- ATP synth, complexes III, IV. Others have I, II components.
Genetic machinery: yeast mt promoters located by ID’ing 5’ ends/transcripts (only primary transcripts have 5’ tri-Pi) by guanylyl transferase capping w/ [a-32P]GTP (transfer guanosine mono-Pi, usually from GTP, releasing pyro-Pi). RNAP is 2 polypeptides- 145kDa core (weak non-specific DNA interaction)+ 43kDa specificity factor w/ seq similarity to bacteriophage T3+ T7 RNAPs, no similarity to E-coli. RNA processing: at least 20 promoters ID’d, most give di-/multi-cistronic transcripts, most contain tRNAs. Cleavage needed to make individual t/mRNAs. No poly-A/capping. Splicing: introns in 21S rRNA, cyt b, COX I genes (#s vary between strains), classified into 2 tyeps based on conserved features of primary seq+ predicted secondary structures. Intron folding suggests 5’+3’ ends brought together:
* Type I- conserved seq elements also in nuclear rRNA from Tetrahymena, found in 21rRNA intron, cyt b introns 2+4, COX I introns 3/6. Splicing requires GTP+ Mg, produces circular intron
* Type II conserved secondary structure, particularly @ 3’ end. Residue bulge in hairpin. Cyt b intron 1, COX I introns 1, 2, 7. Splicing needs Mg, produces lariat.
Animal mt genetic machinery, plants and protists
Animal mt genetic machinery: RNAP similar to yeast, but initiated @ 1 site in D-loop near origin/replication of heavy strand (OH)-> 2 transcripts from heavy strand promoter (HSP1& HSP2)+ 1 transcript from light strand promoter (LSP). Processing- poly-A creates translation stop codons not encoded in mt. Large transcripts cleaved by ribonuclease P (RNaseP) to remove tRNAs, leaving transcripts w/out stop codons, then poly-A introduces stop.
Plant+ protist: relatively few promoters+ long ½ life of RNA-> transcriptional regulators for exp ctrl not sufficient so organelle transcriptomes dependent on RNA binding proteins to reg exp post-transcriptionally+ change seq of transcripts (RNA editing). Most plants+ some protists e.g. slime mould& trypanosomes mtRNAs modified post-transcriptionally. E.g. over 400 C->Us in mRNA/ flowering plants, Ferns+ mosses C->U and U->C almost equal freq- not seen in animals/fungi. Pentatricopeptide repeat (PPR) family similar to tetratricopeptide (TPR) in prokaryotes= major mediator of post-transcriptional ctrl.
RNA editing in trypanosomes, mt protein synthesis
RNA editing more extensive in trypanosomes than plants. Kinetoplasts= mt @ flagellum base of trypanosomes (e.g. T brucei), contain ~50 maxicircles (22kbp, contain r/tRNA genes))+ ~10k minicircles (1kbp) per mt. Protein-coding genes appear to contain extensive frameshifts, transcription+ translation predict truncated proteins, but cDNA seq show editing takes place in 3’->5’ direction post-transcription, involving U deletion and insertion directed by 400 trans-acting guide RNAs (gRNAs) encoded by minicircles. T brucei have ~400 classes/ minicircles encoding 3 diff gRNAs 40-70bp. After transcription, gRNAs have 15-20 UMP residues added to 3’+ base pair mRNA from maxicircle genes to transfer UMPs.
Mt protein synthesis: shares features w/ prokaryotes e.g. genetic code of many mt differs from universal code. Products of mt protein synth ID’d by:
* Protein synth by isolated mt w/ oxidisable substrate E source+ radioactive aa, then ID w/ antibodies+ electrophoresis.
* Effects of specific inhibitors of protein synth like chloramphenicol+ cycloheximide on radiolabelling of proteins w/ aas in vivo, e.g., cyt oxidase subunits: COX I, II, III prevented by chloramphenicol, smaller subunits inhibited by cycloheximide.
Plastid variation
Plastid variation: plastids derived from cyanos, retain some eubacterial features. Plant plastids have 2 mem+ genome+ ribosomes in stroma (also Calvin-Benson-Bassam cycle for CO2 fixation w/ Rubisco). # in plants cells varies w/ tissue+ env, typically 10-100. Carry out other reactions not found elsewhere in cell- fatty acid synth, some aa synth, cofactor synth, etc.
Chromoplasts- yellow, orange, red; Flowers+ fruits; Large carotenoid deposits- attract pollinators, seed dispersants. Leucoplasts colourless, in mature plant cells (e.g. roots. Can mature to amyloplasts, elaioplast), amyloplasts (starch-containing, in roots, seeds, storage tissues- seeds, tubers). Etioplasts (optional intermediate after proplastid)+ light-> Chloroplasts-> gerontoplasts by senescence. All come from:
Proplastid (1-2um) in meristematic stem cells. Differentiate depending on host cell’s developmental programme, types believed to be interconvertible. Normal sev seqs may incl plastid differentiation (e.g. tomato ripening chloroplasts-> chromoplasts). All have identical chloroplast DNA (cpDNA).
Cp diversity and structure
Diversity of metabolic/ morph features in algae+ protists. Surprising lineages contain plasmids, e.g. Plasmodium parasites+ Toxoplasma of apicomplexan lineage contain ‘apicoplasts’- non-photogenic, but genes related to other plastids+ cyanos- enables antibiotic treatments e.g. lincomycin to treat malaria.
Endosymbiosis: original event- cyano engulfed by (mt-containing-mt thought to predate plastids by ~0.5bln yrs)euk-> glaucophyte, red+ green algae, all plants- plastids in this lineage have 2 mem. Almost all other plastid containing lineages obtained them by secondary endosymbiosis events of single celled red/green alga- exact # separate events debated. Many 2o+3o plastids have 3+ membranes.
Chloroplast structure: 3rd internal mem (thylakoid)-> flattened discs, stacks=grana. Site of light dependent reactions. PS II w/ associated oxygen-evolving complex, b6f/cyt complex, PSI, ATP synthase (plus individual proteins w/ plastocyanin, ferredoxin, ferredoxin NADP reductase/FNR). Both chl b6f+ mt bc1 have b-type cyt w/ 2 non-covalently bound haems, a Rieske Fe-S protein, c-type cyt w/cov-attached haem. External acceptor= cyt c in IMS/ plastocyanin in thylakoid lumen.
cp: ecoli free extracts, dna seq and transforming genomes
E-coli free extracts can transcribe chl genes+ translating mRNA. Incubation of cpDNA w/ radiolabelled aa-> synth labelled proteins+ ID’d w/ antibodies. Cell-free protein synth ID’d+ localised ~20 chl encoded genes incl large subunit/ Rubisco, components of PSI/II/ cyt b6f/ ATP synthase. Rubisco small subunit nuclear-encoded. DNA seq-> comparison of complete gene complements. rRNAs 32-33+ tRNAs+~70 proteins’ genes ID’d. several conserved ORFs for proteins of unknown f(x) ID’d (ycf). Deleting/mutating chl genes by producing ‘transplastomic’ plants-> functional characterisation of plastid genes.
Transform genomes by hom recomb- constructs typically delivered w/ biolistic methods+ used for reverse genetics experiments, typically contain aadA aminoglycoside adenylyl transferase to confer spectinomycin resistance. aadA flanked by regions of gene to be disrupted. Trasplastomin plants recovered on spectinomycin media- can take weeks-months to achieve homoplasmy. Used to determine unknown genes’ f(x) e.g. ycf4 is for PSI assembly.
Cp gene heritability
Chl gene existence+ heritability: almost always maternally inherited (except gymnosperms that inherit paternally) as in variegated plant crosses- Baur reported plastids in green+ white shoots different, Correns found leaf colour of progeny depended on which parent had what. Recent evidence that maternal inheritance can break down under specific env conditions, inheritance determined by gene-env interactions.
Ris+ Plaut found “DNase sensitive fibrils”, then Chun visualised DNA in isolates chls, confirming cpDNA existence. Once DNA fragments isolated+ visualised, confirmed non-Mendelian inheritance in higher plants by restriction enzyme digests of plastid DNA from hybrids of diff strains+ species. Maternal inheritance 1st show for cyt f in tobacco+ large subunit of Rubisco (overall 8 large, 8 small). Identity of genes confirmed w/ Northern blotting (RNA extracted, bound to mem, radiolabelled DNA probes w/ known seqs applied-> visualise sizes of transcripts containing cognate RNA)- ID’d rRNAs (23S, 16S, 5S, 4.5S)+ tRNAs.
cp genome variation, machinery and processing
Genome variation: high degree of similarity in unrelated plants. Genomes of parasitic, non-photo plants small, e.g. 35kbp plastid DNA in vestigial non-photo plastids (apicoplasts) of Apicomplexan parasites. Comparing genes in parasitic plant chloroplasts-> non-photo species lost genes for photosynthetic machinery. Diff to colourless amyloplasts/leucoplasts- same genome as chl but not making chlorophyll/PSs.
Genetic machinery: genomes highly compact, some coding seqs overlap, e.g. 50bp overlap psbD+ pcbC.
Transcription- 2 RNAPs, both can transcribe all genes. 1st known plastid encoded polymerase (PEP) similar to prok pols- recognises motifs in promoters (also similar to bacteria)
Processing: primary transcripts cleaved @ both 5’+3’+ internally (endonucleolytic) to cleave polycistronic transcripts. RNaseE cleaves between genes @ A/U rich RNA adjacent to stem loop structures. RNase J (5’->3’ endonuclease) @ 3’ end, e.g., polynucleotide polymerase (PNP). Poly-A promotes degradation of mRNA. RNase P= protein only enzyme, tRNA processing. Intron removal by 6-8 factors, some general+ some specific (cis splicing).
cp rna editing, PPRs, translation
RNA editing: universal code used. RNA editing of a few genes, always C->U, creating start codon in rps2+ psbL via ACG->AUG. specificity determined by RNA binding proteins, many characterized by multiple degenerate repeats (e.g., pentatrico Peptide repeat (PPR) proteins w/ 35 aa repeats, or TPRs (34aa repeats) or OPRs (38aa repeats)- prevent endonucleases processing beyond a certain point.
PPRs also implicated in editing/ splicing/ promoting ribosome binding. PPR repeats form HTH motifs, recognise specific nts (verified w/ gel shift assays) but no catalytic activity. PPRs proliferated in higher plants- >450 in Arabidopsis+ found in mt too. Mammalian genomes encode just 2-6. Participate mostly in seq specific RNA metabolism. Implicated in precursor transcript stability+ processing+ translation. Essential in cytoplasmic male sterility restoration+ chl-> nucleus retrograde signalling.
Translation: own 70S ribosomes. Protein synth of isolated chl w/ s-35-methionine-> light-dependent incorporation of labelled methionine into proteins by isolated pea chloroplasts ID’d Rubisco subunits. Product light dependent, DCMU (PSII inhibitor)+ chloramphenicol CAP (70S inhibitor) inhibited but not cycloheximide (CHX)- 80S inhibitor. mRNAs have ribosome binding sites near 5’, complementary to 3’ end/ 16S rRNA as in E-coli. Initiation a/ N-formyl methionine. Later removed from most proteins. Products ID’d w/ antibodies- all correspond to genes in cpDNA.
Protein import, sorting and assembly in mt and cp
Cyt c, adenine nt transporter, porin, others synth as mature proteins. Most synth as larger precursor forms, (extra 2-10kDa+ mature protein)- N term extensions (presequences/transit peptides)=/= signals targeting ER. Mt preseqs 9->70aa, chl 35->100. No seq identity between seqs from different proteins+ these seqs less conserved between species than mature proteins. Tend to be rich in hydroxyl aas (Ser, Thr), often rich in basic aas (Arg, Lys) w few acidic aas (Glu, Asp). Many mt seqs form regular amphiphilic alpha helices.
Import post- translational. Incubation of radiolabelled precursors w/ isolated chl/mt-> uptake+ processing to mature size. Transit peptides are necessary and sufficient for uptake- no uptake of mature proteins (e.g., cleaved off w/ human peptide deformylase)+ fusing coding info for transit peptides to passenger protein (reporter) allows uptake, indicating highly specific, post-translational transfer of proteins to chl/mt.
Import machinery similar in cp+mt. Both have protein complexes in inner+ outer mems- translocon of outer/inner mem (TIM/TOM) in mt, Translocon of outer/inner chl mem (TIC/TOC) in cp.
Mt import complex: best characterised in yeast. Preproteins cleaved after incubation w/ isolated mt in experiments- effect reduced by pre-treating mt w/ trypsin. Import inhibited if increasing amounts/precursors supplied, suggesting presence of outcompetable receptors- ~600-6000 per organelle. Antibodies raised to proteins in outer mem-> immunogold EM, one of which inhibited transport. Anti-idiotypic antibodies mimic mt transit peptides, inhibit transport-> enables TIM/TOM isolation
mt import
Mt import: nuclear encoded proteins synth on free ribosomes in cytosol, imported:
1) Preseq recognition: complete precursor-> import receptors, often protected by cytosolic chaperones+ co-chaperones (prevent premature folding, false interactions, aggregation). preseq binding factor (PBF)+ a 70kDa heat shock-related protein stabilise translocation competent form/precursor. Mt import stimulating factor (MSF) recgonises transit peptides, forms complex w/ preprotein, keeping them in import competent form, requires ATP, facilitates precursor/ mt outer mem binding
2) Interaction w/ TOM: TOM 20 receptor passes preproteins w/ preseqs to TOM40 channel. Hydrophobic carrier interaction w/ TOM70. TOM22= organiser of TOM complex
:Cleavable pre-proteins pushed through TIM 23 channel. Experiments w/ mitoplasts (mt w/out outer mem)-> precursor proteins can be imported+ cleaved. Threading through TIM+TOM independent
3) Translocation dependent on me potential (200mV across inner mem, abolished w/ valinomycin)- needed only for initial preseq translocation. ATP used for further translocation by preseq translocase associated motor PAM. Hsp70= ATP dependent molecular ratchet, drive translocation
4) Proteolytic processing+ folding: preseq removed by mt processing peptidase MPP in matrix- inhibited by EDTA+ 1,10-phenanthroline metalloprotease. Proteins unfolded-> allow translocation (low temperature import in vitro slows process)
5) Hsp60 aids refolding in matrix
Dysfunction of protein import machinery associated w/ several diseases, w/ mt diseases incl cancers+ neurodegenerative diseases.
plastid import, protein sorting in mt and cp
Plastid import similar to mt- unfolded precursor threaded through 2 mems, TOC/TIC provide receptors+ channels. Involves Chl transit peptide/TOC, then TIC interaction. Uses ATP/GTP, doesn’t require mem potential. 150 kDa stromal processing peptidase (SPP) cleaves. No seq similarity between mt/cp proteins of import. Cp have Hsp70 in IMS not found in mt
Protein sorting: each mt/cp compartment (incl mems) has a distinct protein complement. Mem proteins have specific routes:
1) SAM (sorting assembly machinery)- outer mem proteins (beta-barrels like TOM40, porins) don’t have preseqs. Cross linking studies during in vitro import show they interact SAM, then insert into mem. Some interact TIM9/10 chaperones in IMS. Inner mt mems (e.g. AT/DP translocator) similarly interact TIM9/10, then go via TIM22- has twin VG pores, using mem potential to drive translocation
2) Outer mem alpha-helical proteins interact TOM70- recognises hydrophobic proteins w/ cytosolic chaperones. Mt import (MIM) complex inserts proteins into outer mem.
3) IMS proteins (e.g., chaperones don’t contain preseq. Many have twin Cys motifs (CX3C+ CX9C configs). Enter via TOM, imported+ folded in IMS via oxidating folding system- key component=MIA40 (AKA disulfide relay system.
Assembly of protein complexes
Assembly of protein complexes: crystal structures of all mt+cp complexes well established - e.g. b6f has large subunits and small ones encoded by pet G, L,M, N. plastid- encoded pet G, L, N necessity investigated in homoplasmic KO N tabacum lines. Pet G+N required for major component proper assembly. petL mutants have ~50% wt levels of components, but complex no longer dimer, Rieske encoded by pet C not assembled. Rieske not required for assembly of b6f, components dimerise without it. Loss of Rieske consequence of monomerization (not cause). Minor component (pet M nuclear encoded) essential for assembly. Haem in cyt f essential (mutants can’t bind haem cofactor (CytfH), incorporated into complex, but removing C-term transmem domain (Cytf-MS)-> won’t assemble. If both lost, rapid protein degradation.
regulation of mt/cp biogenesis
Env/dev conditions dictate if new organelles contain functional respiratory/photo apparatus. E.g. no O2-> mt e- transfer can’t occur. In yeast growing in anaerobic conditions by fermentation have small mt w/ few internal mems, no resp complexes (proMt). O2 required for haem biosynth on mt (haem=cofactor for cyts). Mt develop fully w/O2 and/or all glucose consumed.
Nuclear genes (e.g. CYC for cyt c) reg by nuc-encoded HAP1 (haem-activator protein 1) TF. Binds haem-> bind upstream activating seqs (UASs) in CYC promoter. Nuclear genes for cyt oxidase COXIV,V,VI reg by HAP2/3/4/5 complex- may respond to haem levels via HAP1 activation of HAP4 gene.
Angiosperms- dev/ photo-active cp requires light. Chlorophyll not made in dark-> leaves have etioplasts containing paracrystalline mem structure, prolamellar body (dev into thylakoids when light). Other land plants+ algae have enzyme for dark synth/chlorophyll (green in dark). No need to exp photo components in non-green plastids, but genes transcribed in all tissues w/ no large diff in relative transcription rates of individual genes in diff tissues+ no evidence for repressor/activators for individual genes- overall transcription levels depend on RNAP levels+ individual gene transc depends on promoter strength. Then regulated post-transcriptionally (positive regulation, unlike bacteria)- transcripts present in dark but light required to promote translation.
e.g., psbA (D1 of PSII core): stem loop in 5’ UTR. cPABP binds this to activate translation. cPDI modulates binding by reversibly changing cPABP redox status via redox potential or adenosine 5’-diphosphate-pependent Pi-> reversible switch for gene exp, translation of psbA linked to photosynthetic activity.
coordination of biogenesis
Coordination- all characterised protein products of mt+cp genes= components/multisubunit complexes also containing nuc-encoded subunits. Many mt/cp per nucleus-> imbalance (e.g., yeast 50:1, HeLa 8800:1). Yeast nuclear subunit synth can be in mt gene absence (e.g. petite mutants)- exp/ nuclear mt genes not tightly reg.
Rubisco have 8 large/8 small structure. LSU= rbcL cp gene. SSU= RbcS nuc gene, ~50x less abundant. Exp/ nuc genes also not tightly reg. Evidence from:
1) In vivo- cycloheximide inhibits SSU synth, but LSU synth continues for a time. Chloramphenicol inhibits LSU synth, SSU synth continues for a time. SSU doesn’t accumulate, degraded in cp
2) Antisense RNA inhibition of SSU synth in transgenic tobacco containing antisense construct of RbcS cDNA exp from constitutive promoter-> mRNA degrad, SSU synth decrease.
Amount/ Rubisco correlates to amount/ SSU. Reducing SSU-> less LSU accumulation but not transcript level change, suggesting assembly driven/ subunit availability, xs degraded. Coordination by post-transc processes.
nuclear factors for organelle biogenesis
Nuclear factors for organelle biogenesis- nuc genes incl components of RNAP, cp sigma factors, ribosomes. No TFs for specific organelles. Nuclear-encoded proteins also req for stability+ translation/ specific mRNAs in both mt+cp, incl editing- major ctrl point, often 2+ nuc genes needed to make single organelle protein. e.g. removing intron 5b/ COXI required 3 helicase proteins- mutants in these have altered mRNA profiles/ complete transcript loss. In yeast nuclear respiratory deficient mutant screens, >20 extra genes ID’d to be trans-acting factors for COX assembly of 2 types: enzymes (haem biosynth, Cu-homeostasis+ insertion into complex) and proteins for exp/ mt encoded COX genes- stability, splicing, translation.e.g., Petite mutant pet309 impaired in COXI mRNA translation. Complementation rescues, but complement w/ proteins lacking PPR repeat still lack COXI. Human Pet309 defects-> neurodegen Leigh syndrome. Chlamydomonas MCA1 protein req to stabilise cp petA mRNA for cyt f, TCA1 facilitates transl/ petA RNA- both PPR proteins.
signalling between organelles and nucleus
Signalling: nucleus affecting organelle exp= anterograde signals; organelle-> nucleus= retrograde. Nuc genes not transcribed/ transcribed @ basal levels when no functional cp/mt. Yeast petite mutants have reduces TCA cycle+ resp complex gene exp. Barley mutants w/ defective cp ribosomes- reduced RbcS/LHC exp.
LHC= array of protein+cp molecules embedded in thylakoid, transcription ctrled by light. Light perceived by phytochrome (red/far red receptor)- has phytochromobilin cofactor. Activation by red-> translocate cytosol to nucleus, interact repressive TFs that bind promoter/ genes incl LHC.
Ctrl studied by disrupting cp function by:
1) Inhibitors of cp transc/l. targetitoxin inhibits cp RNAP, lincomycin/chloramphenicol inhibit cp protein synth. These prevent nuc gene exp early in seedling dev
2) Photooxidation of cp in carotenoid-deficient mutants/ plants treated w/ norflurazon (inhibit carotenoid synth)-> photobleaching chlorophyll.
This reduced transcripts from Photosynth-assoc nuc genes (PhANGs), e.g. Lhc, RbcS. Genes for mt/cytosolic components unaffected. Treatments of mutant libraries ID’d gun (genomes uncoupled) mutants- continue to exp PhANGs when plastid dev blocked- mainly genes in tetrapyrrole biosynth, incl haem.
mt diseases: human encephalopathies
Mt diseases: Yeast mt mutants grow on non-fermentable substrates, which is impossible for most multicellular euk (obligate aerobes)- so mt mutations often lethal if in all mt, all cells. Can be in only fraction of mt (or mt genomes) per cell, or mutation may affect f(x) of mt in specific tissue:
Human mt encephalomyopathies- neuromuscular diseases w/ mt DNA+ ATP-generating capacity altered. Tissues w/ highest ATP demand (CNS, skeletal muscle) most affected:
1) Mild missense mutations- e.g. Arg-> His point mutation subunit 4/ NADH dehydrogenase-> Leber’s hereditary optic neuropathy (LHON)-> vision loss ~20-24 years old. Decreased mt e- transfer+ ATP synth-> optic nerve cell decline, death. Required homoplasmy (all mtDNA in all mt) to manifest
2) Deleterious point mutations/deletions: lead to loss/ mt protein synth. Myoclonic epilepsy+ ragged-red fibre disease (MERRF)= trnK gene. MELAS= trnL gene. KSS= 2-7kbp deletion. All lethal if homoplasmic.
3) Ageing: specific deletion of 4977bp deletion/ mtDNA in brain correlates w/ ageing. Up to 12%/ mtDNA molecules in brains/ over-80yr olds. Sarcopenia (muscle atrophy) common (up to 40% miscle mass loss by 80) assoc w/ deletions in localised area/ muscle fibres.
mt diseases: incompatibility, cytoplasmic male sterility, cms-T
Nuclear-mt incompatibilities: in yeast, 10 nuc+ 10 mt variants studied-> some combos grow+ respond to stress better. Human mt haplotypes used to study migrations. Mt mutations assoc w/ diff susceptibility/ resistances+ give evidence of nuclear organelle incompatibilities.
Cytoplasmic male sterility in plants: fail to make functional pollen, widely used in plant breeding programmed- prevent self-pollination. Maize- circumvent costly, time-consuming emasculation by hand. Male fertility pf CMS plants can be overcome nuc-encoded gene(s) called restorer-of-fertility (Rf). 1952- male sterile maize (Texas), inheritance cytoplasmic.
Texas/ T cytoplasm (cms-T) incorp in breeding, 85%/ US corn acreage by ’69. Mt genome magged, has new gene T-urf13 arisen by several recombination events between atp6+26S rRNA genes-> hydrophobic protein in inner mt mem- inhibits mt f(x) only in developing anthers-> pollen abortion. Other mt f(x) normal. In ‘69+’70, ;arge part/US corn crop wiped out- Southern corn leaf blight (B maydis fungus)- attacked only T plants. Fungus toxin bound T-urf13 protein-> mt dysfunction on all cells. Some varieties of maize don’t show this, encode “nuclear restorer genes”- supress exp/ T-urf13- often PPR proteins. Hard to experimentally link mt rear.s w/ male sterility. Recently. TALEN-mediated mt genome editing to f(x) characterise ORFs assoc. w/ male sterility.
