How to build organelles Flashcards

1
Q

Do the mitochondria encode their own proteins?

A

Yes however only a handful of the ~300 proteins found in the mitochondria

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2
Q

How can we study mitochondrial import?

A

Many of the original experiments were done in yeast as it is a good model EUK system. It lives happily on glucose - ferments and does not need the mitochondria to do this. However to grow on ethanol/glycerol you need mitochondria function.
Discovered mutations which would only grow on glucose and not ethanol/glycerol and these mutants had impaired mitochondria - either the import of the proteins or the enzymes themselves.
Can test by knocking something out and then reintroducing it on a plasmid

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3
Q

Describe the matrix targeting signals (MITO)

A

Typically N-terminal
Amphipathic helix - charged on one side and hydrophobic on the other
From comparing the cDNAs - not a specific sequence
Discovered in 1980s when cloning and sequencing DNA - found extra sequences on the cDNA which were not present in the protein
Can take it off and put it one different proteins e.g. GFP and visualize where it goes in the cell
Targeting sequences can also have a transmembrane domain sequence if the destination is IM/OM
Internal transmembrane domains with no N-terminal seq are for member of the metabolite carrier family for the IM

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4
Q

How did they test the theory of matrix targeting signals? (MITO)

A

Created a synthetic targeting sequence made of only 3 aa only - tested with a yeast mutant which lacked a subunit of cytochrome oxidase so could only grow on glucose not ethanol/glycerol. Tested different sequence combinations of the targeting sequence which showed the primary sequence was not important rather if it could form an amphipathic helix

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5
Q

What chemical can be used to stop unfolding of DHFR and how can this be useful? (MITO)

A

MTX - anticancer drug which binds tightly to DHFR and prevents unfolding. It can start to be imported however it cans stuck and this allows cross-linking to see what machinery it uses for import

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6
Q

How was TOM 70 identified?

A

By manipulating ATP and membrane potential in vitro. AAC accumulated on the surface and was cross-linked to TOM 70

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7
Q

How was GIP identified?

A

Adding a tag to one component allow the pull-down of interacting partners

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8
Q

Draw the TOM complex

A

See diagram

TOM 20, 22, 6, 40, 7, 5 and 70

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9
Q

How to proteins cross the outer mitochondria membrane?

A

TOM complex

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10
Q

What are the components of the general import pore (GIP)

A

TOM 5, 6, 7, 22, 40

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11
Q

Which TOM forms the central pore in the TOM complex?

A

TOM 40

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12
Q

What is the initial receptor in the import of N-terminal amphipathic helix targeting signals? (MITO)

A

TOM 20

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13
Q

Describe TOM 20

A

Initial receptor for amp. helix signals
Have solved the XRC structure of TOM 20 soluble domain in complex ith amp. targeting signal - showed the cytosolic domain provides a groove lined by hydrophobic residues which interacts with the hydrophobic side of the amp. helix (hydrophobic interactions) and the positive side of the amp. helix extends out into the soluble solution phase - shows why a specific sequence is not necessary

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14
Q

Describe TOM 70

A

Other initial receptor for internal TM targeting signals e.g. metabolite carrier family which have internal hydrophobic repeats.
TOM 70 is abundant in the membrane.
Also receptor for proteins using MSF chaperone and cytosolic hsp70 - Precursor-MSF complex binds TOM 70 and ATP hydrolysis occurs which releases MSF and get the transfer of precursor to TOM 20/22.
Binds multiple copies of TOM 70

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15
Q

Describe the GIP and what each protein does

A

GIP is made up of TOM 5, 6, 7, 22 and 40

  • TOM 22 - receives the proteins recognized by TOM 20/70 and acts as the organizer of the GIP (involved in the assembly of the whole complex). It spans the membrane and has a large cytosolic domain which interacts with charged pre-sequences. Has a IMS domain where it provides a binding site for the pre-sequence as has a negative charged domain which interacts with the positive part of the helix.
  • TOM 5 - acessory receptor which helps in the trasnfer of proteins into the TOM 40 pore and receptor for some IMS such as the tiny TIMs
  • TOM 6 -assembly of TOM 22 with TOM 40
  • TOM 7 - translocase disassembly
  • TOM 40 - import chanell - oligomeric complex
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16
Q

How was TOM 40 visualized?

A
Single particle EM
His-tagged one component and it pulled the whole complex through
TOM 22 sits between 2x TOM 40
Salt bridges may help interactions
Did cross-linking of the TOM complex
See diagram
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17
Q

Explain the binding site or acid chain hypothesis (MITO)

A

The TOM proteins and some TIM proteins provide a sequential set of acidic and hydrophobic binding sites for the positively charged amp. helix pre-sequence
Allows both sides of the helix to interact in an energetically favorable way
Specificity arises from multiple low-affinity interactions (don’t want too high affinity as needs to be transient to allow movement along the chain)

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18
Q

Describe the insertion of beta-barrel proteins in the OM of mitochondria

A

OM proteins which carry an amp. helix followed by a stop-transfer enter through the TOM complex but recognized and segregated into the OM using MIM 1/2.
Beta-barrel proteins also enter via the TOM complex but when they enter and come through the other side they are bound by small TIMs (chaperones)
The SAM machinery inserts them - in yeast it is made up of 4x components ( mdm 10, SAM 50, SAM 37 and SAM 35)
See diagram

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19
Q

Is the machinery for inserting beta-barrels homologous to the machinery in bacteria OM? (MITO)

A

Yes - mitochondria are descents of free-living PRO cells and were engulfed by a EUK cell and over evoution the genes have moved into the nucleus.

SAM 50 is homologous to Omp85 and both have POTRA domains
Tiny TIM chaperones are homologous to Skp/SurA

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20
Q

What is SAM 50 homologous too in bacteria? (MITO)

A

Omp85 - both have POTRA domains and insert OM beta-barrels

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21
Q

What are the tiny TIMs homologous to in bacteria?

A

Skp/SurA

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22
Q

How can we study mitochondria import in vivo?

A

Use yeast mutants which can only grow on glucose no ethanol/glycerol - resp defective
Easy to transform gene in yeast so can look for the ones which grow again - lots of components were identified this way
Some protein knock outs are lethal and therefore they couldn’t be identified this way e.g. TOM 40 therefore had to use thermosensitive mutants which are unable to grow at high temperatures - lots of missense mutants don’t kill the cell but make it less stable

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23
Q

Describe mitochondrial reconstituted import assays

A

In vitro
Easy to isolate mitochondria by differential centrifugation
Use labeled translated proteins and mix with isolated mitochondria with energy and ATP. Split into two and add protease to one. Reisolate and carry out SDS-PAGE/Western blot/Blue-native PAGE for complexes. Can see where the precursor and mature proteins are.
Can manipulate the conditions e.g. collapse membrane potential (no longer see import), study the import of TOM 40 as it needs SAM so K.O. SAM and see the import of TOM 40 is blocked.
See diagram.

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24
Q

All proteins use TOM complex to enter the mitochondria - True or False?

A

True

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25
Q

All proteins use the same TIM machinery - True or false?

A

False

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26
Q

Which proteins use TIM 22 complex?

A

Proteins withOUT a pre-sequence e.g. MCF

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27
Q

Which proteins use TIM 23 complex?

A

Protein with a pre-sequence e.g. matrix proteins. Translocated via TIM23 and processed by matrix processing protease (MPP)

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28
Q

Which proteins use TIM23-SORT?

A

Proteins which carry a matrix targeting signal followed by a stop-transfer signal and they get integrated by a variant of TIM23 called TIM23-SORT

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29
Q

What are the components of the TIM 23 complex?

A

TIM 17/23 - 2x central components, essential for cell viability (need to use temperature sensitive mutants), cation selective voltage-gated channel which responds to the pre-sequence. TIM 17 regulates the opening of the TIM 23 pore.

TIM 50 - Binds the pre-sequence of protein emerging from TOM complex. Closes the TIM 23 channel in the absence of the pre-protein

TIM 21 - links TOM and TIM complexes via the interaction with TOM 22 IMS domain. Disassociates TIM 23 from PAM complex. Role of intermediate in contacting interacting partners of TIM 23. Helps guide protein into the TIM complex.

Gating of the channel is important for maintaining the membrane potential of the IM

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30
Q

Which protein regulates the opening of the TIM 23 pore?

A

TIM 17

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31
Q

Which protein closes the TIM 23 channel?

A

TIM 50

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32
Q

Which TOM does TIM 21 contact?

A

TOM 22 IMS domain

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33
Q

What are the components of the PAM complex?

A

PAM - pre-sequence associated motor - provides the driving force and energetics.

Made up of:
TIM 44 - binds TIM 23 and provides a binding site for mtHsp70. Binds to the translocation peptide first.

mtHsp70 - binds translocation peptide. ATP hydrolysis which is the energy to drive the reaction and movement into the matrix.

Mge1 - nucleotide exchange factor for mtHsp70

Pam 18 - stimulates ATPase activity of mtHsp70

Pam 16 - Controls Pam 18 activity

Pam 17 - Organiser of TIM-23 and PAM interactions. Binds to TIM 23 and dissociates it from TIM 21. Regulates the association of subunits.

See diagram.

34
Q

Draw diagram of TIM 23 complex

A

See diagram in notes.

TIM 17/23, TIM 50, TIM 21, TIM 44, mtHsp70, Mge1, PAM 16/17/18

35
Q

Which PAMs regulate TIM 23

A

PAM 16/17/18

36
Q

What are the two models how mitochondrial import is energised

A

Role of mtHsp70.

2x models:

1) . Hsp70 acts in a single passive fashion - traps the polypeptide as it is coming through - binds it as it comes out of TIM 23 and prevents the backward movement
2) . Hsp70 has a more active role of pulling - when bound by TIM44 - binds chain as it comes through and uses ATP hydrolysis as a power stroke to pull the chain through TIM 23

Most likely a combination of the 2 models and protein-dependent as highly structured proteins may need more active Hsp70

37
Q

What is TIM23-SORT?

A

A variant of TIM 23 for proteins which have a pre-sequence and a stop-transfer and end up in the IM.
When a hydrophobic sorting signal comes after, just behind the amp. helix enter into TIM 23 complex but a different variant is formed. The PAM complex dissociated and instead the TIM complex interacts with protein complex of the ETC (Bc1, COX proteins) and this is mediated by TIM 21.
Allows proteins to enter the IM

38
Q

How do protein enter the IM?

A

TIM23-SORT and an amp. helix followed by a stop-transfer signal

39
Q

Describe the TIM 22 complex

A

For proteins with no amp helix but internal duplicated targeting signals e.g. MCF

TIM 22 - core component which has homology to TIM 23 and 17 for forms a voltage-gated channel which responds to internal pre-sequence peptides

TIM 54 - analogous to TIM 50, large IMS domain which may bind the pre-protein and/or other translocase subunits

TIM 18 - may regulate the assembly of the complex

TIM 12 - link between TIM 9/10 complex and TIM 22 - one of the tiny TIMs involved in inserting beta-barrel into the OM. TIM 12 is special as it is the only tiny TIM which is membrane bound and acts as a link.

See diagram.

40
Q

Draw diagram of TIMM 22 complex

A

See diagram in notes.

TIM 22, 54, 18, 9, 10, 12

41
Q

Describe the 5 stages of mitochondrial carrier proteinsy

A

1) . Chaperone (Hsp70/90) mediated delivery to TOM 70
2) . Interaction between duplicated internal targeting signals and TOM 70
3) . TP-dependent release of protein to GIP and insertion into the GIP as a loop followed by contact with TIM 9/10 complex
4) . The precursors contacts the TIM complex and is further translocated through this complex
5) . Insertion into IM and association into dimer

Integral membrane proteins which are hydrophobic and normally sit in the IM have multiple TM spans - the hydrophobic regions need to be shielded and prevented from aggregation in the cytosol so interact with chaperones and go to TOM 70 then TIM 9/10, TIM12 and finally TIM 22.
Low membrane potential insertion into TIM 22 and high membrane potential protein segregated out of TIM 22 into membrane.

Know the protein form a loop by using DHFR fused to N/C-terminals and MTX to anchor DHFR by preventing unfolding

42
Q

Are the inner membrane complexes structurally and functionally distinct? TIM 22 and TIM 23?

A

Yes - they can function independently from each other. Both use membrane potential as an energy source but TIM 23 also uses ATP.

43
Q

Can you purify a supercomplex of GIP and TIM complexes?

A

Yes - proteins can span both membranes as translocation intermediates allowing the isolation of a supercomplex. May reflect contact sites between inner and outer membranes in EM images.

44
Q

What is the role of membrane potential in the import across the IM of the mitochondria?

A

IT is essential for all proteins to cross or insert into the IM. One function suggested to be an electrophoretic effect on the pre-sequence as the IMS is positive and the matrix is negative (ETC moves protons from matrix into IMS). The other role is to regulate the opening of the channels (keep membrane tight). Without membrane potential the pre-sequence can bind but cannot enter.

45
Q

What are the tiny TIMS?

A

Members include TIM 8, 9, 10, 12 and 13
Contain a conserved twin motif - CX(3)C
Fold by forming two DS bonds between the cysteine of 2 motifs
Hexamers - long extended alpha helices
TIM 9/10 for MCF and TIM 8/13 for other proteins TIM 23
TIM 12 is the only one which is membrane bound

46
Q

Import of proteins containing DS bonds (MITO)

A

DS relay system for introducing DS bonds into IMS proteins. Cross through the TOM complex and enter IMS where they interact with Mia40 bind via oxidation reaction forming a DS bond with it. Mia40 gets re-oxidised by Erv1, DS-change and Erv1 gets regenerated by Cyt C of ETC.

47
Q

What is the the protein which has a C-terminal pre-seq rather than N-terminal (MITO)

A

Hmi1 DNA helicase getting translocated C–>N-terminal so shows the polarity is not important

48
Q

Can you get dual targeting of a protein? (MITO)

A

Yes - Fumerase has a single translocation product of the FUM1 gene and is distributed between the mitochondria and the cytosol. The targeting signal gets cleaved by MPP and then retrograde movement can occur moving the protein back into the cytosol. Hsp70 is meant to stop this but some gets through

49
Q

How are mitochondrially encoded proteins inserted?

A

Mitochondria encode a handful fo their own proteins which are mostly inner membrane proteins part of the resp. chain.
Translated by ribosome in the mitochondrial matrix and need to be inserted into the IM.
Bacteria have multiple pathways but EUK only have one.
Oxa1/Alb3//YidC (Mito/chloroplast/bacteria homoloues)
Oxa1p was discovered because mutant prevented assembly of cytochrome oxidase complex (has both mito and nuclear-encoded subunits) so was resp. deficient. Is responsible for the correct insertion of:
1). mitochondria-encoded IM proteins and one IMS protein cox2
2). some nuclear-encoded protein which first follow TOM-/TIM pathway.
Most are co-translationally inserted from the matrix into the IM
Some proteins which are nuclear encoded go all the way into the matrix then get re-exported from matrix into IMS - evolution perspective at one time the genes would have been int he mitochondria but now int he nucleus so inserted a targeting sequence to bring it back - unusual

50
Q

Do proteins targetted for the chloroplasts have N-terminal targeting signals?

A

Yes - analogous system to mitochondria

51
Q

What is the targeting signal for chloroplasts?

A

Stromal transit peptide is N-terminal and enriched in positive and hydroxylated amino acids - doesn’t have the same hydrophobic nature as the mitochondrial one

Thylakoid transit peptide - hydrophobic after the stromal signal

52
Q

What are the similarities between mitochondrial targeting signals and chloroplast targeting signals?

A

Both have a signal for the matrix/stroma at the N-terminus, followed by hydrophobic stretch and residues with small side chains for protease. If being trafficked to interior destinations then have another targeting sequence.

53
Q

Are chloroplasts descendants of PRO origin?

A

Yes

54
Q

What are the entry requirements across the chloroplast envelope?

A

Chloroplasts can make their own ATP in the light if all of the substrates are present - if add un-couplers they collapse the membrane potential on thylakoids and import is inhibited not because membrane potential is needed but as no ATP is produced because if you do the same experiment in the dark and you need to ATP and you still get import even in the presence of un-couplers.

Membrane potential is important for ATP synthesis

55
Q

How to protein cross the outer envelope of the chloroplasts?

A

TOC 159/34/75
Pre-protein is P and interacts with GTP bound TOC 34 –> GTP hydrolysis –> TOC 159 –> GTP hydrolysis __> transfer through the TOC 75 channel
GTP hydrolysis drives conformational change whereby TOC 159 pushes the precursor into the translocation channel.
TOC 159 is both a receptor and a motor

56
Q

Describe the cytosol-guidance complex (CHLORO)

A

It is not essential but highly stimulatory for most pre-cursors in vitro
P on a specific serine or threonine by cytosolic kinase specific for transit peptides
The P-protein binds complex consisting of 14-3-3 protein and Hsp 70. 14-3-3 subunit recognises the P-transit peptide. Guidance complex docks at the TOC complex
Precursor de-P preceeds import
Importance in vivo is uncertain

57
Q

What 2 proteins make up the guidance complex (CHLORO)

A

14-3-3 protein and Hsp 70

58
Q

TIC110

A

Inner envelope protein
Major component which binds to 2x stroma chaperones involved in driving translocation/folding of incoming proteins
Analogous to TIM 23
Single gene in Arabidopsis essential for viability - can only study with antisense experiments - down-regulate expression get albino phenotype

59
Q

How were the components of the import machinery identified?

A

Similar approaches to the mitochondria import but is harder to use genetic approaches as plants are more complex.
Biochemical approaches came first - generating translocation intermediates (in mitochondria this was done by MTX or lower temp) but in chloroplast were 2 ways:
1). Limited ATP concentration so complex translocation occurs slowly or not at all - need low [ATP] for OM but high [ATP] for IM mainly due to the chaperones
2). Using purified outer envelope vesicles. As inner envelope os missing the translocation cannot be completed as no TIC machinery

Can then do cross-linking of the translocon intermediates to adjacent proteins followed by solubilization and SDS-PAGE and use antibodies to see what protein it is. Or can solubilize the protein complex use native conditions and isolate the proteins associated with the translocation intermediate and use detergents and native PAGE/immunoprecipitation/gel filtration. The second method is less harsh.

60
Q

What are the 4 components of the TOC machinery?

A

TOC 75, 159, 34 and 64

61
Q

What species was used for genetics of chloroplasts?

A

Arabidopsis as the genome sequence is known

62
Q

What species did they isolate chloroplasts from and why?

A

Pea leaf plants as was easy to get lots of chloroplasts

63
Q

Describe TOC 159

A

Major import receptor
Bind ATP and GTP
Acidic N-terminal domain likely binds positive amino acid transit peptide in stroma
Antibodies against TOC 159 inhibit early intermediate formation
Arabidopsis has 4 TOC 159 like proteins - AtTOC159, AtTOC132, AtTOC120 and AtTOC90 which are all related but are not the same.

64
Q

What happens if you knock out AtTOC159?

A

Get albino seedlings with defective chloroplasts but they still have many of the housing keeping enzymes

65
Q

What happens if you knock out AtTOC132

A

Have reduced house-keeping enzymes

66
Q

What happens if you knock-out both AtTOC159 and AtTOC132

A

Lethal

67
Q

Describe TOC 34

A

Binds GTP and sequence homology to TOC 159
Forms a stable complex with other TOCs in absence of the precursor
GTP-regulated in vitro binding to precursor
TOC 34 becomes P and may have a role in regulating import
Arabidopsis homologues - AtTOC33 and AtTOC34

68
Q

What are the Arabidopsis homologues of TOC 34?

A

AtTOC 33 and AtTOC 34

69
Q

What happens if you knock-out AtTOC33?

A

Yellow colour due to lower chlorophyll

70
Q

What happens if you knock-out both AtTOC33 and AtTOC34

A

Lethal

71
Q

Describe TOC 75

A

Predicted to be a 16 TM beta-barrel protein which forms a voltage-gated cation selective channel
Cross-link to intermediates which span both membranes
Probably the principle component of the protein-conducting channel through the outer envelope
TOC 75 is essential for viability
Multiple variants of TOC but cannot cover function

72
Q

Do TOM 40 and TOC 75 share sequence homology?

A

No they do not and are evolutionary distinct from each other

73
Q

TOC 64

A

Stable association with other TOCs and interacts with early import intermediate
Proposed binding site for the guidance complex
TOC 64 knock-outs do not have a detectable phenotype

74
Q

TIC 40

A

Associated with precursor proteins - arrest protein import with TIC 110
N-terminal membrane anchor
Most of the protein faces the stroma
C-terminal homology to Hsp70 interacting protein (Hip) - likely involved in recruiting chaperones to stromal side of membrane
Knock-out gives pale phenotype

75
Q

TIC 20

A

Hydrophobic and membrane embedded
Could form a channel
Antisense plants give pale phenotypes

76
Q

TIC 22

A

Cross-link to precursor protein
Peripheral and faces inter-membrane space
May be a receptor for TIC complex or mediate association of TIC and TOC complexes at contact site
Analogous to mitochondria TIM 22

77
Q

Draw the model for import into chloroplasts

A

See diagram

78
Q

What in chloroplasts acts as the PAM in mito?

A

Hsp 93 and Hsp 70

79
Q

What can the TOC complex be inactivated by?

A

Inactivates by oxidation but activates by reduction of DS bonds in the TOX complex

80
Q

Redox regulation of import (CHLORO)

A

Can recruit redox sensitive proteins to the TIC complex. When NADP+ is high them import will be activated as this means the rate of e- transport is limiting. NADP+ gets reduced during photosymethetic e- transport.
Activity of the ETC can regulate the import of pre-cursor proteins

81
Q

How to proteins get into the outer envelope of chloroplasts?

A

Some proteins will be resident members of outer envelope. They also have N-terminal targetting signals bound by chaperone called Akr2 (chaperone for OEP7 and OEP64). Also require the TOC machinary but are not translocated in the same way - probarbly mor ethan one mechanism

82
Q

Do plant cells contain mitochondria and chloroplasts?

A

Yes so have to differentiate between them:
Chloroplast transit peptides are generally rather specific but there are examples of proteins targeting to both and this may be a rule rather than an exception for some classes of proteins e.g. amino acyl tRNA synthetases where 17/25 are dual targetted.Dual targetted proteins can be due to different reasons for example ambiguous protein targeting signals or alternative transcription/translation start sites or splicing variations