Lecture 15 - mitochondria and chloroplasts part 3

Question 1

What is the chloroplast?

Answer 1

semi-autonomous double membraned plant cell organelle - derived from photosynthetic cyanobacterium

Question 2

What are the functions of chloroplast?

Answer 2

• site of photosynthesis
• also involved in several other important metabolic processes

Question 3

Are chloroplasts highly mobile?

Answer 3

yes, move along cytoskeleton elements via molecular motors

Question 4

What are the eight subdomains of the chloroplast?

Answer 4

1. envelope - consists of outer and inner membranes
2. outer membrane - contains porins, not as permeable to ions/small molecules as outer membrane of mitochondria
3. intermembrane space
4. inner membrane - highly impermeable, contains various transporters
5. thylakoids - flattened discs arranged in stacks
6. thylakoid membranes - site of ATP synthase; maintain H+ gradient in thylakoid lumen
7. thylakoid lumen - aqueous interior of thylakoid high [H+]
8. aqueous interior (space inside of envelope and outside of thylakoids)

Question 5

what does the stroma contain?

Answer 5

enzymes involved in carbohydrate synthesis and plastid genome
also contains ribosomes - translation of plastid genome encoded proteins

Question 6

What are some characteristics of the plastid genome?

Answer 6

circular, size and and copy/gene number varies between species
encodes ribosomal proteins some Ps proteins including a few located in the thylakoid, tRNA and rRNAs and some RNA pol subunits

Question 7

Are most chloroplast proteins nuclear encoded?

Answer 7

yes

Question 8

How are chloroplasts connected?

Answer 8

stromules

Question 9

What are stromules?

Answer 9

long, stroma filled membrane tubules - branched dynamic rapidly extend and contract, allow for efficient metabolite transfer, communication etc. between chloroplast and other organelle

Question 10

How do chloroplast multiply?

Answer 10

fission - divide in response to environmental stimuli or developmental signals

Question 11

What are the two protein machineries involved in chloroplast division?

Answer 11

FtsZ and PD - both form ring like structures either on inside or outside of chloroplast envelope - tighten and pinch organelle into two daughter chloroplasts

Question 12

Where is the FtsZ division machinery located?

Answer 12

stromal side of inner membrane

Question 13

What does FtsZ machinery include?

Answer 13

FtsZ1 and FtsZ2

Question 14

What do FtsZ1/2 do in chloroplast fission?

Answer 14

• spontaneously assemble into long, filamentous polymers at midway point on inner membrane surface
• form FtsZ ring at future chloroplast division, linkage mediated by ARC6
  
  • this position is mediated by ARC3,
    MinD, MinE
  • integral inner membrane spanning
    protein localized to equator in
    dividing chloroplast
  • stromal facing domain of ARC6 links
    to and stabilizes Z-ring

Question 15

What is the function of ARC6

Answer 15

1. linkage of z-ring to inner membrane at chloroplast division site mediated by ARC6 - stromal facing domain of ARC6 links to and stabilizes Z-ring
2. also controls tightening of Z-ring, intermembrane space facing domain of ARC6 binds to PD machinery in chloroplast outer membrane

Question 16

What does PD machinery include?

Answer 16

PDV1 and PDV2

Question 17

What do IMS facing domains PDV1/2 interact with?

Answer 17

IMS facing domain of ARC6
also bind to ARC5

Question 18

What do PD division machinery do in chloroplast fission?

Answer 18

• bind to ARC5 - assembles into spiral like structures to form PD ring
• PD ring wraps around outside of chloroplast: tightening of ring via GTP hydrolysis causes constriction of outer envelope

Question 19

How do FtsZ and PD machineries work together to fully fission chloroplast?

Answer 19

continued constriction and eventually scission of chloroplast membrane involves concerted action of both internal z-ring and external PD ring, results in formation of two daughter chloroplasts

Question 20

What do chloroplast proteins possess?

Answer 20

unique targeting sequence that serves as zipcode?

Question 21

How many subchloroplast targeting pathways are there?

Answer 21

6, vary depending on proteins final location in the chloroplast

Question 22

What are the steps for chloroplast protein targeting to the stroma?

Answer 22

1. Stromal-destined proteins possess stromal import sequence - located at N-term, cleaved following import into stroma
2. in cytoplasm, precursor protein synthesized on free ribosomes and recognized by cytoplasmic molecular chaperones which maintain a conformation of nascent protein in partially unfolded import competent state
3. at surface of chloroplasnt, proteins stromal import sequence recognized by toc complex, binding of toc requires GTP
4. precusor proteins transferred through toc complex and then through adjacent tic complex
   the tic and toc complexes are adjacent to each other at contact sites, the membranes pressed closely together
5. precursor protein translocation occurs across both membranes sequentially (unlike mitochondria, not mediated by H+ gradient)
6. N-term end of import sequence exits tic complex channel into stroma, cleaved by stromal processing enzyme
   emerging precursor protein also recognized by stromal chaperone protein hsp93, this acts as a molecular motor (ratchet), it undergoes ATP dependent and conf. changes that tpulls protein into stroma and prevents in back sliding . Hsp70 requires ATP to ensure protein is properly folded

Question 23

What is the function of the stromal import sequence?

Answer 23

This sequence is responsible for targeting nascent stromal protein to cytoplasmic surface of chloroplast and subsequent translocation across outer and inner membranes

Question 24

What are some characteristics of the toc complex?

Answer 24

multi-protein complex, includes receptors and accessory proteins. binding of the stromal import sequence to toc receptor requires GTP

Question 25

What is the function of hsp93 in the chloroplast protein targeting?

Answer 25

hsp93, this acts as a molecular motor (ratchet), it undergoes ATP dependent and conf. changes that pulls protein into stroma and prevents in back sliding . Hsp70 requires ATP to ensure protein is properly folded

Question 26

What are toc and tic complexes involved in?

Answer 26

import or insertion of other chloroplast proteins in addition to the stroma

Question 27

How is targeting proteins to the thylakoid lumen the same/different as stroma import?

Answer 27

precursor protein imported from cytosplasm into stroma as described above except, removal of stromal import sequence by stromal protease revels thylakoid targeting sequence, precusor protein then engages one of several different thylakoid import pathways either the SRP dependent pathway or the pH dependent pathway

Question 28

What are the two pathways that the thylakoid import can involve?

Answer 28

SRP dependent pathway
pH dependent pathway

Question 29

What is the SRP dependent pathway?

Answer 29

1. precursor protein in stroma maintained in partially unfolded import competent state by stromal chaperones (involves ATP)
2. thylakoid targeting sequence recognized by chloroplast signal recognition particle (SRP)
3. SRP binds chloroplast SRP receptor (GTP) on thylakoid membrane and protein translocated into lumen via thylakoid sec61 like translocon
4. thylakoid targeting sequence removed by thylakoid lumenal protease
5. molecular chaperones in thylakoid lumen (requires ATP) mediate folding of mature protein

Question 30

What are the steps to the pH-dependent pathway?

Answer 30

1. precursor protein fully folded in stroma
2. folded protein imported into thylakoid lumen via di-arginine containing thylakoid targeting sequence and unique receptor/translocon complex at thylakoid membrane (relies on energy from proton gradient between stroma and lumen [H+] thylakoid lumen > [H+] stroma
3. thylakoid targeting sequence on mature protein removed by lumenal protease