Mitochondria + Chloroplast Part 2 Flashcards
What are chloroplasts?
Semi-autonomous plant cell organelle – derived from the photosynthetic cyanobacterium
Site of photosynthesis (Ps)
CO2, H2O + sun (light energy)
Ps in chloroplasts
O2 + energy-rich carbohydrates (sugars)
Aerobic respiration in mitochondria
CO2 + ATP (chemical energy)
Also involved in several other important metabolic processes
e.g., fatty acid and amino acid biosynthesis, nitrogen and sulfur assimilation
Chloroplasts are often highly mobile in cells
Move along cytoskeleton elements via molecular motors
What is chloroplast morphology?
Double-membrane-bound organelle
Complex organization
Envelope: consists of outer and inner membranes
Outer membrane: contains porins, not as permeable to ions/small molecules as outer membrane of mitochondria
Intermembrane space
Inner membrane: highly impermeable; contains various transporters
Outer and inner membranes differ in terms of overall protein and lipid composition – functionally distinct membranes
Thylakoids – 3rd (internal) membrane system
Flattened membranes discs arranged in stacks – grana thylakoids - or between stacks – stroma thylakoids
Thylakoid membranes: site of ATP synthase; maintain H+ gradient in the thylakoid lumen
Thylakoid lumen: the aqueous interior of thylakoid; high [H+]
Functionally equivalent to mitochondrial intermembrane space
Stroma: aqueous interior (space inside of envelope and outside of thylakoids)
Contains enzymes involved in carbohydrate synthesis and plastid genome
Circular: size and copy/gene number vary between plant species
Encodes ribosomal proteins, some Ps proteins, including a few located in thylakoid, tRNA, and rRNAs, and some RNA polymerase subunits
All other 3000 plastid proteins nuclear-encoded
Chloroplasts are often connected via stromules
Long, stroma-filled membrane tubules that are highly dynamic – branched, and rapidly extend and contract
Allows for efficient metabolite transfer, communication, etc. between chloroplasts and/or other organelles (e.g., ER-mitochondria, etc)
What are the chloroplast dynamics?
Chloroplasts multiply by fission: divide (in synchronization) in response to certain environmental stimuli and/or developmental signals
e.g., fission of chloroplasts at end of G1 – organelle duplication prior to S phase
Two sets of protein machinery involved in chloroplast division: FtsZ and PD
Both form ring-like structures either on inside or outside of the chloroplast envelope
Rings ‘tightened’ and ‘pinch’ organelle into two daughter chloroplasts
How do Chloroplasts divide?
FtsZ Division Machinery
Internal machinery – located on the stromal side of the inner membrane
Derived from cyanobacterial endosymbiont from which chloroplasts evolved
Includes FtsZ1 and FtsZ2
Soluble stromal proteins; homologs of bacterial division protein FtsZ
FtsZ1/2 spontaneously assemble into long, filamentous polymers at the midway point (equator) on the inner membrane surface
Form FtsZ-ring (Z-ring) at the future chloroplast division site
Initial positioning of FtsZ proteins at the equator is mediated by several other soluble, stromal proteins – ARC3, MinD, and MinELinkage of Z-ring to the inner membrane at chloroplast division site mediated by ARC6
Integral inner membrane-spanning protein localized to the equator in dividing chloroplast
The stromal-facing domain of ARC6 links to and stabilizes FtsZ-ring; mutation in ARC6 gene disrupts fission process – results information of 1-2 giant chloroplasts
ARC6 also controls ‘tightening’ of FtsZ-ring: ARC6’s interaction with Z-ring signals start of chloroplast constriction process
Intermembrane space (IMS)-facing domain of ARC6 binds to Plastid Dividing (PD) machinery in chloroplast outer membrane
Includes PDV1 and PDV2
Heterodimeric, integral outer transmembrane proteins recruited (via binding to ARC6) to the midway point in dividing chloroplast
IMS-facing domains of PDV1/2 interact with IMS-facing domain of ARC6
What is the PD division machinery?
PDV1 and PDV2 also binds to ARC5
Cytoplasmic-facing domains of PDV1 & PDV2 bind to ARC6 at the site of division on chloroplast outer membrane surface
ARC5 is a cytoplasmic, soluble dynamin-related GTPase protein
ARC5 assembles into ‘spiral-like’ structures that form PD ring
PD ring wraps around outside of chloroplast – ‘tightening’ of PD-ring (via GTP hydrolysis) causes constriction of the outer envelope
Dynamins also involved in mitochondrial fission (Drp1) and the formation of clathrin-coated transport vesicles at TGN and plasma membrane
How does FtsZ and PD Division machinery work together?
Continued constriction and eventual scission (separation) of chloroplast membranes involves the concerted action of both ‘internal’ Z-ring and ‘external’ PD-ring
Results in the formation of two ‘daughter’ chloroplasts
What is chloroplast protein targetting?
Like mitochondria, the majority of chloroplast proteins (soluble & membrane-bound) are nuclear-encoded, synthesized on ‘free’ ribosomes in the cytoplasm, and targeted post-translationally (after they are translated) to the organelle
A highly efficient, but complex process – chloroplasts contain 6 sub-compartments
A number of key similarities (and differences) with mitochondrial protein targeting
Destinations:
The outer membrane, intermembrane space, inner membrane, stroma, thylakoid membrane, thylakoid lumen
All chloroplast proteins possess unique targeting sequences
Specific sequences of amino acids that serve as ‘zip codes’ to mediate protein targeting from cytoplasm to chloroplast surface and to specific sub chloroplast compartment
Multiple (6) sub chloroplast targeting pathways
Vary depending on protein’s final location in chloroplast
The outer or inner membrane
Intermembrane space
Stroma
Thylakoid lumen or membrane
Different pathways rely on different sub chloroplast targeting signals and shared and/or unique import machinery
Targeting and import of chloroplast stromal proteins is the best-understood process
Most stromal-destined proteins possess a stromal import sequence
Located at protein’s N-terminus
Enriched in hydroxylated residues (S/T) and small hydrophobic residues
Cleaved following protein import into the stroma
Responsible for targeting the nascent stromal protein to the cytoplasmic surface of chloroplast and its subsequent translocation across outer and inner membranes
Targeted from Cytoplasm to Stroma
In the cytoplasm, the precursor (stromal-destined) protein synthesized on free ribosomes and recognized by cytoplasmic molecular chaperones [+ATP]
Cytosolic Hsp70
Maintain conformation of the nascent protein in partially unfolded import-competent state
At the surface of the chloroplast, protein’s stromal import sequence recognized (bound) by Toc complex (Relies on GTP for energy)
The multi-protein complex includes receptor(s) subunits, transmembrane channel, and various accessory proteins
Binding of stromal import sequence to Toc receptor involves GTP (energy requirement for import)
Precursor protein transferred through Toc complex and then through adjacent Tic complex
Toc and Tic complexes adjacent to each other at contact sites – outer and inner membranes appressed against each other
Precursor protein translocation occurs across both membranes sequentially
Unlike mitochondria, protein translocation across outer and inner chloroplast membranes not mediated by H+ electrochemical potential
N-terminal stromal-import sequence of precursor protein exists Tic complex channel intro stroma
Stromal-import sequence cleaved by stromal processing enzyme
Emerging precursor protein also recognized and bound by stromal chaperone protein Hsp93
Hsp93 acts as a molecular motor (‘ratchet’)
Undergoes ATP-dependent conformational changes that ‘pulls’ protein into the stroma and prevents ‘backsliding’ of protein back into cytoplasm and assists with initial protein folding (mitochondrial matrix Hsp70)
Molecular chaperones in the stroma [+ATP] ensure cleaved, mature protein is properly folded
TOC and TIC complexes also involved in important or insertion of other chloroplast proteins
All nuclear-encoded chloroplast proteins (soluble and membrane-bound) access chloroplast through Toc and/or Tic complexes
Targeting proteins to thylakoid lumen:
Precursor protein imported from the cytoplasm into stroma as described above, except
Removal of the stromal-important sequence by stromal protease reveals a thylakoid-targeting sequence
Precursor protein then engages one of several different thylakoid import pathways:
SRP- dependent pathway
pH-dependent pathway
What is the SRP-dependent pathway?
Precursor protein in stroma maintained in partially unfolded, import-competent state by stromal chaperones [+ATP]
Thylakoid-targeting sequence recognized by chloroplast signal recognition particle (SRP)
SRP-binds to chloroplast SRP receptor [+GTP] on thylakoid membrane and protein translocated into the lumen via Sec61-like translocon
Thylakoid-targeting sequence removed by luminal protease
Molecular chaperones in thylakoid lumen [+ATP] mediate the folding of the mature protein
What is the pH-dependent pathway?
Precursor protein fully folded in the stroma
Folded protein imported into thylakoid lumen via di-arginine-containing thylakoid-targeting sequence and unique receptor/translocon complex at the thylakoid membrane
The mechanism for how a large, folded protein is translocated across the thylakoid membrane is not well understood
Thylakoid-targeting sequence on mature protein removed by luminal protease
