Targeting and assembly of proteins destined for chloroplasts and mitochondria

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Targeting and assembly of proteins destined for
chloroplasts and mitochondria

• How are proteins targeted to chloroplasts and
  mitochondria from the cytoplasm?
• How do they get through the membranes?

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Two types of cytosolic ribosomes free and
membrane-bound. They synthesize proteins with
different destinations.
Fig. 4.3, Buchanan et al.
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1. Peptide domains for targeting to different
organelles
Targeting Domain
Organelle
ER Signal peptide (SP)
Chloroplast Transit peptide (TP)
Mitochondrion Pre-sequence
Nucleus Nuclear localization signal (NLS)
Peroxisome Peroxisomal targeting signal(s) (PTS1 and PTS2)
Vacuole Vacuolar sorting signal (VSS)
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2. Chaperonins play roles in membrane transport
on both sides of the membrane.
Fig. 4.2 Buchanan et al.
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Transport into organelles can be carried out in
cell-free systems using in vitro-synthesized
precursors.
SS- rbcS
Fig. 4.5, Buchanan et al.
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Maturation intermediate seen mainly with proteins
destined for the inner (i.e., thylakoid membrane
and lumen) compartments
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Features of chloroplast protein import (into the
organelle)

1. Post-translational
2. Proteins synthesized as precursors with an Amino
   (N)-terminal extension.
3. The N-terminal extension acts as the zip code,
   and often called transit peptide. It is removed
   during or soon after import.

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• 4. Chaperonins bind to precursor before, during
  and after membrane translocation. Hsp70-type
  chaperonins maintain partially folded state in
  cytoplasm, whereas Hsp60 (cpn60) and Hsp70
  promote folding inside organelle.
• 5. ATP and GTP are also required for envelope
  membrane translocation.

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• 6. Import receptors and translocation complexes
  (i.e., Tocs and Tics) assemble at envelope
  membrane contact sites.
• Proteins of the outer membrane complex are called
  Tocs
• 159, 75 and 34 kDa (159 and 34 kDa proteins bind
  GTP)
• Toc75 is the main pore (a beta-barrel protein)
• HSP70 IAP (or import intermediate associated
  protein) - functions between IM and OM)
• Inner membrane translocon complex proteins are
  called Tics
• Tic20, Tic21, and Tic110 (kDa) proteins form
  channel
• 7. After import, specific endoproteases in stroma
  remove transit peptide sequences.

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Chaperones Tocs Tics
Fig. 4.6, Buchanan et al.
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Targeting to inner chloroplast compartments
thylakoid membrane- spanning and lumen proteins

• Proteins destined to the inner compartments
  (i.e., thylakoid-membrane spanning and lumen
  proteins) have longer Transit Peptides with 2 zip
  codes.
• They are removed in two steps
• cleave cleave
• Precursor ? Intermediate ? Mature

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• the first cleavage unmasks a second sorting
  signal (zip code)
• the intermediate goes to the inner compartment
• the second cleavage generates mature protein

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Chaperones Tocs Tics Bipartite TP on
lumen-targeted protein.
Fig. 4.6, Buchanan et al.
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3 pathways for protein targeting into and across
thylakoid membranes (to lumen)

• 3 pathways, but may share some components
• secA-dependent
• pH gradient-dependent (or Tat pathway)
• SRP-dependent

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SecA-Dependent Pathway

• Involves a soluble, secA (bacterial gene)
  homologue
• requires ATP
• pH gradient stimulates
• Examples of proteins transported this way
• Plastocyanin
• OE33 33 kDa protein of the oxygen evolving
  component of PSII (OEC)

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OEC (or OE) proteins of PSII mediate water
splitting Found in thylakoid lumen
Yamamoto, Plant Cell Physiol. 2001
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pH Gradient-dependent (or Tat) pathway

• Requires the pH gradient across thylakoid
  membrane (generated by photosynthesis)
• Examples of proteins transported by this
  pathway
• OE24 and OE17 subunits of the OEC
• Transit peptides of these proteins have
  twin- arginine (Tat) motif that is essential for
  transport across thylakoids
• also occurs in bacteria

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SRP-Dependent Pathway

• Involves a signal recognition particle (SRP)-like
  protein (cSRP54)
• SRP occurs in prokaryotes and eukaryotes, where
  its composed of an RNA (7SL) and several proteins
• Green plant chloroplast SRP does not have an RNA
  subunit
• requires GTP
• pH gradient stimulates
• Examples of proteins transported by this pathway
• LHCPs light-harvesting chlorophyll proteins
  (cab genes)

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Role of SRP and its receptor in targeting to ER
Fig. 4.15, Buchanan et al.
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P. Jarvis (2008) New Phytol 179257-285
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Differences in Mitochondrial vs Chloroplast
targeting/import

• Many similarities between mitochondrial and
  chloroplast targeting/import mechanisms, but
  also important differences
• Mitochondria have 1 less membrane and 1 less
  soluble compartment
• the proteins in the mito. membrane import
  machinery are not homologous to the Toc or Tic
  proteins
• import into the mito. matrix requires an
  electrochemical potential across the IM

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Proteins targeted to multiple organelles

• There are many targeted to both chloroplasts and
  mitochondria
• Example Most (18 out of 20) of the organellar
  aminoacyl-tRNA synthetases in Arabidopsis are
  targeted to both organelles
• Some proteins found in both the chloroplast and
  the ER (or Golgi)
• Some ER ? Golgi ? Chloroplast protein targeting
  (Carbonic anhydrase 1 of A.t.)
• Prominent in algae with a Chloroplast ER

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Bioinformatic Predictions of Protein Subcellular
Locations from Sequences

• Target P
• Predicts whether protein is Chloro., Mito.,
  Secreted (Signal pep.) or Cytosolic
• Signal P
• Predicts whether protein has signal peptide
• ChloroP
• Predicts whether protein has a Transit peptide
• (Locating proteins in the cell using TargetP,
  SignalP, and related tools. 2007. O. Emanuelsson,
  S. Brunak, G. von Heijne, H. Nielsen. Nature
  Protocols 2, 953-971)
• Psort
• Mitoprot., Predotar