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Targeting and assembly of proteins destined for chloroplasts and mitochondria

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Targeting to inner chloroplast compartments: thylakoid membrane and lumen ... OEC (or OE) proteins of PSII mediate water splitting: Found in thylakoid lumen ... – PowerPoint PPT presentation

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Title: Targeting and assembly of proteins destined for chloroplasts and mitochondria


1
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?

2
Two types of cytosolic ribosomes free and
membrane-bound. They synthesize proteins with
different destinations.
Fig. 4.3, Buchanan et al.
3
Peptide domains for targeting to different
organelles
Targeting Domain
Organelle
4
Chaperonins play roles in membrane transport on
both sides of the membrane.
Fig. 4.2 Buchanan et al.
5
Transport into organelles can be carried out in
cell-free systems using in vitro-synthesized
precursors.
SS- rbcS
Fig. 4.5, Buchanan et al.
6
Maturation intermediate seen mainly with proteins
destined for the inner (i.e., thylakoid membrane
and lumen) compartments
7
Features of chloroplast protein import (into the
organelle)
  • Post-translational
  • Proteins synthesized as precursors with an Amino
    (N)-terminal extension.
  • The N-terminal extension acts as the zip code,
    and often called transit peptide. It is removed
    during or soon after import.

8
  • 4. Chaperonins bind to precursor before 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.

9
  • 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)
  • Only 1 inner membrane translocon complex (Tic)
    protein identified so far (110 kDa)
  • 7. After import, specific endoproteases in stroma
    remove transit peptide sequences.

10
Chaperones Tocs Tics
Fig. 4.6, Buchanan et al.
11
Targeting to inner chloroplast compartments
thylakoid membrane and lumen
  • Proteins destined to the inner compartments
    (I.e., thylakoid membrane and lumen) have longer
    Transit Peptides with 2 zip codes.
  • They are removed in two steps
  • cleave cleave
  • Precursor ? Intermediate ? Mature

12
  • the first cleavage unmasks a second sorting
    signal (zip code)
  • the intermediate goes to the inner compartment
  • the second cleavage generates mature protein

13
Chaperones Tocs Tics Bipartite TP on
lumen-targeted protein.
Fig. 4.6, Buchanan et al.
14
Targeting into and across thylakoid membranes (to
lumen)
  • 3 pathways, but may share some components
  • secA-dependent
  • pH gradient-dependent
  • SRP-dependent

15
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)

16
OEC (or OE) proteins of PSII mediate water
splitting Found in thylakoid lumen
Yamamoto, Plant Cell Physiol. 2001
17
pH Gradient-dependent 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 motif that is essential for
    transport across thylakoids

18
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
  • requires GTP
  • pH gradient stimulates
  • Examples of proteins transported by this pathway
  • LHCPs light-harvesting chlorophyll proteins
    (cab genes)

19
Role of SRP and its receptor in targeting to ER
Fig. 4.15, Buchanan et al.
20
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

21
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., etc.
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