Targeting and Assembly of proteins Destined for Chloroplasts and

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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?
Two types of cytosolic
ribosomes: free and
membrane-bound.
They synthesize proteins
with different destinations.
Fig. 4.3, Buchanan et al.
1. Peptide domains for targeting to different organelles
Organelle
Targeting Domain
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)
2. Chaperonins
play roles in
membrane
transport on
both sides of
the membrane.
Fig. 4.2 Buchanan et al.
Transport into organelles
can be carried out in cellfree systems using in
vitro-synthesized
precursors.
SS- rbcS
Fig. 4.5, Buchanan et al.
Maturation
intermediate
seen mainly
with proteins
destined for
the inner (i.e.,
thylakoid
membrane
and lumen)
compartments
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.
4. Chaperonins bind to precursor before, during
and after membrane translocation. Hsp70type 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.
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.
Chaperones
Tocs
Tics
Fig. 4.6, Buchanan et al.
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
• the first cleavage unmasks a second sorting
signal (zip code)
• the intermediate goes to the inner compartment
• the second cleavage generates mature protein
Chaperones
Tocs
Tics
Bipartite TP
on lumentargeted
protein.
Fig. 4.6, Buchanan et al.
3 pathways for protein targeting into
and across thylakoid membranes (to
lumen)
3 pathways, but may share some
components:
1. secA-dependent
2. pH gradient-dependent (or Tat pathway)
3. SRP-dependent
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)
OEC (or OE) proteins of PSII mediate water
splitting: Found in thylakoid lumen
Yamamoto, Plant Cell Physiol. 2001
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 twinarginine (Tat) motif that is essential for
transport across thylakoids
– also occurs in bacteria
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)
Role of SRP and its receptor in targeting to ER
Fig. 4.15, Buchanan et al.
P. Jarvis (2008) New Phytol 179:257-285
Differences in Mitochondrial vs Chloroplast
targeting/import
•
Many similarities between mitochondrial and
chloroplast targeting/import mechanisms, but
also important differences:
1. Mitochondria have 1 less membrane and 1
less soluble compartment
2. the proteins in the mito. membrane import
machinery are not homologous to the Toc
or Tic proteins
3. import into the mito. matrix requires an
electrochemical potential across the IM
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
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
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