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. 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)
PTS1 is at the C-terminus; PTS2 is at the N-terminus.
VSSs can be near or in the N-terminal signal peptide, or can be C-terminal peptides.

4. 2. Chaperonins play roles in membrane transport on both sides of the membrane.
Chaperones: Hsp70 type
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
Pre-SS is about 24 kDa, and mature SS is about 18 kDa.
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, 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.

9. 6. Import receptors and translocation complexes (i. e
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.
The 75 kDa protein is the pore, and is unusual as it is a Beta-barrel proteins (spans membrane 7 times but with beta strand structures in stead of alpha helices.

10. Chaperones
Tocs
Tics
Fig. 4.6, Buchanan et al.

11. 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

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. Bipartite TP on lumen-targeted protein.
Chaperones
Tocs
Tics
Bipartite TP on lumen-targeted protein.
Fig. 4.6, Buchanan et al.

14. 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

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)
Also requires other sec genes besides secA.

16. OEC (or OE) proteins of PSII mediate water splitting: Found in thylakoid lumen
Yamamoto, Plant Cell Physiol. 2001

17. 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
SRRxFLK is the bacterial signal (consensus) peptide.

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
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)
Most chloroplast SRPs don’t’ seem to have the RNA component like bacteria.

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

20. P. Jarvis (2008) New Phytol 179:257-285
Carbonic Anhydrase 1 is imported via the ER-Golgi system; this pathway may be an evolutionary remnant that was maintained for dual targeting (ER-Golgi and Chloroplasts) of a few proteins. The PDI of Chlamy that binds the psbA mRNA 5’ UTR is dual targeted to ER and chloroplasts.
P. Jarvis (2008) New Phytol 179:

21. 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

22. 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

23. 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 O. Emanuelsson, S. Brunak, G. von Heijne, H. Nielsen. Nature Protocols 2, )
Psort
Mitoprot., Predotar