1. Translocation into mitochondria/plastids/peroxisome 1.The question and complexity Both mitochondria and plastids have their own genome but a large number of proteins in these organelles are encoded in the nucleus, made in the cytosol, and eventually cross the organelle membrane into the right compartment to do their job. Complexity: look at the complexity of these organelles—there are at least 6 different sub-organelle space in plastid/chloroplast and 4 in the mitochondria

2. The coordination of Protein targeting: Both plastid-made and cytosol-made proteins need to go to 6 spaces inside the chloroplasts! How do they know where to go and what are the signals?

3. 2. Signal sequences and receptors in chloroplast targeting 1)Signal sequence of chloroplast proteins is called transit peptide A precursor made in the cytosol contains N-terminal sequence that is required and sufficient for chloroplast targeting. After translocation, the transit peptide is cleaved by a peptidase. required and sufficient: If you delete the sequence off the chloroplast proteins, these proteins can not be transported into chloroplasts anymore; if you attach the transit peptide to other non-chloroplast proteins, then the sequence can direct these “foreign” proteins into chloroplasts. 2) How do you measure chloroplast import?

4. Chloroplasts and protein substrates: chop the leaves and isolate chloroplasts by centrifugation. The protein substrate can be made by in vitro transcription and translation in the presence of Met-S-35 to label the proteins radioactive.

5. The re-purification of chloroplast and treatment with protease (why?).

6. The gel picture: lane 1 is the precursor substrate before mixing with the chloroplasts, lane 2 contains the proteins from chloroplasts without protease treatment; lane 3 is the protein after protease treatment of the chloroplasts. Small subunit of Rubisco as example.

7. More recent studies using fusion proteins: fuse certain protein with an easy-to-detect markers such as GFP (green fluorescence protein) or GUS. Transform the constructed gene fusion into the plants to produce transgenic plants or do the transient assay but bombardment of t he gene into a plant tissue---measure the marker protein location. One can fuse just the transit peptide to a marker and see this sequence direct the transport of the marker into the chloroplast. Ssu protein Ssu transit peptide-fused to GUS Ssu transit peptide-fused to GFP

8. 3) Chloroplast proteins are delivered across the envelope through protein channels a)The chaperones hsp70 type I helps to keep the proteins unfolded before delivery through the channel. b)Several proteins inside the outer and inner membrane envelope form a “channel’-like structure and help substrate proteins go through the double membranes. The details involved in the interaction between the substrate and channel proteins are not understood. The outer membrane proteins called TOC (translocon of outer chloroplast envelope) and the inner membrane proteins are called TIC (translocon of inner chloroplast envelope). c)Both ATP and GTP are consumed. d)Once delivered into the stroma, a peptidase cleaves the transit sequence off the precursor. e)The proteins are then folded by chaperones (type I or type II). f)Proteins are assembled into complexes if needed.

9. Across the double envelope membranes

10. 4) The signal for thylakoid targeting requires two regions of N-terminal sequence---a bipartite transit sequence Was discovered by study of plastocyanin, a thylakoid lumen protein (in the electron transport chain between the PSII and PSI). A copper-binding protein that appears bright blue color. Envelope/thylakoid stroma thylakoid 1 2 3 4 m I P

11. 5) Trans-thylakoid pathways At least three different mechanisms are involved in protein targeting into thylakoid lumen/membrane. Different pathway help different substrate proteins. The “signals” for each pathway is not very well understood. a)Sec pathway--- because this pathway consists of proteins similar to those in the bacterial secretory pathway. This pathway is stimulated by pH gradient and ATP (plastocyanin takes this pathway) b)SRP pathway---consists of proteins with similarity to the ER translocation pathway. Stimulated by pH gradient and GTP. The light harvesting protein takes this pathway to get integrated into the thylakoid membrane. c)Delta pH pathway—only requires pH gradient (eg, oxygen evolution complex protein)

13. Summary Where and the how are proteins targeted?

14. 3. Protein targeting into the mitochondria---quite similar to the chloroplast mechanisms: 1)Also involves N-terminal sequences as signals; in fact the nature of the peptide sequence is also similar to the chloroplast targeting sequence although they are not exchangable. 2)Also involve outer and inner membrane proteins as translocons (they are called TOM and TIM instead of TOC/TIC). 3)Also requires ATP and chaperone type I (hsp70) 4)Requires more than one sequence signal for targeting to the intermembrane space; multiple pathways for targeting 5)Also involves peptidase to cleave precursors 6)Also involve chaperone to re-fold the proteins inside the organelle.

15. Model of TOM/TIM: Further enforce the idea of protein channels formed by some of the proteins in he membranes