1. “Folding quality control in the export of proteins by the bacterial twin-arginine translocation pathway” DeLisa MP, Tullman D, Georgiou G. Proc Natl Acad Sci. 13;100(10):6115-20. Geoffrey Meyerson Tarak Shah October 13, 2004 Microbiology & Biotechnology

2. Question of the Day What are the two known prerequisites necessary for the translocation of a protein through the Tat pathway? What are the two known prerequisites necessary for the translocation of a protein through the Tat pathway?

3. Agenda Introduction & background Introduction & background Sec pathwaySec pathway Tat pathwayTat pathway Overview of experiment Overview of experiment Discussion Discussion Applications to biotechnology Applications to biotechnology

4. Sec and Tat Pathways Robinson and Bolhuis, Nature, May 2001

5. Recognition Sequences Tat and Sec have similar peptide recognition sequences Tat and Sec have similar peptide recognition sequences Both have positively charged N-terminusBoth have positively charged N-terminus Followed by a region of hydrophobic amino acidsFollowed by a region of hydrophobic amino acids More polar C-terminus before cleavage site (leading to the rest of the active protein)More polar C-terminus before cleavage site (leading to the rest of the active protein) Tat pathway contains an arginine-arginine motif surrounded by a mostly conserved peptide sequence Tat pathway contains an arginine-arginine motif surrounded by a mostly conserved peptide sequence Sec does not contain arginine-arginine sequence Sec does not contain arginine-arginine sequence Typically Tat exhibits a less hydrophobic region than Sec Typically Tat exhibits a less hydrophobic region than Sec

6. Peptide Signal Sequences Note: Big arrows designate signal peptidase cleavage sites. Sec Signal Sequence Hydrophobic region N-domainC-domainH-domain Tat Signal Sequence Hydrophobic region Pohlschröder, FEMS Microbiology Review, September 2003

7. Green Fluorescent Protein GFP formed in cytoplasm Translocated through Tat GFP formed in cytoplasm Not translocated Robinson and Bolhuis, Nature, May 2001

8. The Tat Pathway What is it? What is it? How does it function? How does it function? Tat vs. Sec? Tat vs. Sec? Membrane Robinson and Bolhuis, Nature, May 2001

9. Leadup to Tat Clark and Theg (1997) Clark and Theg (1997) unfolding is not a ubiquitous requirement for protein translocationunfolding is not a ubiquitous requirement for protein translocation at least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplastsat least some domains of targeted proteins can maintain a nonlinear structure during their translocation into and within chloroplasts Hynds et al (1998) Hynds et al (1998) Unfolded proteins can be translocated either through the Sec or Tat pathway “Correct folding is not a prerequisite for translocation”

10. The Experiment Objective: To examine relationship between protein folding and export competence Objective: To examine relationship between protein folding and export competence How It Was Accomplished: By analyzing subcellular localization of proteins with structural disulfide bonds in the context of oxidizing and reducing cytoplasm and periplasm How It Was Accomplished: By analyzing subcellular localization of proteins with structural disulfide bonds in the context of oxidizing and reducing cytoplasm and periplasm

11. Main Concept Proteins do not form disulfide bonds in a reducing cytoplasm thus inhibiting Tat pathway Proteins do not form disulfide bonds in a reducing cytoplasm thus inhibiting Tat pathway Tat pathway was utilized in oxidizing cytoplasm and reducing periplasm Tat pathway was utilized in oxidizing cytoplasm and reducing periplasm In oxidizing cytoplasm and periplasm, no Tat transport occurs In oxidizing cytoplasm and periplasm, no Tat transport occurs Wild type ControlExperiment

12. The Experiment Fuse to AP 8 leader peptides that direct export via Tat pathway Fuse to AP 8 leader peptides that direct export via Tat pathway Treat cell samples to prevent the formation of disulfide bonds during fractionation by osmotic shock Treat cell samples to prevent the formation of disulfide bonds during fractionation by osmotic shock Determine the AP enzymatic activities in the cytoplasmic and periplasmic fractions Determine the AP enzymatic activities in the cytoplasmic and periplasmic fractions

13. Western Blotting WTExperiment Note: Grey = periplasm White = cytoplasm Tat mutant Periplasm Cytoplasm

14. Trypsin Resistance Note: periplasmic fraction

15. Tat Export of F AB F AB fusion of heavy chain and a leader peptide recognized by the Tat pathway led to tranlocation of entire antibody F AB fusion of heavy chain and a leader peptide recognized by the Tat pathway led to tranlocation of entire antibody Hitchhiker mode of export allows a leaderless polypeptide to be exported via association with a second polypeptide that can engage Tat Hitchhiker mode of export allows a leaderless polypeptide to be exported via association with a second polypeptide that can engage Tat

16. Tat Leader Sequences } } Class I Class II } } Class I Class II Tat sequence

17. Discussion Tat export requires specific Tat leader peptides as well as folding of the protein in the cytoplasm Tat export requires specific Tat leader peptides as well as folding of the protein in the cytoplasm ONLY folded proteins allowed to be exported through TatONLY folded proteins allowed to be exported through Tat Class I leader peptides required use of Tat pathway Class I leader peptides required use of Tat pathway Class II leader peptides were pathway agnostic Class II leader peptides were pathway agnostic The state of the protein (folding) determined which pathway was usedThe state of the protein (folding) determined which pathway was used

18. Discussion Inability to export AP and F AB proteins in the absence of disulfide bonds indicates the existence of a quality control mechanism Inability to export AP and F AB proteins in the absence of disulfide bonds indicates the existence of a quality control mechanism A cytoplasmic domain of Tat can function as a chaperone to the Tat membrane site A cytoplasmic domain of Tat can function as a chaperone to the Tat membrane site

19. Biotechnology Applications Proteins of interest could be fused with a Tat leader sequence. Proteins of interest could be fused with a Tat leader sequence. Proteins that would normally remain folded in the cytoplasm can now be induced to translocate through the Tat pathway. Proteins that would normally remain folded in the cytoplasm can now be induced to translocate through the Tat pathway. This facilitates protein separation, the most costly step in bioreactor processes This facilitates protein separation, the most costly step in bioreactor processes Way to engineer proteins for transport without needed to change structure of protein Way to engineer proteins for transport without needed to change structure of protein