Quantitative Analysis of
Chloroplast Protein Targeting Pathways

Work as a PhD Student

My work as a MOAC PhD Student brings together work from Molecular Cell Biology and Numerical Methods for Partial Differential Equations. My supervisors are Colin Robinson (Molecular Cell Biology, Department of Biological Sciences), and Markus Kirkilionis (Mathematics Institute and the Centre for Scientific Computing). The main chloroplast protein targeting pathway under investigation in Colin's lab is the Twin-Arginine Translocation (Tat) pathway and my work will be directed at a quantitative analysis of this pathway in vitro; involving spatial (finite element) and non-spatial (compartmental) models based on schematic biological models in the literature.

Twin-Arginine Translocation

The Tat pathway is involved in the movement of proteins across the Escherichia coli cytoplasmic membrane and the Pisum sativum (pea) thylakoid membranes inside chloroplasts. Gene products targeted to this pathway are encoded in chromosomal DNA and exhibit a cleavable amino-terminal peptide sequence (the presequence) with a twin-arginine motif found just before the hydrophobic middle region of this peptide sequence. The peptide sequence is cleaved at the final destination to give the smaller mature form of the gene product; prior to this processing, we refer to the gene product as the preprotein.

Experimental Techniques

Sodium docdecyl sulphate (SDS) polyacylamide gel electrophoresis (PAGE) is used to locate and quantify translocated and untranslocated proteins and preproteins. Hydrogen bonds are broken by high temperatures (70+ degrees centrigrade) and the reducing agent beta-mercaptoethanol is used to break disulphide bridges. The anionic detergent SDS binds to the unfolded protein (specifically, to the peptide backbone) to prevent refolding and so that the polypeptide chain has a negative charge roughly proportional to the length of the chain. The polypeptide chains are separated on the gels and probed by silver staining, Coomassie staining, or immuno-blotting.

The import substrates of choice are the 16K and 23K Tat-substrate subunits of the thylakoid integral membrane photosystem II oxygen-evolving complex, and fluorescent proteins (variants of green fluorescent protein - GFP).

References

Protein Translocation Across Biological Membranes
William Wickner and Randy Schekman
Science 310:1452-1456 (Dec 2005) [Part of the "Getting Across the Membrane" section]

A good introduction to protein translocation.

Function and Evolution of Grana
Conrad W. Mullineaux
Trends in Plant Science 10(11):521-525 (Nov 2005)

Article putting thylakoid membranes in a wider context.

Location and Mobility of Twin Arginine Translocase Subunits in the Escherichia coli Plasma Membrane
Nicola Ray, Anja Nenninger, Conrad W. Mullineaux, and Colin Robinson
Journal of Biological Chemistry 280(18):17961-17968 (May 2005)

Probing of membrane-bound components of the Tat pathway.

The Escherichia coli Twin-arginine Translocation Apparatus Incorporates a Distinct Form of TatABC Complex, Spectrum of Modular TatA Complexes and Minor TatAB Complex
Joanne Oates, Claire M.L. Barrett, James P. Barnett, Katheryne G. Byrne, Albert Bolhuis, and Colin Robinson
Journal of Molecular Biology 346(1):295-305 (Feb 2005)

Interactions between subunits of the Tat translocase in E. coli.

The Core TatABC Complex of the Twin-arginine Translocase in Escherichia coli: TatC Drives Assembly Whereas TatA is Essential for Stability
Dorothea Mangels, Joanne Mathers, Albert Bolhuis, and Colin Robinson
Journal of Molecular Biology 345(2):415-423 (Jan 2005)

Outline of the Tat pathway in E. coli.

Available Files

Seminar 1.6 MB

Poster 212KB

Lab Talk 335KB