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Isolation of Physiologically Active Thylakoids and Their Use in Energy-Dependent Protein Transport Assays
Published on: September 28, 2018
Prerequisites for terminal processing of thylakoidal Tat substrates
Stefan Frielingsdorf1, Ralf Bernd Klösgen
1Institute of Biology-Plant Physiology, Martin Luther University Halle-Wittenberg, Weinbergweg 10, 06120 Halle (Saale), Germany.
The Journal of Biological Chemistry
|June 22, 2007
Summary
The twin arginine translocation (Tat) system moves proteins across membranes. Signal peptide removal requires charged residues near cleavage sites, not hydrophobic ones, and can occur after protein release from Tat complexes.
Area of Science:
- Molecular Biology
- Cell Biology
- Protein Transport
Background:
- The twin arginine translocation (Tat) system facilitates the transport of folded proteins across biological membranes in bacteria and chloroplasts.
- Tat pathway substrates are translocated via signal peptides containing a twin arginine motif, which are typically removed post-translocation by specific peptidases.
Purpose of the Study:
- To investigate the molecular requirements for signal peptide processing by thylakoidal processing peptidase in the Tat pathway.
- To determine the role of amino acid composition at the cleavage site in Tat substrate maturation.
Main Methods:
- Site-directed mutagenesis of thylakoidal Tat substrate signal peptides and N-terminal mature protein regions.
- Thylakoid transport assays to analyze protein translocation and processing.
- Analysis of unprocessed transport intermediates within membrane complexes and the lipid bilayer.
Main Results:
- Signal peptide removal is dependent on the presence of charged or polar amino acid residues in either the C-terminal region of the signal peptide or the N-terminal region of the mature protein.
- Increased hydrophobicity in these regions inhibits signal peptide processing.
- Unprocessed Tat substrates exist as stable intermediates, either within large membrane complexes or free in the lipid bilayer, indicating dissociation from the Tat machinery prior to processing.
Conclusions:
- Tat pathway substrate maturation is governed by the physicochemical properties of amino acids flanking the peptidase cleavage site.
- Protein transport via the Tat system and subsequent signal peptide processing are separable events, with substrates potentially released from translocation complexes before maturation.
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