ABSTRACT
The Tat machinery enables folded proteins to be translocated across biological membranes. In vitro studies have shown that Tat substrates can interact with membranes prior to translocation. In this study we investigated the initial states of this interaction with thylakoid lipid monolayers at the air-water interface by using monolayer techniques combined with infrared reflection-absorption spectroscopy (IRRAS). We used enhanced green fluorescent protein (EGFP) as a model substrate and the signal peptide SP16 from the 16 kDa protein of the spinach oxygen-evolving complex (OEC16). We found that the signal peptide is essential for the interaction of the model substrate with lipid monolayers. IRRA spectroscopy showed an increased amount of α-helical secondary structure elements for the chimeric model substrate i16/EGFP (SP16 fused to EGFP) compared with EGFP; this can be attributed to the signal peptide.
Subject(s)
Gene Products, tat/chemistry , Gene Products, tat/metabolism , Lipids/chemistry , Protein Sorting Signals , Signal Transduction , Thylakoids/chemistry , Water/chemistry , Adsorption , Air , Green Fluorescent Proteins/chemistry , Green Fluorescent Proteins/metabolism , Models, Biological , Protein Folding , Spectrophotometry, Infrared , Thylakoids/metabolism , Unilamellar Liposomes/chemistry , Unilamellar Liposomes/metabolismABSTRACT
The twin arginine translocation (Tat) machinery which is capable of transporting folded proteins across lipid bilayers operates in the thylakoid membrane of plant chloroplasts as well as in the cytoplasmic membrane of bacteria. It is composed of three integral membrane proteins (TatA, TatB, and TatC) which form heteromeric complexes of high molecular weight that accomplish binding and transport of substrates carrying Tat pathway-specific signal peptides. Western analyses using affinity purified antibodies showed in both, juvenile and adult tissue from Arabidopsis thaliana, an approximately equimolar ratio of the TatB and TatC components, whereas TatA was detectable only in minor amounts. Upon Blue Native-PAGE, TatB and TatC were found in four heteromeric TatB/C complexes possessing molecular weights of approximately 310, 370, 560 and 620 kDa, respectively, while TatA was detected only in a molecular weight range below 200 kDa. The implications of these findings on the currently existing models explaining the mechanism of Tat transport are discussed.
Subject(s)
Arabidopsis Proteins/metabolism , Arabidopsis/metabolism , Protein Subunits/metabolism , Arabidopsis Proteins/chemistry , Arabidopsis Proteins/isolation & purification , Intracellular Membranes/metabolism , Pisum sativum/metabolism , Protein Structure, Tertiary , Protein Subunits/chemistry , Protein Subunits/isolation & purification , Protein Transport , Solubility , Species Specificity , Thylakoids/metabolismABSTRACT
The chloroplast is an organelle of prokaryotic origin that is situated in an eukaryotic cellular environment. As a result of this formerly endosymbiotic situation, the chloroplast houses a unique set of protein transport machineries. Among those are evolutionarily young transport pathways which are responsible for the import of the nuclear-encoded proteins into the organelle as well as ancient pathways operating in the 'export' of proteins from the stroma (the former cyanobacterial cytosol) across the thylakoid membrane into the thylakoid lumen. In this review, we have tried to address the main features of these various transport pathways.
