Influence of tat mutations on the ribose-binding protein translocation in Escherichia coli Nathalie Pradel, a Claire-Lise Santini, a Chang-Yun Ye, a L ena Fevat, a,1 Fabien G erard, a,2 Meriem Alami, b and Long-Fei Wu a, * a Laboratoire de Chimie Bact erienne, UPR9043, Institut de Biologie Structurale et Microbiologie, CNRS, 31 chemin Joseph Aiguier, F-13402 Marseille cedex 20, France b Institut f€ ur Biochemie und Molekularbiologie and Fakulat€ atf€ ur Biologie, Universit€ at Freiburg, Hermann-Herder-Strasse 7, D-79104 Freiburg, Germany Received 16 May 2003 Abstract Proteins are exported across the bacterial cytoplasmic membrane either as unfolded precursors via the Sec machinery or in folded conformation via the Tat system. The ribose-binding protein (RBP) of Escherichia coli is a Sec-pathway substrate. Intriguingly, it exhibits fast folding kinetics and its export is independent of SecB, a general chaperone protein dedicated for protein secretion. In this study, we found that the quantity of RBP was significantly reduced in the periplasm of tat mutants, which was restored by in trans expression of the tatABC genes. Pulse-chase experiments showed that significant amount of wild-type RBP was processed in a secY mutant in the presence of azide (SecA inhibitor), whereas the processing of a slow folding RBP derivative was almost com- pletely blocked under the same conditions. These results would suggest that under the Sec-defective conditions the export of a portion of folded RBP could be rescued by the Tat system. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Ribose-binding protein; Folding; Translocation; Sec; Tat system Two major export systems, Sec and Tat, are involved in the translocation of proteins into the periplasm of gram-negative bacteria. The Sec apparatus seems to thread exported proteins in a locally unfolded state across membrane [1]. In contrast, the hallmark of the Tat system is that it is capable of translocating folded proteins or enzyme complexes across the plasma mem- brane [2]. The signal peptides of the proteins exported by the Tat pathway share similar overall structures with the Sec-dependent signal peptides, but possess a twin- arginine motif in the n-region, a weakly hydrophobic h-region, and a positively charged Sec-avoidance signal around the h-region [3]. Using these criteria, less than 30 Escherichia coli proteins have been predicted to be ex- ported via the Tat pathway and about a dozen of them were experimentally demonstrated [2,4]. While most studies are in agreement with these criteria, an increas- ing number of exceptions have been observed. Naturally occurring KR motif, or engineered KR, RK, and KK motifs preserve the capacity of mediating Tat pathway transport under certain sequence contexts [5,6]. Besides the signal peptide, studies with fusion proteins showed that the folding state of passenger proteins plays an important role in determination of pathway to be used for their export [3,7,8]. Extensive tertiary structure makes precursor protein translocation incompetent with the Sec pathway [9]. It is likely that during export via the Sec machinery a kinetic competition exists between productive translocation and folding of precursor intracellularly into a stable con- formation that is not compatible with translocation [9– 11]. Signal peptide and SecB protein retard the rate of folding; hence increase the translocation efficiency Biochemical and Biophysical Research Communications 306 (2003) 786–791 www.elsevier.com/locate/ybbrc BBRC * Corresponding author. Fax: +33-4-9171-8914. E-mail address: wu@ibsm.cnrs-mrs.fr (L.-F. Wu). 1 Present address: Department of Biological Sciences, University of Essex, Wivenhoe Park, Colchester CO4 3SQ, UK. 2 Present address: Horticultural Sciences and Plant Molecular and Cellular Biology, University of Florida, Gainesville, FL 32611, USA. 0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)01043-X