ARCHAEAL BINDING PROTEIN-DEPENDENT ABC TRANSPORTER - MOLECULAR AND BIOCHEMICAL-ANALYSIS OF THE TREHALOSE MALTOSE TRANSPORT-SYSTEM OF THE HYPERTHERMOPHILIC ARCHAEON THERMOCOCCUS LITORALIS/

We report the cloning and sequencing of a gene cluster encoding a malt ose/trehalose transport system of the hyperthermophilic archaeon Therm ococcus litoralis that is homologous to the malEFG cluster encoding th e Escherichia coli maltose transport system. The deduced amino acid se quence of the malE product, the trehalose/maltose-binding protein (TMB P), shows at its N terminus a signal sequence typical fur bacterial se creted proteins containing a glyceride lipid modification at the N-ter minal cysteine, The T. litoralis malE gene was expressed in E. coli un der control of an inducible promoter with and without its natural sign al sequence, In addition, in one construct the endogenous signal seque nce was replaced by the E. coli MalE signal sequence, The secreted, so luble recombinant protein was analyzed for its binding activity toward s trehalose and maltose, The protein bound both sugars at 85 degrees C with a K-d of 0.16 mu M. Antibodies raised against the recombinant so luble TMBP recognized the detergent-soluble TMBP isolated from T. lito ralis membranes as well as the products from all other DNA constructs expressed in B. coli. Transmembrane segments 1 and 2 as well as the N- terminal portion of the large periplasmic loop of the E. coli MalF pro tein are missing in the T. litoralis MalF. MalG is homologous througho ut the entire sequence, including the six transmembrane segments. The conserved EAA loop is present in both proteins, The strong homology fo und between the components of this archaeal transport system and the b acterial systems is evidence for the evolutionary conservation of the binding protein-dependent ABC transport systems in these two phylogene tic branches.