Cloning, expression, and purification of functional Sec11a and Sec11b, type I signal peptidases of the archaeon Haloferax volcanii.
Across evolution, type I signal peptidases are responsible for the cleavage of secretory signal peptides from proteins following their translocation across membranes. In Archaea, type I signal peptidases combine domain-specific features with traits found in either their eukaryal or bacterial counterparts. Eukaryal and bacterial type I signal peptidases differ in terms of catalytic mechanism, pharmacological profile, and oligomeric status. In this study, genes encoding Sec11a and Sec11b, two type I signal peptidases of the halophilic archaeon Haloferax volcanii, were cloned. Although both genes are expressed in cells grown in rich medium, gene deletion approaches suggest that Sec11b, but not Sec11a, is essential. For purification purposes, tagged versions of the protein products of both genes were expressed in transformed Haloferax volcanii, with Sec11a and Sec11b being fused to a cellulose-binding domain capable of interaction with cellulose in hypersaline surroundings. By employing an in vitro signal peptidase assay designed for use with high salt concentrations such as those encountered by halophilic archaea such as Haloferax volcanii, the signal peptide-cleaving activities of both isolated membranes and purified Sec11a and Sec11b were addressed. The results show that the two enzymes differentially cleave the assay substrate, raising the possibility that the Sec11a and Sec11b serve distinct physiological functions.
Dept. of Life Sciences, Ben Gurion University, P.O. Box 653, Beersheva 84105, Israel.
Amir
A
Fine
Vered
V
Irihimovitch
Idit
I
Dahan
Zvia
Z
Konrad
Jerry
J
Eichler
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D020745:Genome, Archaeal; D019622:Haloferax volcanii / Q000201:enzymology*; D008565:Membrane Proteins / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012697:Serine Endopeptidases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*
D019843:Archaeal Proteins; D008565:Membrane Proteins; D012697:Serine Endopeptidases; C026515:type I signal peptidase