Functional and structural characterization of the minimal Sec translocase of the hyperthermophile Thermotoga maritima.
The genome of the hyperthermophilic bacterium Thermotoga maritima contains the genes that encode core subunits of the protein translocase, a complex consisting of the molecular motor SecA and the protein conducting pore SecYE. In addition, we identified an erroneous sequence in the genome encoding for a putative secG gene. The genes of the T. maritima translocase subunits were overexpressed in Escherichia coli and purified to homogeneity. T. maritima SecA showed a basal thermostable ATPase activity that was stimulated up to 4-fold by phospholipids with an optimum at 74 degrees C. Membrane vesicles and proteoliposomes containing SecYE or SecYEG supported 2- to 4-fold stimulation of the precursor dependent SecA ATPase activity. Imaging of small two-dimensional crystals of the SecYE complex using electron microscopy showed square-shaped particles with a side-length of about 6 nm. These results demonstrate that in T. maritima a highly thermostable translocase complex is operational.
Pretz
Monika G
MG
Department of Molecular Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Remigy
Hervé
H
Swaving
Jelto
J
Albers
Sonja V
SV
Garrido
Victoria G
VG
Chami
Mohamed
M
Engel
Andreas
A
Driessen
Arnold J M
AJ
D000251:Adenosine Triphosphatases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D003001:Cloning, Molecular; D003460:Crystallization; D026901:Membrane Transport Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D021381:Protein Transport; D000069816:SEC Translocation Channels; D000081416:SecA Proteins; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics
D001426:Bacterial Proteins; D026901:Membrane Transport Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D000069816:SEC Translocation Channels; D000251:Adenosine Triphosphatases; D000081416:SecA Proteins