Occurrence of two plastidic ATP/ADP transporters in Arabidopsis thaliana L.--molecular characterisation and comparative structural analysis of similar ATP/ADP translocators from plastids and Rickettsia prowazekii.
Recently, we sequenced a cDNA clone from Arabidopsis thaliana L. encoding an ATP/ADP transporter protein (AATP1) located in the plastid envelope membrane. The deduced amino acid sequence of AATP1 exhibits a high degree of similarity (> 66%) to the ATP/ADP transporter from the obligate intracellular gram-negative bacterium Rickettsia prowazekii. Here we report a second plastidic ATP/ADP carrier from A. thaliana (AATP2). As deduced from the amino acid sequence, AATP2 exhibits 77.6% identity to AATP1 and 36% to the rickettsial protein. Hydropathy analysis indicates that all three translocators are highly hydrophobic membrane proteins, which exhibit marked similarities and differences. The AATP1 translocator lacks the sixth transmembrane domain that is present in AATP2 and the bacterial transporter in R. prowazekii. In contrast to AATP1 and the bacterial transport protein, only AATP2 exhibits a truncated C-terminal end. To compare the general biochemical properties of AATP2 with the known transport properties of AATP1 we cloned the entire AATP2 cDNA into plasmid pJT118, leading to the presence of an additional N-terminal histidine tag of 10 amino acids. For heterologous expression of His10-AATP2 we chose the Escherichia coli strain C43, which was reported recently to allow overproduction of eucaryotic membrane transport proteins. After transformation and subsequent induction by isopropylthio-2-D-galactopyranoside intact E. coli cells harbouring plasmid pJT118 showed import of radioactively labelled ATP and ADP. As deduced from a Lineweaver-Burk analysis His10-AATP2 exhibited apparent Km values for ATP and ADP of 22 microM and 20 microM, respectively. Import of ADP into His10-AATP2-expressing E. coli cells occurred at a rate of 24 nmol x mg protein(-1) x h(-1), which was about threefold faster than import of ATP. These biochemical characteristics are similar to transport properties of the heterologously expressed His10-AATP1 protein.
Möhlmann
T
T
Pflanzenphysiologie, Universität Osnabrück, Germany.
Tjaden
J
J
Schwöppe
C
C
Winkler
H H
HH
Kampfenkel
K
K
Neuhaus
H E
HE
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D007700:Kinetics; D000226:Mitochondrial ADP, ATP Translocases / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism; D018087:Plastids / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D012283:Rickettsia prowazekii / Q000378:metabolism*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
C106084:AATP1 protein, Arabidopsis; C111793:AATP2 protein, Arabidopsis; D029681:Arabidopsis Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D000226:Mitochondrial ADP, ATP Translocases