Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1.
NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex-complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated 'NDH-1MS', was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.
Zhang
Pengpeng
P
Department of Biology, Plant Physiology and Molecular Biology, University of Turku, Turku, FI-20014, Finland.
Battchikova
Natalia
N
Paakkarinen
Virpi
V
Katoh
Hirokazu
H
Iwai
Masako
M
Ikeuchi
Masahiko
M
Pakrasi
Himadri B
HB
Ogawa
Teruo
T
Aro
Eva-Mari
EM
D000595:Amino Acid Sequence; D002846:Chromatography, Affinity; D000458:Cyanobacteria / Q000166:cytology / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D042967:Electron Transport Complex I / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D005798:Genes, Bacterial / Q000235:genetics; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes / Q000737:chemistry / Q000378:metabolism; D009532:Nickel / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D020524:Thylakoids / Q000378:metabolism
D046912:Multiprotein Complexes; D021122:Protein Subunits; D009532:Nickel; D042967:Electron Transport Complex I