Isolation and structural characterization of the Ndh complex from mesophyll and bundle sheath chloroplasts of Zea mays.
Complex I (NADH: ubiquinone oxidoreductase) is the first complex in the respiratory electron transport chain. Homologs of this complex exist in bacteria, mitochondria and chloroplasts. The minimal complex I from mitochondria and bacteria contains 14 different subunits grouped into three modules: membrane, connecting, and soluble subcomplexes. The complex I homolog (NADH dehydrogenase or Ndh complex) from chloroplasts from higher plants contains genes for two out of three modules: the membrane and connecting subcomplexes. However, there is not much information about the existence of the soluble subcomplex (which is the electron input device in bacterial complex I) in the composition of the Ndh complex. Furthermore, there are contrasting reports regarding the subunit composition of the Ndh complex and its molecular mass. By using blue native (BN)/PAGE and Tricine/PAGE or colorless-native (CN)/PAGE, BN/PAGE and Tricine/PAGE, combined with mass spectrometry, we attempted to obtain more information about the plastidal Ndh complex from maize (Zea mays). Using antibodies, we detected the expression of a new ndh gene (ndhE) in mesophyll (MS) and bundle sheath (BS) chloroplasts and in ethioplasts (ET). We determined the molecular mass of the Ndh complex (550 kDa) and observed that it splits into a 300 kDa membrane subcomplex (containing NdhE) and a 250 kDa subcomplex (containing NdhH, -J and -K). The Ndh complex forms dimers at 1000-1100 kDa in both MS and BS chloroplasts. Native/PAGE of the MS and BS chloroplasts allowed us to determine that the Ndh complex contains at least 14 different subunits. The native gel electrophoresis, western blotting and mass spectrometry allowed us to identify five of the Ndh subunits. We also provide a method that allows the purification of large amounts of Ndh complex for further structural, as well as functional studies.
Darie
Costel C
CC
Brookdale Department Molecular, Cell and Developmental Biology, Mount Sinai School of Medicine, New York, NY 10029-6574, USA. costel.darie@mssm.edu
Biniossek
Martin L
ML
Winter
Veronika
V
Mutschler
Bettina
B
Haehnel
Wolfgang
W
D000818:Animals; D002736:Chloroplasts / Q000378:metabolism*; D004579:Electron Transport; D015180:Electrophoresis, Gel, Two-Dimensional; D018087:Plastids / Q000378:metabolism; D011808:Quinone Reductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011994:Recombinant Proteins / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D003313:Zea mays / Q000201:enzymology*
D011994:Recombinant Proteins; C041831:NADH dehydrogenase (quinone); D011808:Quinone Reductases