Topology of the plastid Ndh complex and its NDH-F subunit in thylakoid membranes.
We have investigated the topologies of Ndh (a plastid complex with NADH dehydrogenase activity) and its NDH-F subunit in thylakoids by trypsin and proteinase V8 digestion of both intact and Triton X-100-permeabilized barley thylakoids and identification of the products with antibodies against specific sequences of the NDH-A, NDH-K and NDH-F subunits. Antibody binding and protection against proteinases were also assayed. The analysis of the digestion products of NDH-F by immunodetection and matrix-assisted laser-desorption ionization-time-of-flight allowed us to propose its membrane topology and to compare it with bioinformatic predictions and with that of the homologous subunit (ND5/NuoL/NQO12) of the respiratory complex I. Results indicate that the thylakoid Ndh complex may have an L-shaped structure, similar to that of respiratory complex I, with the hydrophilic arm orientated towards the stroma and the hydrophobic arm inserted into the thylakoid. NDH-A and NDH-K may be located at the bridge between the two arms. Similar to ND5/NuoL/NQO12 of complex I, NDH-F must be distally located in the hydrophobic arm. NDH-F would include up to 15 transmembrane helices and 14 hydrophilic regions. A conserved His-349 in the X transmembrane helix could be involved in H+ pumping. The conserved Thr-181 NDH-F, whose probable phosphorylation increases the activity of the Ndh complex, is located within the hydrophilic region between the V and VI transmembrane helices.
Casano
Leonardo M
LM
Departamento de Biología Vegetal, Universidad de Alcalá, Alcalá de Henares, 28871 Madrid, Spain. leonardo.casano@uah.es
Lascano
H Ramiro
HR
Martín
Mercedes
M
Sabater
Bartolomé
B
D000595:Amino Acid Sequence; D000906:Antibodies / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000737:chemistry; D001467:Hordeum / Q000737:chemistry*; D007425:Intracellular Membranes / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009245:NADH Dehydrogenase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D010455:Peptides / Q000378:metabolism; D045322:Photosynthetic Reaction Center Complex Proteins / Q000737:chemistry; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D018087:Plastids / Q000737:chemistry*; D011237:Predictive Value of Tests; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry* / Q000378:metabolism; D012269:Ribosomal Proteins / Q000737:chemistry; D016415:Sequence Alignment / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D020524:Thylakoids / Q000737:chemistry* / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D000906:Antibodies; D029968:Escherichia coli Proteins; D010455:Peptides; D045322:Photosynthetic Reaction Center Complex Proteins; D010940:Plant Proteins; D021122:Protein Subunits; D012269:Ribosomal Proteins; C036004:ribosomal protein S15; C489904:NdhF protein, plant; D009245:NADH Dehydrogenase; C479743:NuoL protein, E coli; D014357:Trypsin