First published online April 3, 2003; 10.1104/pp.102.018325
Plant Physiol, May 2003, Vol. 132, pp. 318-330
Identification of Novel Mitochondrial Protein Components of
Chlamydomonas reinhardtii. A Proteomic
Approach1
Robert
van Lis,
Ariane
Atteia,
Guillermo
Mendoza-Hernández, and
Diego
González-Halphen1*
Departamento de Genética Molecular, Instituto de
Fisiología Celular (R.v.L., A.A., D.G.-H.) and Departamento de
Bioquímica, Facultad de Medicina (G.M.-H.), Universidad
Nacional Autónoma de México, 04510 Mexico D.F.,
Mexico
Pure mitochondria of the photosynthetic alga
Chlamydomonas reinhardtii were analyzed using blue
native-polyacrylamide gel electrophoresis (BN-PAGE). The major
oxidative phosphorylation complexes were resolved:
F1F0-ATP synthase, NADH-ubiquinone
oxidoreductase, ubiquinol-cytochrome c reductase, and
cytochrome c oxidase. The oligomeric states of these
complexes were determined. The F1F0-ATP synthase runs exclusively as a dimer, in contrast to the C.
reinhardtii chloroplast enzyme, which is present as a monomer
and subcomplexes. The sequence of a 60-kD protein, associated with the
mitochondrial ATP synthase and with no known counterpart in any other
organism, is reported. This protein may be related to the strong
dimeric character of the algal F1F0-ATP
synthase. The oxidative phosphorylation complexes resolved by BN-PAGE
were separated into their subunits by second dimension sodium dodecyl
sulfate-PAGE. A number of polypeptides were identified mainly on the
basis of their N-terminal sequence. Core I and II subunits of complex
III were characterized, and their proteolytic activities were
predicted. Also, the heterodimeric nature of COXIIA and COXIIB subunits
in cytochrome c oxidase was demonstrated. Other
mitochondrial proteins like the chaperone HSP60, the alternative
oxidase, the aconitase, and the ADP/ATP carrier were identified.
BN-PAGE was also used to approach the analysis of the major chloroplast
protein complexes of C. reinhardtii.
1
This work was supported by Consejo
Nacional de Ciencia y Tecnología (grant no. 27754N), by
Dirección General de Asuntos para el Personal Académico
(Mexico; grant no. IN204595), by Dirección General de Estudios de
Posgrado-Universidad Nacional Autónoma de México (PhD
student fellowship to R.v.L.), and by the National Science Foundation
(grant no. MCB-9975765 to the Chlamydomonas genome project).
*
Corresponding author; e-mail dhalphen{at}ifisiol.unam.mx; fax
5255-56-22-56-11.
© 2003 American Society of Plant Biologists
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