Characterization of the cardiolipin synthase from arabidopsis thaliana

Cardiolipin (CL) is an anionic membrane phospholipid of a unique tetraacyl structure, consisting of two phosphatidyl moieties linked by a glycerol bridge. It constitutes the cytoplasmic membrane of eubacteria and represents the marker lipid of mitochondria in eukaryotes, where CL is predominantly found in the inner membrane. In all eukaryotes, CL biosynthesis occurs in the inner mitochondrial membrane via phosphatidylglycerol (PG) as conserved intermediate and the CL synthase (CLS) catalyzes the transfer of the phosphatidyl motiety from cytidinediphosphate-diacylglycerol onto PG. The analysis of yeast and mammalian mutants deficient in CL or PG biosynthesis provided evidence that these anionic lipids are required for proper biogenesis and function of mitochondria and, thus, for maintenance of cell integrity. In contrast to yeast and mammalian cells, little is known about the CL biosynthesis and functions in plants. To gain access to the CLS protein of plants, the respective gene from Arabidopsis thaliana that encodes a hydrophobic protein of 38 kDa with a cleavable transit peptide for the import into mitochondria was cloned and its identity was confirmed by functional expression studies in Escherichia coli and yeast. The plant CLS was shown to exhibit an alkaline pH optimum, a strict requirement for divalent cations, and a distinctly lower Km value for CDP-DAG than for PG. It displayed a preference for both its substrates esterified with unsaturated acyl groups and the activity of the enzyme increased concomitantly with the unsaturation level of the substrates. Similarly to the mammalian enzyme, but unlike the yeast one, the plant CLS was highly sensitive towards detergents. Solubilization and purification experiments revealed that the protein requires a defined phospholipid environment, particularly the presence of CL, to acquire its catalytically active conformation. To investigate the functional role of CL in plant mitochondria, A. thaliana mutants carrying a T-DNA insertion in four different positions of the CLS gene were analysed. Plants homozygous with respect to the insertion in either the CLS promoter region or the intron 7 of the CLS gene showed no significant alterations in the CL levels. On the other hand, only heterozygous insertional mutants were obtained for plants with a T-DNA insertion in exons 1 and 5, respectively, of the CLS gene. These data suggest that the T-DNA insertion in the exons represent loss-of-function mutations, and that these mutations are lethal for A. thaliana plants.

Grantor dc:publisher

Publikationsserver der RWTH Aachen University

Year dc:date

2006

Author dc:creator

• Novicki, Marcin

Contributors dc:contributor

• Frentzen, Margarete

• info:eu-repo/classification/ddc/570
• Biowissenschaften, Biologie
• Cardiolipin
• Lipide
• Mitochondrium
• Ackerschmalwand
• lipid biosynthseis
• CDP-DAG
• anionic phospholipid
• T-DNA mutant

Statement dc:rights

• info:eu-repo/semantics/openAccess

Language dc:language

eng

Identifier

https://publications.rwth-aachen.de/search?p=id:%22RWTH-CONV-123124%22

OAI identifier oai:identifier

oai:publications.rwth-aachen.de:61462