6533b858fe1ef96bd12b63da

RESEARCH PRODUCT

Tonoplast subcellular localization of maize cytochrome b5 reductases

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Plant cytochrome b 5 reductases (b 5 R) are assumed to be part of an ER-associated redox chain that oxidizes NADH to provide electrons via cytochrome b5 (cyt b 5 ) to ER-associated fatty acyl desaturase and related hydroxylases, as in mammalian cells. Here we report on cDNA cloning of a novel maize b 5 R, NFR II, strongly related to a previously cloned cDNA, NFR I (Bagnaresi et al., 1999, Biochem, J. 338, 499-5051. Maize b 5 R isoforms are produced by a small multi-gene family. The NFR cDNAs were shown to encode active b 5 Rs by heterologous expression in yeast. Both reductases, in addition to Fe 3+ -chelates, efficiently reduced Cu 2+ -chelates. Using a polyclonal antibody able to recognize both NFR I and NFR II isoforms, no ER or mitochondrial localization could be detected in maize roots. Unexpectedly, maize b 5 Rs were found to be targeted to the tonoplast. Using the most specific assay to measure NFR activity, we confirmed that the highest NFR specific activity is associated with tonoplast-enriched maize root fractions. Tonoplast targeting is not consistent with a role in desaturase reactions or with the other functions ascribed to date to plant b 5 R. This indicates that alternative ER-associated electron donors for desaturases need to be sought, and that plant b 5 Rs may have previously unexpected functions.