title: Subcellular localization and tissue specific expression of amidase 1 from Arabidopsis thaliana.
abstract: Amidase 1 (AMI1) from Arabidopsis thaliana converts indole-3-acetamide (IAM), into indole-3-acetic acid (IAA). AMI1 is part of a small isogene family comprising seven members in A. thaliana encoding proteins which share a conserved glycine- and serine-rich amidase-signature. One member of this family has been characterized as an N-acylethanolamine-cleaving fatty acid amidohydrolase (FAAH) and two other members are part of the preprotein translocon of the outer envelope of chloroplasts (Toc complex) or mitochondria (Tom complex) and presumably lack enzymatic activity. Among the hitherto characterized proteins of this family, AMI1 is the only member with indole-3-acetamide hydrolase activity, and IAM is the preferred substrate while N-acylethanolamines and oleamide are not hydrolyzed significantly, thus suggesting a role of AMI1 in auxin biosynthesis. Whereas the enzymatic function of AMI1 has been determined in vitro, the subcellular localization of the enzyme remained unclear. By using different GFP-fusion constructs and an A. thaliana transient expression system, we show a cytoplasmic localization of AMI1. In addition, RT-PCR and anti-amidase antisera were used to examine tissue specific expression of AMI1 at the transcriptional and translational level, respectively. AMI1-expression is strongest in places of highest IAA content in the plant. Thus, it is concluded that AMI1 may be involved in de novo IAA synthesis in A. thaliana.
authors.affiliation: Department of Plant Physiology, Ruhr-University Bochum, Universitaetsstrasse 150, ND 2/27, 44801 Bochum, Germany. stephan.pollman@rub.de
authors.forename: Stephan
authors.initials: S
authors.lastname: Pollmann
authors.forename: Daniel
authors.initials: D
authors.lastname: Neu
authors.forename: Thomas
authors.initials: T
authors.lastname: Lehmann
authors.forename: Oliver
authors.initials: O
authors.lastname: Berkowitz
authors.forename: Tina
authors.initials: T
authors.lastname: Schäfer
authors.forename: Elmar W
authors.initials: EW
authors.lastname: Weiler
mesh_terms: D000581:Amidohydrolases / Q000737:chemistry / Q000378:metabolism*; D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D008969:Molecular Sequence Data; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013347:Subcellular Fractions / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D017931:DNA Primers; D000581:Amidohydrolases; C019665:amidase