The branched-chain amino acid transaminase gene family in Arabidopsis encodes plastid and mitochondrial proteins

Abstract

Branched-chain amino acid transaminases (BCATs) play a crucial role in the metabolism of leucine, isoleucine, and valine. They catalyze the last step of the synthesis and/or the initial step of the degradation of this class of amino acids. In Arabidopsis, seven putative BCAT genes are identified by their similarity to their counterparts from other organisms. We have now cloned the respective cDNA sequences of six of these genes. The deduced amino acid sequences show between 47.5% and 84.1% identity to each other and about 30% to the homologous enzymes from yeast (Saccharomyces cerevisiae) and mammals. In addition, many amino acids in crucial positions as determined by crystallographic analyses of BCATs from Escherichia coli and human (Homo sapiens) are conserved in the AtBCATs. Complementation of a yeast Deltabat1/Deltabat2 double knockout strain revealed that five AtBCATs can function as BCATs in vivo. Transient expression of BCAT:green fluorescent protein fusion proteins in tobacco (Nicotiana tabacum) protoplasts shows that three isoenzymes are imported into chloroplasts (AtBCAT-2, -3, and -5), whereas a single enzyme is directed into mitochondria (AtBCAT-1).

Figure 1

Alignment of the amino acid sequences deduced from the seven AtBCAT cDNA sequences. Amino acid residues conserved in at least five AtBCATs are highlighted by black boxes. The conserved Lys suggested to bind pyridoxal phosphate is indicated by a gray box. Numbered arrowheads indicate important amino acids as determined by the crystallization of the respective enzymes from E. coli and human. Positions of the introns are given by gray vertical arrows.

Figure 2

Complementation analysis of the AtBCATs in the yeast double knockout mutant Δbat1/Δbat2. To examine the suggested BCAT function in vivo, the different AtBCAT cDNA sequences were cloned into pRS425GPD vectors and transformed into Δbat1/Δbat2. Restored growth on medium lacking Val, Leu, and iso-Leu indicates the competent BCAT activity of AtBCAT-1 (A), -2 (B), -3 (C), -5 (D), and -6 (E) in yeast. Complementation was also observed with ScBAT-1 as positive control. No growth is detected without transformation (Δbat1/Δbat2) or after transformation of the vector without insert (pRS425GPD).

Figure 3

Subcellular localization of different AtBCATs. A, Constructs used for the transient transformation of tobacco protoplasts. cDNA fragments representing the 47 (AtBCAT-1), 126 (AtBCAT-2), 413 (AtBCAT-3), and 156 (AtBCAT-5) N-terminal amino acids were cloned in frame upstream of the green fluorescent protein (GFP) reading frame. Expression of the fusion proteins is under control of the cauliflower mosaic virus 35S promoter and the nopaline synthase terminator. B, Fluorescence images of the protoplasts taken with the following filter sets: GFP, D395x/440 DLCP/HQ510/50 and GG 475 LP; this set allows the specific detection of the GFP fluorescence. Fluorescein isothiocyanate (FITC), HQ 470/40/Q 495 LP/HQ 500 LP; this filter set allows the simultaneous detection of GFP and chlorophyll autofluorescence, and chloroplasts containing the GFP fusion protein will appear yellow. MitoTracker, HQ545/30/Q570 LP/HQ 610/75; this filter set is optimized for the specific visualization of the MitoTracker red dye. Rhodamine, BP530–585/FT600/LP615; this filter allows the detection of the chlorophyll autofluorescence. Space bars correspond to 10 μm. The GFP fluorescence of the AtBCAT-1:GFP fusion protein coincides with the pattern of Mitotracker red-stained mitochondria. The GFP fluorescences of AtBCAT-2, -3, and -5 fusion proteins are congruent with the autofluorescence of chlorophyll, as seen in the yellow FITC images, indicating their localization in chloroplasts.