Compartmentation of GABA metabolism raises intriguing questions Barry J. Shelp 1 , Robert T. Mullen 2 and Jeffrey C. Waller 3 1 Department of Plant Agriculture, University of Guelph, Guelph, ON, Canada N1G 2W1 2 Department of Molecular and Cellular Biology, University of Guelph, Guelph, ON, Canada N1G 2W1 3 Department of Chemistry & Biochemistry, Mount Allison University, Sackville, NB, Canada E4L 1G8 This synopsis covers the compartmentation of g-amino- butyrate (GABA) metabolism, highlighting recent prog- ress with Arabidopsis (Arabidopsis thaliana) and raising questions about mitochondrial GABA and succinic semi- aldehyde (SSA) transport, the fate of succinic semialde- hyde once it exits mitochondria, and biochemical interactions between GABA metabolism and related processes such as photorespiration. GABA metabolism and compartmentation in the pre- Arabidopsis era GABA is a ubiquitous four-carbon, non-protein amino acid that has been linked to stress, signaling and storage in plants [1]. It has long been known that GABA is derived from glutamate and then converted to SSA and succinate, which enters the tricarboxylic acid cycle. These three reactions were thought to be catalyzed by glutamate de- carboxylase (GAD), pyruvate- and 2-oxoglutarate-depen- dent GABA transaminase (GABA-T) and SSA dehydrogenase (SSADH), respectively, and referred to collectively as the GABA shunt pathway. In the pre-Ara- bidopsis era, research led to a model of the intracellular compartmentation of the GABA shunt, whereby GAD is located in the cytosol and GABA-T and SSADH are located in mitochondria, and thus implicates the transport of GABA across the mitochondrial membranes [2]. More recently, various aspects of this model have been corrobo- rated and extended by studies with Arabidopsis, including the characterization of the mitochondrial targeting signals for GABA-T and SSADH, the discovery of a mitochondrial GABA transporter, and a branch point for SSA catabolism that involves plastid and cytosolic isoforms of SSA reduc- tase (SSAR). In this Spotlight article, we briefly discuss these recent findings and how, despite their contribution to our overall understanding of the intracellular compart- mentation of GABA metabolism, several important ques- tions about this pathway remain unanswered. Localization and substrate specificity of GAD, GABA-T and SSADH GAD activity is restricted to the cytosol, specific for gluta- mate, maximally active at a pH of approximately 5.8, and regulated by pH and Ca 2+ -calmodulin. It is believed that this proton-consuming reaction can limit cytosolic acidosis in certain species during exposure to various stress con- ditions such as hypoxia. Consistent with the mitochondrial localization of soy- bean (Glycine max) GABA-T based on subcellular fractio- nations, a mitochondrial matrix targeting signal for Arabidopsis GABA-T was predicted to be located in the N-terminal 36 amino acids of the protein, and this was supported by in vivo targeting analysis of Arabidopsis GABA-T-green fluorescent protein (GFP) fusions [2]. Re- combinant Arabidopsis GABA-T activity has a pH opti- mum between 9.0 and 9.5 and is highly specific for GABA in the forward reaction but, unlike GABA-Ts from non-plant systems, appears to use pyruvate and glyoxylate as amino donors, rather than 2-oxoglutarate [2]. Interestingly, the glyoxylate-dependent reaction is irreversible, and glycine acts as a competitive inhibitor for the reaction. The prima- ry source of glyoxylate in green leaves originates from the oxygenation of ribulose-1, 5-bisphosphate and the oxida- tion of the resultant glycolate, the so-called photorespira- tory pathway. Therefore, it has been suggested that photorespiration can interact with GABA metabolism [2]. The previously reported mitochondrial location for soy- bean SSADH has also been supported by the identification of a putative 33-amino acid N-terminal mitochondrial targeting presequence in Arabidopsis SSADH, as well as allied subcellular fractionation studies, demonstrating that the protein is localized exclusively in the mitochon- drial matrix [3]. The Arabidopsis SSADH activity has a pH optimum between 9.0 and 10.0 and is essentially irrevers- ible, highly specific for SSA and NAD, and regulated by the ratio of NADH to NAD + . GABA transport Efficient growth of Arabidopsis on GABA has provided evidence of GABA transporters in plants, and heterologous complementation of a GABA-transport-deficient yeast mutant has identified that AtProT2 (Arabidopsis thaliana proline transporter 2) transports GABA (K m = 1.7 mM), although with less efficiency than for other stress-related compounds such as glycine, betaine and proline [4]. Nota- bly, similar substrate specificity was observed with three other plasma membrane-located members of the AtProT family [4]. Based on sequence homology to the ProT family overall, a member of the Arabidopsis amino acid transport- er family (AtGAT1) was functionally characterized in yeast (Saccharomyces cerevisiae) and frog (Xenopus laevis) oocytes and found to act as a proton-driven, high-affinity transporter of GABA (K m = 47 mM) and other related Spotlight Corresponding author: Shelp, B.J. (bshelp@uoguelph.ca) 1360-1385/$ – see front matter ß 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tplants.2011.12.006 Trends in Plant Science, February 2012, Vol. 17, No. 2 57