Nine 3-ketoacyl-CoA thiolases (KATs) and acetoacetyl-CoA thiolases (ACATs) encoded by five genes in Arabidopsis thaliana are targeted either to peroxisomes or cytosol but not to mitochondria

Abstract

The sub-cellular location of enzymes of fatty acid beta-oxidation in plants is controversial. In the current debate the role and location of particular thiolases in fatty acid degradation, fatty acid synthesis and isoleucine degradation are important. The aim of this research was to determine the sub-cellular location and hence provide information about possible functions of all the putative 3-ketoacyl-CoA thiolases (KAT) and acetoacetyl-CoA thiolases (ACAT) in Arabidopsis. Arabidopsis has three genes predicted to encode KATs, one of which encodes two polypeptides that differ at the N-terminal end. Expression in Arabidopsis cells of cDNAs encoding each of these KATs fused to green fluorescent protein (GFP) at their C-termini showed that three are targeted to peroxisomes while the fourth is apparently cytosolic. The four KATs are also predicted to have mitochondrial targeting sequences, but purified mitochondria were unable to import any of the proteins in vitro. Arabidopsis also has two genes encoding a total of five different putative ACATs. One isoform is targeted to peroxisomes as a fusion with GFP, while the others display no targeting in vivo as GFP fusions, or import into isolated mitochondria. Analysis of gene co-expression clusters in Arabidopsis suggests a role for peroxisomal KAT2 in beta-oxidation, while KAT5 co-expresses with genes of the flavonoid biosynthesis pathway and cytosolic ACAT2 clearly co-expresses with genes of the cytosolic mevalonate biosynthesis pathway. We conclude that KATs and ACATs are present in the cytosol and peroxisome, but are not found in mitochondria. The implications for fatty acid beta-oxidation and for isoleucine degradation in mitochondria are discussed.

Identification and Characterization of Arabidopsis Indole-3-Butyric Acid Response Mutants Defective in Novel Peroxisomal Enzymes

“…In addition, the enzyme acting in the thiolase step ( Figure 8, step 5) has not been established. Although PED1 is the best-characterized and most active thiolase, two other thiolases (KAT1 and KAT5) are encoded in the Arabidopsis genome (Germain et al 2001;Carrie et al 2007) and could act on IBA intermediates. Furthermore, we expect that an enzyme to remove the CoA moiety (Figure 8, step 6) will be required; several thioesterases with unknown substrates are predicted to reside in the peroxisome (Reumann et al 2004) and may catalyze this reaction.…”

Peroxisome Function, Biogenesis, and Dynamics in Plants

“…These analyses have uncovered noncanonical PTS1 signals and revealed the importance of residues upstream of the PTS1 for targeting (Chowdhary et al, 2012). As not all predicted targeting signals confer peroxisomal localization (Ching et al, 2012), fusions of fluorescent reporters to candidate matrix proteins can be used to visualize localization in transgenic plants (Mano et al, 1999;Cassin-Ross and Hu, 2014;Wu et al, 2016) or following transient transfection of tobacco leaves (Reumann et al, 2009;Quan et al, 2013), cell culture (Mano et al, 1999;Carrie et al, 2007), or onion epidermal cells (Chowdhary et al, 2012;Skoulding et al, 2015). PTS1 proteins are recognized by PEX5 (van der Leij et al, 1993;Zolman et al, 2000), and PTS2 proteins are recognized by PEX7 ( Fig.…”

Función del gen 3-cetoacil-CoA tiolasa 2 (KAT2) en defensa y desarrollo. Participación de los peroxisomas en las respuestas a herida en Arabidopsis thaliana.

“…El genoma de Arabidopsis también contiene otros dos genes KAT (Germain et al, 2001). El gen KAT5, llamado previamente PEROXISOMAL 3-KETOACYL-COA THIOLASE 1 (PKT1) oPKT2, da lugar a dos mRNA diferentes por procesamiento alternativo (Carrie et al, 2007) y el gen KAT1, presenta alta homología de secuencia con el gen PED1/KAT2. De los diferentes genes KAT, el mayor nivel de expresión del gen KAT2, los fenotipos observados en el mutante ped1/kat2 y la coexpresión de este gen con otros del metabolismo de β-oxidación, a diferencia de KAT5 (Carrie et al, 2007), sugieren que este gen codifica la proteína KAT mas importante de β-oxidación en Arabidopsis (Footitt et al, 2007a).…”

“…Las enzimas KAT están incluidas en la superfamilia de tiolasas, que está ampliamente distribuida en todos los organismos y que en eucariotas se localiza en peroxisomas, mitocondrias y citosol (Peretó et al, 2005 intervienen en la síntesis y degradación de acetoacetil-CoA y otras rutas principalmente anabólicas con variedad de substratos (Peretó et al, 2005;Carrie et al, 2007). En el genoma de Arabidopsis hay dos genes que codifican enzimas ACAT (Carrie et al, 2007).…”

“…Recientemente se ha comprobado la localización peroxisomal in vivo de todas las proteínas KAT de Arabidopsis, así como su ausencia en mitocondrias mediante fusiones a GFP (Carrie et al, 2007). Referente a la especificidad en la longitud de cadena del substrato, aunque se han descrito tiolasas vegetales que si presentan especificidad, se ha sugerido que en Arabidopsis, al menos KAT2, no presenta especificidad ya que esta proteína parece actuar tanto con substratos cortos como con substratos largos (Germain et al, 2001;Castillo et al, 2004;Afitlhile et al, 2005).…”