Mitochondrial fatty acid synthase 1 Dual-localized enzymatic components constitute the fatty acid synthase systems 2 in mitochondria and plastids

Plant fatty acid biosynthesis occurs in both plastids and mitochondria. Here, we report 27 the identification and characterization of Arabidopsis ( Arabidopsis thaliana ) genes 28 encoding three enzymes shared between the mitochondria- and plastid-localized Type II 29 fatty acid synthase systems (mtFAS and ptFAS, respectively). Two of these enzymes, β- 30 ketoacyl-acyl carrier protein (ACP) reductase (pt/mtKR) and enoyl-ACP reductase 31 (pt/mtER) catalyze two of the reactions that constitute the core four-reaction cycle of the 32 FAS system, which iteratively elongates the acyl-chain by two carbon atoms per cycle. 33 The third enzyme, malonyl-CoA:ACP transacylase (pt/mtMCAT) catalyzes the reaction 34 that loads the mtFAS system with substrate by malonylating the phosphopantetheinyl 35 cofactor of ACP. Green fluorescent protein (GFP) fusion experiments revealed that the 36 these enzymes localize to both chloroplasts and mitochondria. This localization was 37 validated by characterization of mutant alleles, which were rescued by transgenes 38 expressing enzyme variants that were retargeted only to plastids or only mitochondria. 39 The singular retargeting of these proteins to plastids rescued the embryo-lethality 40 associated with disruption of the essential ptFAS system, but these rescued plants 41 displayed phenotypes typical of the lack of mtFAS function, including reduced lipoylation 42 of the H subunit of the glycine decarboxylase complex, hyperaccumulation of glycine, 43 and reduced growth. However, these latter traits were reversible in an elevated CO 2 44 atmosphere, which suppresses mtFAS-associated photorespiration-dependent 45 chemotypes. Sharing enzymatic components between mtFAS and ptFAS systems constrains the evolution of these non-redundant fatty acid biosynthetic machineries.


INTRODUCTION
9 particularly the case for the proteins encoded by AT2G05990, AT2G30200 and 148 AT1G24360. The full-length proteins encoded by these three genes guide the 149 expression of the fused GFP to both mitochondria and plastids ( Figure 1C to 1E). In the 150 case of AT2G05990-encoded protein, the N-terminal pre-sequence directs GFP to 151 plastids (row 2 of Figure 1E), but the segment which lacks this N-terminal pre-sequence 152 guides GFP to mitochondria (row 3 of Figure 1E). In contrast, the N-terminal pre-153 sequences of AT2G30200 and AT1G24360 direct the GFP-fusions to mitochondria (row 154 2 of Figures 1C and 1D), whereas upon removal of these N-terminal pre-sequences from 155 each protein, the remaining mature segments direct the accumulation of the fused GFP 156 protein to plastids (row 3 of Figures 1C and 1D).

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Subcellular targeting information of the AT3G45770-encoded protein is simpler to 158 interpret, with the full-length AT3G45770-protein (rows 1 of Figure 1B) and the N-159 terminal pre-sequence (rows 2 of Figure 1B) directing GFP to mitochondria, whereas 160 removal of the N-terminal pre-sequence from the AT3G45770-encoded protein guides 161 GFP to the cytosol (rows 3 of Figure 1B). In summary therefore, in contrast to the 162 AT3G45770-encoded protein, proteins encoded by AT2G30200, AT1G24360 and 163 AT2G05990 encode dual localization signals, for both mitochondria and plastids. One of 164 these signals resides in the N-terminal pre-sequence and the other in the mature portion  for genetic complementation. The expression of each candidate Arabidopsis protein was 172 accurately targeted to yeast mitochondria, by genetically fusing the mitochondrial pre-173 sequence (MP) of the yeast COQ3 protein (Hsu et al., 1996) to the N-terminus of the 174 mature Arabidopsis proteins, and their expression in yeast was under the transcriptional 175 control of the constitutive PGK promoter (de Moraes et al., 1995).

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The yeast mutant strains that lack mtFAS functions cannot utilize glycerol as a sole 177 carbon source because they are deficient in respiration (Torkko et al., 2001). On media 178 that use glycerol as the sole carbon source, the yeast mutant strains lacking individual 179 mtFAS components (i.e., mct1, oar1 or etr1) showed no growth unless they expressed

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In the case of the ER components of the mtFAS system, recombinant purified proteins 187 encoded by AT2G05990 or AT3G45770 (expressed in E. coli) were also evaluated in 188 vitro for their ability to catalyze the expected chemical reaction. Because enoyl-ACP 189 reductases are active with both enoyl-ACP (their native substrate) and enoyl-CoA (Chen 190 et al., 2008), in these experiments each protein was tested for the ability to reduce 191 enoyl-CoA substrates. These assays were conducted with Δ2 trans -10:1-CoA and Δ2 trans 192 -16:1-CoA, and activity was monitored by the decrease in A340 due to the coupled 193 oxidation of the pyrimidine nucleotides (NADH or NADPH). Both AT2G05990 and 194 AT3G45770 proteins were capable of reducing the enoyl-CoA substrates, and they 195 exhibited comparable K m , V max and catalytic efficiency (k cat /K m ) with both tested substrates ( Figure 2B). Moreover, these assays established that AT2G05990 is an 197 NADH-dependent reductase, and its activity with NADPH was undetectable. In contrast,

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AT3G45770 is an NADPH-dependent reductase, and its activity with NADH was 199 undetectable.

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In combination therefore, the GFP-transgenic fluorescence data, the yeast genetic 201 complementation experiments and the biochemical characterizations of purified proteins 202 indicated that three Arabidopsis genes (AT2G30200, AT1G24360 and AT2G05990) 203 encode proteins that are dual targeted to plastids and mitochondria, and they have the 204 ability to catalyze the MCAT, KR and ER reactions, respectively. We therefore labeled 205 these proteins as pt/mtMCAT (AT2G30200), pt/mtKR (AT1G24360) and pt/mtER 206 (AT2G05990) indicating their dual localizations. In contrast, AT3G45770 encoded a 207 mitochondrially-localized ER enzyme, which we labeled as mtER, indicating its 208 functionality in the sole organelle.

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The in planta roles of pt/mtMCAT and pt/mtKR in mtFAS

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The role of pt/mtMCAT (AT2G30200) and pt/mtKR (AT1G24360) as enzymatic 211 components of the mtFAS system was further evaluated by characterizing Arabidopsis 212 plants carrying T-DNA-tagged mutant alleles at each locus (details in the Materials and 213 Methods section). As previously described (Bryant et al., 2011)

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Therefore, we designed transgenic complementation experiments to further confirm that 223 these dual localized gene products are also components of the mtFAS system.

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Specifically, plants that were heterozygous mutants at the AT2G30200 locus 225 (pt/mtMCAT) or the AT1G24360 locus (pt/mtKR) were transformed with transgenes that 226 express two versions of the pt/mtMCAT or pt/mtKR proteins, respectively. One version of 227 these transgenes expressed the ORF that encodes the full-length proteins, and as

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In contrast, the transgenic plants expressing the N-terminally truncated pt/mtMCAT or 244 pt/mtKR proteins (i.e., these proteins were predicted to be plastid localized but not 245 targeted to mitochondria) exhibited reduced size ( Figure 3A). Most significantly, when 246 these plants were grown in a 1% CO 2 atmosphere, where photorespiration deficiency is 247 suppressed, the stunted growth morphology was reversed ( Figure 3A). Further

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Two enoyl-ACP reductase isozymes for mtFAS 272 Two genetic loci appear to encode proteins that catalyze the enoyl-ACP reduction 273 reaction of mtFAS, the AT2G05990 locus, which encodes an NADH-dependent 274 reductase, and the AT3G45770 locus, which encodes an NADPH-dependent reductase.

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The former protein was dual targeted (i.e., pt/mtER), and the latter was targeted to 276 mitochondria (i.e., mtER). We genetically dissected the roles of these two enoyl-ACP

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In contrast, biochemical analyses that evaluated the metabolic status of these mutant 297 plants indicated that both mtER and pt/mtER contribute to mtFAS. The evidence that 298 supports this conclusion includes: a) immunoblot analysis that indicated the lipoylation 299 states of GDC in mter and pt/mter-rnai single mutants was reduced to between 60% to 300 80% of the wild-type levels, and this protein was even further under-lipoylated to about 301 10% of the wild-type level in the double mutant plants ( Figure 4B); b) accompanied with 302 the reduction in lipoylation status of the H protein, glycine accumulation was increased 303 by about 15-fold in these double mutant lines; and c) this latter attribute was completely 304 reversed when these double mutant plants were grown in the 1% CO 2 atmosphere, 305 which suppresses photorespiration ( Figure 4C). Other changes in amino acid levels 306 were detected in these double mutants (Supplemental Table 1). Collectively therefore, 307 we conclude that the mtFAS system appears to be redundantly enabled by two enoyl-308 ACP reductases, a dual localized enoyl-ACP reductase (pt/mtER; AT2G05990) and a 309 mitochondrially localized enoyl-ACP reductase (mtER; AT3G45770).  mitochondria, referred to as "dual-targeted", and thus they contribute to both mtFAS and 337 ptFAS capabilities. In addition, the mitochondrial ER reaction appears to be catalyzed by 338 redundant enzymes, an NADPH-dependent mtER and a dual-targeted NADH-dependent 339 pt/mtER. The pt/mtER and mtER belong two distinct families of enzymes; pt/mtER

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Complementation tests were performed as previously described (Torkko et al., 2001).      transcriptionally controlled by the phosphoglycerate kinase promoter (pPGK) and 548 terminator (tPGK). Mitochondrial pre-sequence of yeast COQ3 protein was fused 549 to N-terminus of each protein to ensure the mitochondrial localization. All yeast 550 strains, carrying the indicated expression cassettes were grown on media 551 containing either glycerol or glucose as the sole carbon source, and a dilution 552 series served as the inoculum for each strain.