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The formation of a camalexin-biosynthetic metabolon

ABSTRACT: Arabidopsis thaliana efficiently synthesizes the antifungal phytoalexin camalexin without apparent release of bioactive intermediates, such as indole-3-acetaldoxime, suggesting channeling of the biosynthetic pathway involving an enzyme complex. To identify such protein interactions, two independent untargeted co-immunoprecipitation (Co-IP) approaches with the biosynthetic enzymes CYP71B15 and CYP71A13 as baits were performed and the camalexin biosynthetic P450 enzymes were co-purified. These interactions were confirmed by targeted Co-IP and förster resonance energy transfer measurements based on fluorescence lifetime imaging (FLIM-FRET). Furthermore, interaction of CYP71A13 and Arabidopsis P450 reductase 1 (ATR1) and recruitment of cytosolic gamma-glutamyl peptidase 1 (GGP1) to the endoplasmic reticulum (ER) was observed. An increased substrate affinity of CYP79B2 in presence of CYP71A13 was shown, indicating substrate channeling by the complex. During camalexin biosynthesis, indol-3-acetonitrile (IAN) is activated by CYP71A13 and glutathionylated to GS-IAN. To clarify whether this is mediated by a specific glutathione transferase (GST), CYP71A13 was expressed together with each of the 54 Arabidopsis GSTs in S. cerevisiae and GS-IAN-synthesis was observed for GSTU2. Corresponding studies of knock-out and overexpression plants did not show an altered camalexin content. However, an influence of the closely related GSTU4 on camalexin biosynthesis was demonstrated. GSTU4 is co-expressed with tryptophan- and camalexin specific enzymes and physically interacts with CYP71A13. Surprisingly, camalexin concentrations were reduced in knock-out and elevated in GSTU4 overexpressing plants. This shows that GSTU4 is not directly involved in camalexin biosynthesis but rather has a regulatory role or is involved in transport or a competing mechanism.

INSTRUMENT(S): LTQ Orbitrap Elite, Q Exactive

ORGANISM(S): Arabidopsis Thaliana (mouse-ear Cress)

TISSUE(S): Rosette

SUBMITTER: Stephanie Wilhelm

LAB HEAD: Bernhard Kuster

PROVIDER: PXD008812 | Pride | 2019-08-26

REPOSITORIES: Pride

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			Action	DRS
	00576_F09_P004336_B00_A00_R1.raw	Raw
	00576_G09_P004337_B00_A00_R1.raw	Raw
	00611_G02_P04673_B00_A00_R1.raw	Raw
	00611_H02_P04674_B00_A00_R1.raw	Raw
	00645_A10_P004888_B00_A00_R1.raw	Raw

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Project description:Glucose-6-phosphate dehydrogenase is the rate-limiting enzyme of the oxidative pentose-phosphate pathway (OPPP). The OPPP mainly provides NADPH and sugar-phosphate building blocks for anabolic pathways and is present in all eukaryotes. In plant cells, the irreversible part of the OPPP is found in several compartments. Among the isoforms catalyzing the first OPPP step in Arabidopsis, G6PD1 to G6PD4 target plastids (with G6PD1 being also directed to peroxisomes), whereas G6PD5 and G6PD6 operate in the cytosol. We noticed that alternative splice forms G6PD5.4 and G6PD5.5 may encode N-terminally extended proteoforms. Compared to cytosolic G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts showed accumulatedion at in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms accumulate inlabel different subdomains of the endoplasmic reticulum (ER), and analysis of C-terminal roGFP fusions showed that their catalytic domains face the cytosol. In g6pd5-1 g6pd6-2 Arabidopsis mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms G6PD5.4 and G6PD5.5 were both active, and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases isoforms (catalyzing the second OPPP step), we noticed that isoform Arabidopsis PGL2 carries a C-terminal CaaX motif that may be prenylated for membrane attachment. Reporter-PGL2 fusions co-localized with G6PD5.4 in ER subdomains, which was abolished by Cys-to-Ser exchange in the 256CSIL motif. For Among the Arabidopsis 6-phosphogluconate dehydrogenase isoforms (catalyzing the third OPPP step) S-acylated peptides were detected ofor all three isoforms three PGD isoforms were detected for all three Arabidopsis PGD isoforms in a recent palmitoylome, listing with dual cytosolic/peroxisomal PGD2 displaying three sites sites for PGD2. Co-expression of GFP-PGD2 co-expression occasionally diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2’s presence. Upon GFP-based pull-down of GFP-G6PD5.4, uUnlabeled PGD2 and PGD2 PGL2 (but not PGL2) co-purified withwere enriched by GFP-G6PD5.4, but also enzymes that depend on NADPH provision at the ER, indicative ofupon GFP-based pull-down, demonstrating that the physical interaction with the two dehydrogenaseshree OPPP enzymes physically interact. When the membrane-bound G6PD5.5 and 5.4 reporter fusionvariants were co-expressed with enzymes that depend on NADPH provision at the ER, co-localization in ER subdomains was found for KCR1 (ketoacyl-CoA reductase, involved in fatty acidthe elongation of fatty acids), and ATR1 (NADPH: cytochrome-P450 oxidoreductase), but also withor pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as the indirectly (via ATR) NADPH-dependent cyto¬chrome P450 enzymes, like C4H/CYP73A5 (cinnamate 4-hydroxylase), co-localization in ER subdomains was observed. Thus, alternative splicing of G6PD5 can direct the NADPH producing OPPP reactions to the cytosolic face of the ER, where they may operate as membrane-bound metabolon to support several important biosynthetic pathways of plant cells.

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The formation of a camalexin-biosynthetic metabolon

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