Dataset Information

Alternative splicing of Arabidopsis G6PD5 can recruits the NADPH-producing OPPP reactions to the endoplasmic reticulum

ABSTRACT: 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.

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

SUBMITTER: Iris Finkemeier

PROVIDER: PXD035221 | JPOST Repository | Mon Jul 11 00:00:00 BST 2022

REPOSITORIES: jPOST

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			Action	DRS
	220330_C22_011_OE480_Thermo_Rev06_%2322_019_PAP22_055.raw	Raw
	220330_C22_014_OE480_Thermo_Rev06_%2322_019_PAP22_055.raw	Raw
	220330_C22_014_OE480_Thermo_Rev06_%2322_019_PAP22_055_2.raw	Raw
	220330_C22_014_OE480_Thermo_Rev06_%2322_019_PAP22_070.raw	Raw
	220330_C22_014_OE480_Thermo_Rev06_%2322_019_PAP22_070_2.raw	Raw

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Publications

Alternative splicing of <i>Arabidopsis G6PD5</i> recruits NADPH-producing OPPP reactions to the endoplasmic reticulum.

Linnenbrügger Loreen L Doering Lennart L Lansing Hannes H Fischer Kerstin K Eirich Jürgen J Finkemeier Iris I von Schaewen Antje A

Frontiers in plant science 20220902

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 ...[more]

PMID: 36119606

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