We studied the localization of 6-phosphogluconate dehydrogenase (PGD) isoforms of Arabidopsis (Arabidopsis thaliana). Similar polypeptide lengths of PGD1, PGD2, and PGD3 obscured which isoform may represent the cytosolic and/or plastidic enzyme plus whether PGD2 with a peroxisomal targeting motif also might target plastids. Reporter-fusion analyses in protoplasts revealed that, with a free N terminus, PGD1 and PGD3 accumulate in the cytosol and chloroplasts, whereas PGD2 remains in the cytosol. Mutagenesis of a conserved second ATG enhanced the plastidic localization of PGD1 and PGD3 but not PGD2. Amino-terminal deletions of PGD2 fusions with a free C terminus resulted in peroxisomal import after dimerization, and PGD2 could be immunodetected in purified peroxisomes. Repeated selfing of pgd2 transfer (T-)DNA alleles yielded no homozygous mutants, although siliques and seeds of heterozygous plants developed normally. Detailed analyses of the C-terminally truncated PGD2-1 protein showed that peroxisomal import and catalytic activity are abolished. Reciprocal backcrosses of pgd2-1 suggested that missing PGD activity in peroxisomes primarily affects the male gametophyte. Tetrad analyses in the quartet1-2 background revealed that pgd2-1 pollen is vital and in vitro germination normal, but pollen tube growth inside stylar tissues appeared less directed. Mutual gametophytic sterility was overcome by complementation with a genomic construct but not with a version lacking the first ATG. These analyses showed that peroxisomal PGD2 activity is required for guided growth of the male gametophytes and pollen tube-ovule interaction. Our report finally demonstrates an essential role of oxidative pentose-phosphate pathway reactions in peroxisomes, likely needed to sustain critical levels of nitric oxide and/or jasmonic acid, whose biosynthesis both depend on NADPH provision.
The metabolite transporter GPT1 is important for NADPH provision in both plastid and peroxisomes, mostly during pollen tube growth towards ovules, and is thus essential for fertilization in Arabidopsis.
Recent work revealed that PGD2, an Arabidopsis 6-phosphogluconate dehydrogenase (6-PGD) catalysing the third step of the oxidative pentose-phosphate pathway (OPPP) in peroxisomes, is essential during fertilization. Earlier studies on the second step, catalysed by PGL3, a dually targeted Arabidopsis 6-phosphogluconolactonase (6-PGL), reported the importance of OPPP reactions in plastids but their irrelevance in peroxisomes. Assuming redundancy of 6-PGL activity in peroxisomes, we examined the sequences of other higher plant enzymes. In tomato, there exist two 6-PGL isoforms with the strong PTS1 motif SKL. However, their analysis revealed problems regarding peroxisomal targeting: reporter–PGL detection in peroxisomes required construct modification, which was also applied to the Arabidopsis isoforms. The relative contribution of PGL3 versus PGL5 during fertilization was assessed by mutant crosses. Reduced transmission ratios were found for pgl3-1 (T-DNA-eliminated PTS1) and also for knock-out allele pgl5-2. The prominent role of PGL3 showed as compromised growth of pgl3-1 seedlings on sucrose and higher activity of mutant PGL3-1 versus PGL5 using purified recombinant proteins. Evidence for PTS1-independent uptake was found for PGL3-1 and other Arabidopsis PGL isoforms, indicating that peroxisome import may be supported by a piggybacking mechanism. Thus, multiple redundancy at the level of the second OPPP step in peroxisomes explains the occurrence of pgl3-1 mutant plants.
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 encode N-terminally extended proteoforms. Compared to G6PD5.1, RT-PCR signals differed and fluorescent reporter fusions expressed in Arabidopsis protoplasts accumulated in distinct intracellular sites. Co-expression with organelle-specific markers revealed that the G6PD5.4 and G6PD5.5 proteoforms label 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 mutant protoplasts lacking cytosolic G6PDH activity, the ER-bound proteoforms were both active and thus able to form homomers. Among the Arabidopsis 6-phosphogluconolactonases (catalyzing the second OPPP step), we noticed that isoform 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. Among the Arabidopsis 6-phosphogluconate dehydrogenases (catalyzing the third OPPP step), S-acylated peptides were detected for all three isoforms in a recent palmitoylome, with dual cytosolic/peroxisomal PGD2 displaying three sites. Co-expression of GFP-PGD2 diminished crowding of OFP-G6PD5.4 at the ER, independent of PGL2's presence. Upon pull-down of GFP-G6PD5.4, not only unlabeled PGD2 and PGL2 were enriched, but also enzymes that depend on NADPH provision at the ER, indicative of physical interaction with the OPPP enzymes. When membrane-bound G6PD5.5 and 5.4 variants were co-expressed with KCR1 (ketoacyl-CoA reductase, involved in fatty acid elongation), ATR1 (NADPH:cytochrome-P450 oxidoreductase), or pulled C4H/CYP73A5 (cinnamate 4-hydroxylase) as indirectly (via ATR) NADPH-dependent cytochrome P450 enzyme, 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.
Former studies on Arabidopsis glucose-6-phosphate/phosphate translocator isoforms GPT1 and GPT2 reported viability of gpt2 mutants, however an essential function for GPT1, manifesting as a variety of gpt1 defects in the heterozygous state during fertilization/seed set. Among other functions, GPT1 is important for pollen and embryo-sac development. Since previous work on enzymes of the oxidative pentose phosphate pathway (OPPP) revealed comparable effects, we investigated whether GPT1 might dually localize to plastids and peroxisomes. In reporter fusions, GPT2 was found at plastids, but GPT1 also at the endoplasmic reticulum (ER) and around peroxisomes. GPT1 contacted oxidoreductases and also peroxins that mediate import of peroxisomal membrane proteins from the ER, hinting at dual localization. Reconstitution in yeast proteoliposomes revealed that GPT1 preferentially exchanges glucose-6-phosphate for ribulose-5-phosphate. Complementation analyses of heterozygous gpt1 plants demonstrated that GPT2 is unable to compensate for GPT1 in plastids, whereas genomic GPT1 without transit peptide (enforcing ER/peroxisomal localization) increased gpt1 transmission significantly. Since OPPP activity in peroxisomes is essential during fertilization, and immuno-blot analyses hinted at unprocessed GPT1-specific bands, our findings suggest that GPT1 is indispensable at both plastids and peroxisomes. Together with the G6P-Ru5P exchange preference, dual targeting explains why GPT1 exerts functions distinct from GPT2 in Arabidopsis.One sentence summaryIn contrast to plastidial GPT2, GPT1 exhibits slightly different exchange preferences and alternatively targets the ER, from where the protein can be relocated to peroxisomes on demand.
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