Evidence for dual targeting of Arabidopsis plastidial glucose-6-phosphate transporter GPT1 to peroxisomes via the ER

Abstract

ABSTRACTFormer 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.