title: Alternative targeting of Arabidopsis plastidic glucose-6-phosphate dehydrogenase G6PD1 involves cysteine-dependent interaction with G6PD4 in the cytosol.
abstract: Arabidopsis peroxisomes contain an incomplete oxidative pentose-phosphate pathway (OPPP), consisting of 6-phosphogluconolactonase and 6-phosphogluconate dehydrogenase isoforms with peroxisomal targeting signals (PTS). To start the pathway, glucose-6-phosphate dehydrogenase (G6PD) is required; however, G6PD isoforms with obvious C-terminal PTS1 or N-terminal PTS2 motifs are lacking. We used fluorescent reporter fusions to explore possibly hidden peroxisomal targeting information. Among the six Arabidopsis G6PD isoforms only plastid-predicted G6PD1 with free C-terminal end localized to peroxisomes. Detailed analyses identified SKY as an internal PTS1-like signal; however, in a medial G6PD1 reporter fusion with free N- and C-terminal ends this cryptic information was overruled by the transit peptide. Yeast two-hybrid analyses revealed selective protein-protein interactions of G6PD1 with catalytically inactive G6PD4, and of both G6PD isoforms with plastid-destined thioredoxin m2 (Trx(m2) ). Serine replacement of redox-sensitive cysteines conserved in G6PD4 abolished the G6PD4-G6PD1 interaction, albeit analogous changes in G6PD1 did not. In planta bimolecular fluorescence complementation (BiFC) demonstrated that the G6PD4-G6PD1 interaction results in peroxisomal import. BiFC also confirmed the interaction of Trx(m2) with G6PD4 (or G6PD1) in plastids, but co-expression analyses revealed Trx(m2) -mediated retention of medial G6PD4 (but not G6PD1) reporter fusions in the cytosol that was stabilized by CxxC¹¹³S exchange in Trx(m2) . Based on preliminary findings with plastid-predicted rice G6PD isoforms, we dismiss Arabidopsis G6PD4 as non-functional. G6PD4 orthologs (new P0 class) apparently evolved to become cytosolic redox switches that confer thioredoxin-relayed alternative targeting to peroxisomes.
authors.affiliation: Institut für Biologie und Biotechnologie der Pflanzen, Westfälische Wilhelms-Universität Münster, Schlossgarten 3, 48149 Münster, Germany.
authors.forename: Tanja
authors.initials: T
authors.lastname: Meyer
authors.forename: Christian
authors.initials: C
authors.lastname: Hölscher
authors.forename: Christian
authors.initials: C
authors.lastname: Schwöppe
authors.forename: Antje
authors.initials: A
authors.lastname: von Schaewen
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D003545:Cysteine / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism*; D017930:Genes, Reporter; D005816:Genetic Complementation Test; D005954:Glucosephosphate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D009154:Mutation; D019697:Onions / Q000235:genetics / Q000378:metabolism; D020675:Peroxisomes / Q000378:metabolism; D010802:Phylogeny; D018087:Plastids / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D013879:Thioredoxins / Q000235:genetics / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism; D020798:Two-Hybrid System Techniques
chemical_list: D029681:Arabidopsis Proteins; D007527:Isoenzymes; D011993:Recombinant Fusion Proteins; D013879:Thioredoxins; D005954:Glucosephosphate Dehydrogenase; D003545:Cysteine