Analysis of homo- and hetero-dimerization among the three 6-phosphogluconate dehydrogenase isoforms of Arabidopsis

Abstract

We recently described that all three 6-phosphogluconate dehydrogenase (6PGDH) isoforms of Arabidopsis (PGD) with similar length show dual localization: PGD1 and PGD3 in the cytosol and in plastids, and PGD2 in the cytosol and in peroxisomes. We set out to investigate heterodimer formation, however due to only weak homodimerization of all Arabidopsis PGD isoforms in yeast cells, we conducted further protein-protein interaction studies in planta to investigate homomer versus heteromer formation and their sub-cellular localization. Bimolecular fluorescence complementation (BiFC) analyses in co-transfected Arabidopsis protoplasts demonstrated that all PGD isoforms may form homo- and heterodimers. Notably, with free N-terminal ends, PGD1-PGD3 heterodimers were detected both in the cytosol and in the plastid stroma, but heterodimers with PGD2 only in the cytosol. This independently confirmed that PGD2 cannot enter plastids. On the other hand, with free C-terminal ends, PGD1-PGD2 and PGD3-PGD2 heterodimers were confined to the cytosol, indicating that only PGD2 homodimers are imported by peroxisomes. Together these findings suggest functional redundancy of PGD1 and PGD3 inside plastids, and relevance of PGD1-PGD2 or PGD3-PGD2 heterodimer formation in the cytosol: this could retain sufficient 6PGDH activity needed for NADPH provision, especially during stress defense and initiation of developmental responses.

Keywords: A. thaliana; OPPP; PGD isoforms; dimerization; sub-cellular targeting.

Figure 1.

Extent of dimer formation among the 3 Arabidopsis PGD isoforms. A, Dimerization analyses of the indicated binding (BD) and activation domain (AD) construct combinations in yeast strain SMY3. With the empty vector, none of the PGD isoforms showed significant growth (Y2H, - signs) on selective drop-out medium (SD-LWH) in the presence of 3-Amino-1,2,4-triazole (3-AT, top; successive 1:10 dilutions are depicted as triangles). Weak growth of PGD1-PGD1, PGD2-PGD2, and PGD3-PGD3 combinations detected on 2.5 mM 3-AT plates indicates homo-dimerization (Y2H, + signs). B-C, Bimolecular fluorescence complementation (BiFC) analyses upon co-expression of the schematically indicated split YFP combinations (top, driven by the CaMV 35S promoter) in Arabidopsis protoplasts isolated from tissue-culture plants (grown on solidified half-strength Murashige & Skoog medium, pH 5.7 with 1% sucrose). PGD isoforms listed on the left were fused to the C-terminal half and those listed above the columns to the N-terminal half of split YFP. B, With free N-terminal ends, PGD1 and PGD3 formed homodimers (a and f) as well as heterodimers (c and d) in the cytosol and inside plastids, but with PGD2 only heterodimers in the cytosol (b and e). Two representative cells are shown for each combination; the images were recorded 24-48 h after transfection. C, With free C-terminal ends, all full-length split YFP-PGD combinations labeled the cytosol (data not shown). For the mature versions (that allow peroxisome import of PGD2) heterodimers were formed in the cytosol, but none of them co-localized with the peroxisome (Per) marker (PGD2-PGD1, panel b; PGD2-PGD3, panel e), by contrast to PGD2 homodimers used as positive control (not shown) or in combination with G6PD1 used as negative control (panels g-i). The images were recorded 48-72 h after transfection. Only merged channels are shown (maximal projections of approximately 35 single sections): reconstituted YFP signals in yellow, peroxisome marker (PGL3_short-OFP) in magenta, and chlorophyll auto-fluorescence in blue. Co-localization or very close signals (less than 200 nm) appear whitish. Homo, homo-dimers; hetero, hetero-dimers. Bars = 3µm.