Bacterial‐ and plant‐type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria

Abstract

SUMMARYPhosphoenolpyruvate carboxylase (PEPC) from developing castor oil seeds (COS) exists as two distinct oligomeric isoforms. The typical class-1 PEPC homotetramer consists of 107-kDa plant-type PEPC (PTPC) subunits, whereas the allosterically desensitized 910-kDa class-2 PEPC hetero-octamer arises from the association of class-1 PEPC with 118-kDa bacterial-type PEPC (BTPC) subunits. The in vivo interaction and subcellular location of COS BTPC and PTPC were assessed by imaging fluorescent protein (FP)-tagged PEPCs in tobacco suspension-cultured cells. The BTPC-FP mainly localized to cytoplasmic punctate/globular structures, identified as mitochondria by co-immunostaining of endogenous cytochrome oxidase. Inhibition of respiration with KCN resulted in proportional decreases and increases in mitochondrial versus cytosolic BTPC-FP, respectively. The FP-PTPC and NLS-FP-PTPC (containing an appended nuclear localization signal, NLS) localized to the cytosol and nucleus, respectively, but both co-localized with mitochondrial-associated BTPC when co-expressed with BTPC-FP. Transmission electron microscopy of immunogold-labeled developing COS revealed that BTPC and PTPC are localized at the mitochondrial (outer) envelope, as well as the cytosol. Moreover, thermolysin-sensitive BTPC and PTPC polypeptides were detected on immunoblots of purified COS mitochondria. Overall, our results demonstrate that: (i) COS BTPC and PTPC interact in vivo as a class-2 PEPC complex that associates with the surface of mitochondria, (ii) BTPC's unique and divergent intrinsically disordered region mediates its interaction with PTPC, whereas (iii) the PTPC-containing class-1 PEPC is entirely cytosolic. We hypothesize that mitochondrial-associated class-2 PEPC facilitates rapid refixation of respiratory CO 2 while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.

Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds

“…All tissues were frozen in liquid N 2 and stored at Ϫ80°C until used. Tobacco (Nicotiana tabacum L.) Bright Yellow-2 suspensioncultured cells were maintained and prepared for biolistic bombardment as described previously (34).…”

“…Transient transformation of tobacco cells with RcSUS1-EYFP and mCherry-RcPPC3 (encoding cytosolic targeted plant-type phosphoenolpyruvate (PEP) carboxylase from developing COS) (34) was performed with 5 g of each plasmid DNA using a Bio-Rad Biolistic particle delivery system. Bombarded cells were incubated for 8 h to allow for gene expression and protein sorting, fixed in 4% (w/v) formaldehyde, and imaged using epifluorescence microscopy as previously described (34).…”

“…Epifluorescence microscopy demonstrated that RcSUS1-EYFP was targeted exclusively to the cytosol (Fig. 6), as evidenced by its co-localization with co-expressed, fluorescent protein-tagged plant-type PEP carboxylase from developing COS (mCherry-RcPPC3), a well characterized cytosolic marker fusion protein (34). This result is consistent with bioinformatics analyses of the RcSUS1 sequence, which failed to predict the presence of any membrane-spanning domains or mitochondrial or plastid targeting peptides in its N terminus.…”

Reciprocal Control of Anaplerotic Phosphoenolpyruvate Carboxylase by in Vivo Monoubiquitination and Phosphorylation in Developing Proteoid Roots of Phosphate-Deficient Harsh Hakea

“…In contrast, BTPC genes encode larger 116-to 118-kD polypeptides owing to a unique intrinsically disordered region that mediates BTPC's tight interaction with coexpressed PTPC subunits. This association results in the formation of unusual Class-2 PEPC heterooctameric complexes that are largely desensitized to allosteric effectors and that dynamically associate with the surface of mitochondria in vivo (O'Leary et al, 2009(O'Leary et al, , 2011aIgawa et al, 2010;Park et al, 2012).…”

Regulatory Phosphorylation of Bacterial-Type PEP Carboxylase by the Ca2+-Dependent Protein Kinase RcCDPK1 in Developing Castor Oil Seeds

“…Subsequent research revealed that the native BTPC exists as a p118, but that upon COS extraction it is rapidly cleaved by an endogenous thiol endopeptidase into 54-kD and 64-kD polypeptides (p54 and p64; Gennidakis et al, 2007). BTPC's proteolytic cleavage site (Lys-446/Ile-447) as well as its in vivo Ser-425 and Ser-451 phosphorylation sites all occur within an approximate 10-kD intrinsically disordered region, a distinctive feature of green algal and vascular plant BTPCs that mediates their tight interaction with coexpressed PTPC subunits to form the Class-2 PEPC heteromeric complex (O'Leary et al, 2011b;Park et al, 2012).…”