title: Bacterial- and plant-type phosphoenolpyruvate carboxylase isozymes from developing castor oil seeds interact in vivo and associate with the surface of mitochondria.
abstract: Phosphoenolpyruvate 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of respiratory CO(2) while sustaining a large anaplerotic flux to replenish tricarboxylic acid cycle C-skeletons withdrawn for biosynthesis.
authors.affiliation: Department of Biology, Queen's University, Kingston, Ontario K7L 3N6, Canada.
authors.forename: Joonho
authors.initials: J
authors.lastname: Park
authors.forename: Nicholas
authors.initials: N
authors.lastname: Khuu
authors.forename: Alexander S M
authors.initials: AS
authors.lastname: Howard
authors.forename: Robert T
authors.initials: RT
authors.lastname: Mullen
authors.forename: William C
authors.initials: WC
authors.lastname: Plaxton
mesh_terms: D000595:Amino Acid Sequence; D002367:Ricinus communis / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D018929:Cell Culture Techniques; D019295:Computational Biology; D056625:Endosperm / Q000166:cytology / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D010730:Phosphoenolpyruvate Carboxylase / Q000235:genetics / Q000378:metabolism*; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D025941:Protein Interaction Mapping; D021381:Protein Transport; D011993:Recombinant Fusion Proteins; D012639:Seeds / Q000166:cytology / Q000201:enzymology* / Q000235:genetics; D016415:Sequence Alignment; D014026:Nicotiana / Q000166:cytology / Q000201:enzymology / Q000235:genetics
chemical_list: D007527:Isoenzymes; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D010730:Phosphoenolpyruvate Carboxylase