Reversible Phosphorylation of Photosynthetic PEP Carboxylase: Studies on C4-Leaf PP2A and Recombinant PEPC-Kinase from CAM-Induced Mesembryanthemum crystallinum

Authors

L. Dong, University of Nebraska - Lincoln
N. V. Ermolova, University of Nebraska - Lincoln
M. A. Cushman, University of Nevada, Reno, NV
T. Taybi, University of Newcastle, Newcastle-upon- Tyne NE1 7RU, UK
J. C. Cushman, University of Nevada, Reno, NV
Raymond Chollet, University of Nebraska - LincolnFollow

Document Type

Article

Date of this Version

7-2001

Comments

From: Abstracts of the 12th International Congress on Photosynthesis (Brisbane, Australia, August 18–23, 2001), published in Photosynthesis Research 69:1-3 (July, 2001), p. 149. Copyright © Elsevier B.V. Used by permission.

Abstract

The activity and allosteric properties of plant PEPC are controlled posttranslationally by specific reversible phosphorylation of a strictly conserved Ser residue near the N-terminus. This up/down-modulation is catalyzed by a transcriptionally regulated, seemingly dedicated Ser/Thr kinase (PpcK) and an opposing Ser/Thr phosphatase (PP2A). We have now partially purified and characterized the native form of this largely “neglected” PP2A from maize leaves using various chromatographic and affinity matrices, and C₄ [³²P]PEPC as substrate (Dong et al., 2001, Planta [in press]). The results indicate that the C₄-leaf holoenzyme is analogous to yeast and mammalian PP2As in regards to its heterotrimeric structure (~170 kDa), comprised of a ~103-kDa core PP2Ac- A heterodimer complexed with a ~74-kDa B-type subunit, and its sensitivity to free Me²⁺ and various inhibitors, activators and anionic metabolites. Notably, this native PP2A (a) lacks any strict phosphoprotein specificity in that it dephosphorylates C₄ PEPC, mammalian phosphorylase a, and casein in vitro, and (b) displays, at best, only modest light/dark effects in vivo on its apparent Mr, component core subunits, and activity against C₄ PEPC-SerP. In addition, we will also report new findings on a recombinant form of CAM PpcK from M. crystallinum (Taybi et al., 2000, Plant Physiol.) produced as a highly soluble, active fusion with the ~55-kDa NusA carrier protein in E. coli. This NusA—PpcK fusion protein has been purified by sequential IMAC and FPLC, used for detailed analysis of its target-protein specificity and other kinetic properties, and cleaved “on-bead” by thrombin to yield free PpcK for antibody production.