In Vivo Regulatory Phosphorylation Site in C 4 - Leaf Phosphoenolpyruvate Carboxylase from Maize and Sorghum 1

Reversible seryl-phosphorylation contributes to the light/dark regulation of C4-leaf phosphoenolpyruvate carboxylase (PEPC) activity in vivo. The specific regulatory residue that, upon in vitro phosphorylation by a maize-leaf protein-serine kinase(s), leads to an increase in catalytic activity and a decrease in malatesensitivity of the target enzyme has been recently identified as Ser-15 in 32P-phosphorylated/activated dark-form maize PEPC (J-A Jiao, R Chollet [1990] Arch Biochem Biophys 283: 300-305). In order to ascertain whether this N-terminal seryl residue is, indeed, the in vivo regulatory phosphorylation site, [32P]phosphopeptides were isolated and purified from in vivo 32P-labeled maize and sorghum leaf PEPC and subjected to automated Edman degradation analysis. The results show that purified light-form maize PEPC contains 14-fold more 32P-radioactivity than the corresponding dark-form enzyme on an equal protein basis and, more notably, only a single N-terminal serine residue (Ser-15 in maize PEPC and its structural homolog, Ser-8, in the sorghum enzyme) was found to be 32P-phosphorylated in the light or dark. These in vivo observations, combined with the results from our previous in vitro phosphorylation studies (J-A Jiao, R Chollet [1989] Arch Biochem Biophys 269: 526-535; [1990] Arch Biochem Biophys 283: 300-305), demonstrate that an N-terminal seryl residue in C4 PEPC is, indeed, the regulatory site that undergoes light/dark changes in phosphorylation-status and, thus, plays a major, if not cardinal role in the light-induced changes in catalytic and regulatory properties of this cytoplasmic C4-photosynthesis enzyme in vivo. PEPC3 (EC 4.1.1.31) is the initial carboxylating enzyme in the C4 pathway of photosynthesis (6). Its catalytic activity, regulatory properties (e.g. sensitivity to feedback inhibition 'This research was supported in part by grants DMB-8704237 and DCB-9017726 from the National Science Foundation and is published as Journal Series No. 9369 of the University of Nebraska Agricultural Research Division. 2 Permanent address: Laboratorio de Fisiologia Vegetal, Facultad de Biologia, Universidad de Sevilla, Avenida de la Reina Mercedes s/n, 41012, Sevilla, Spain. 3Abbreviations: PEPC, phosphoenolpyruvate carboxylase; PEP, phosphoenolpyruvate; TPCK, L-1-tosylamido-2-phenylethyl chloromethyl ketone; IDA, iminodiacetic acid; FPLC, fast-protein liquid chromatography. by L-malate and allosteric activation by glucose 6-phosphate) and seryl-phosphorylation status undergo concomitant in vivo changes upon light/dark transitions of the parent leaf tissue in a variety of C4 plants, including, among others, maize (7, 10, 15, 18), sorghum (5, 21), Portulaca oleracea (9), and Setaria verticillata (16). Recent studies with a reconstituted in vitro phosphorylation system, comprised of purified darkform maize leaf PEPC, a partially purified protein-serine kinase(s) from light-adapted leaves and ATP. Mg, established that the protein kinase-mediated changes in the catalytic activity and malate sensitivity of C4-PEPC are directly correlated with the concomitant changes in the seryl-phosphorylation status ofthe target enzyme in vitro (1 1). The amino acid sequence of the single in vitro regulatory phosphorylation site and its flanking region have been determined as His-HisSer(P)-Ile-Asp-Ala-Gln-Leu-Arg (12, 19). This N-terminal nonapeptide corresponds exactly to residues 13 to 21 in the deduced primary structure of maize leaf PEPC (8), with Ser15 being the regulatory phosphorylation site (12, 19). This specific seryl residue is far removed from a species-invariant, active-site lysine residue (Lys-606 in maize PEPC) in the Cterminal region of the -1 I0-kD subunit polypeptide (14). In this report, detached maize and sorghum leaves were fed [32P]orthophosphate, and the 32P-labeled PEPC was subsequently purified from lightand dark-adapted tissue for isolation and amino acid sequence analysis of the in vivo [32p]_ phosphopeptides. The results indicate that only a single, Nterminal seryl residue is 32P-labeled in vivo. This specific seine is located at position 15 and 8 in maize and sorghum leaf PEPC, respectively. More importantly, the phosphorylation status of this N-terminal seryl residue undergoes changes in response to light/dark transitions; it is much more phosphorylated in the light than in the dark. These observations, together with previous homologous (1 1, 12) and heterologous (12, 19) reconstituted in vitro phosphorylation studies, demonstrate that the regulatory phosphorylation of a single, Nterminal seryl residue plays a key, if not cardinal role in the light-induced changes in catalytic activity and malate sensitivity of C4-leaf PEPC in vivo. MATERIALS AND METHODS