Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation.

PM28A is a major intrinsic protein of the spinach leaf plasma membrane and the major phosphoprotein. Phosphorylation of PM28A is dependent in vivo on the apoplastic water potential and in vitro on submicromolar concentrations of Ca2+. Here, we demonstrate that PM28A is an aquaporin and that its water channel activity is regulated by phosphorylation. Wild-type and mutant forms of PM28A, in which putative phosphorylation sites had been knocked out, were expressed in Xenopus oocytes, and the resulting increase in osmotic water permeability was measured in the presence or absence of an inhibitor of protein kinases (K252a) or of an inhibitor of protein phosphatases (okadaic acid). The results indicate that the water channel activity of PM28A is regulated by phosphorylation of two serine residues, Ser-115 in the first cytoplasmic loop and Ser-274 in the C-terminal region. Labeling of spinach leaves with 32P-orthophosphate and subsequent sequencing of PM28A-derived peptides demonstrated that Ser-274 is phosphorylated in vivo, whereas phosphorylation of Ser-115, a residue conserved among all plant plasma membrane aquaporins, could not be demonstrated. This identifies Ser-274 of PM28A as the amino acid residue being phosphorylated in vivo in response to increasing apoplastic water potential and dephosphorylated in response to decreasing water potential. Taken together, our results suggest an active role for PM28A in maintaining cellular water balance.     

Reconstitution of water channel function of an aquaporin overexpressed and purified from Pichia pastoris

The aquaporin PM28A is one of the major integral proteins in spinach leaf plasma membranes. Phosphorylation/dephosphorylation of Ser274 at the C-terminus and of Ser115 in the first cytoplasmic loop has been shown to regulate the water channel activity of PM28A when expressed in Xenopus oocytes. To understand the mechanisms of the phosphorylation-mediated gating of the channel the structure of PM28A is required. In a first step we have used the methylotrophic yeast Pichia pastoris for expression of the pm28a gene. The expressed protein has a molecular mass of 32462 Da as determined by matrix-assisted laser desorption ionization-mass spectrometry, forms tetramers as revealed by electron microscopy and is functionally active when reconstituted in proteoliposomes. PM28A was efficiently solubilized from urea- and alkali-stripped Pichia membranes by octyl-beta-D-thioglucopyranoside resulting in a final yield of 25 mg of purified protein per liter of cell culture.     

Structural characterization of two aquaporins isolated from native spinach leaf plasma membranes

Two members of the aquaporin family, PM28A and a new one, PM28C, were isolated and shown to be the major constituents of spinach leaf plasma membranes. These two isoforms were identified and characterized by matrix-assisted laser desorption ionization-mass spectrometry. Edman degradation yielded the amino acid sequence of two domains belonging to the new isoform. PM28B, a previously described isoform, was not found in our preparations. Scanning transmission electron microscopy mass analysis revealed both PM28 isoforms to be tetrameric. Two types of particles, a larger and a smaller one, were found by transmission electron microscopy of negatively stained solubilized proteins and by atomic force microscopy of PM28 two-dimensional crystals. The ratio of larger to smaller particles observed by transmission electron microscopy and single particle analysis correlated with the ratio of PM28A to PM28C determined by matrix-assisted laser desorption ionization-mass spectrometry. The absence of PM28B and the ratio of PM28A to PM28C indicate that these plasma membrane intrinsic proteins are differentially expressed in spinach leaves. These findings suggest that differential expression of the various aquaporin isoforms may regulate the water flux across the plasma membrane, in addition to the known mechanism of regulation by phosphorylation.     

蜡梅Chimonanthus praecox (L.) Link COR413蛋白基因(Cpcor413pm1)的分子特性与表达分析

为研究蜡梅冬季开花过程的抗寒分子机理,在构建蜡梅花cDNA文库及EST分析的基础上,通过随机克隆测序,克隆了1个蜡梅COR413蛋白的cDNA基因,命名为Cpcor413pm1.Cpcor413pm1 cDNA长946　bp,推测的编码蛋白CpCOR413PM1包含201个氨基酸.CpCOR413PM1蛋白N端保守性差,没有信号肽,具有5个保守的跨膜区,1个潜在的GPI锚点,具有可能对蛋白结构或活性十分重要的位置保守的7个Pro、1个Cys, 1个被富Gly区一分为二的α螺旋区域,以及Tyr、Thr、Ser磷酸化位点各1个.序列比较分析表明,Cpcor413pm1是首次从蜡梅中克隆到的COR413蛋白基因.RT-PCR分析表明, 该基因在蜡梅的萌动期、蕾期、露瓣期、初开期、盛开期、衰老期均有表达,但在初开期和盛开期表达丰度更高,推测Cpcor413pm1与蜡梅花的抗冻性有密切关系.     

Spinach Leaf Sucrose-Phosphate Synthase and Nitrate Reductase Are Phosphorylated/Inactivated by Multiple Protein Kinases in Vitro

The regulation of sucrose-phosphate synthase (SPS) and nitrate reductase (NR) activities from mature spinach (Spinacia oleracea L.) leaves share many similarities in vivo and in vitro. Both enzymes are light/dark modulated by processes that involve, at least in part, reversible protein phosphorylation. Experiments using desalted crude extracts show that the ATP-dependent inactivation of spinach SPS and NR is sensitive to inhibition by glucose-6-phosphate. Also, a synthetic peptide homolog of the spinach SPS phosphorylation site inhibits the ATP-dependent inactivation of both enzymes with a similar concentration dependence. We have addressed the possibility that SPS and NR are regulated by the same protein kinase by partially purifying the protein kinases involved. Three unique kinase activities can be separated by anion-exchange and size-exclusion chromatography. Each peak of activity has a different substrate specificity. By gel filtration, they have apparent molecular masses of approximately 45, 60, and 150 kD. Additionally, the activities of the two smaller kinases are dependent on micromolar concentrations of Ca2+, whereas the 150-kD kinase is not. Finally, the 150-kD kinase has a subunit molecular mass of about 65 kD as determined by renaturing the kinase activity in situ following sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Plants have isoforms for acyl carrier protein that are expressed differently in different tissues

Two closely related isoforms of acyl carrier protein (I and II) have been purified from spinach leaves. Differences in the N-terminal amino acid sequence and amino acid composition indicate that these proteins are coded by different genes. The two spinach leaf isoforms have been resolved and characterized by ion-exchange high-performance liquid chromatography, by thin layer isoelectric focusing, and by differences in mobility upon polyacrylamide gel electrophoresis. Both isoforms are effectively bound by antibodies raised to acyl carrier protein I. However, in competition experiments isoform II is only about 40% effective in blocking isoform I binding to antibody. Therefore, the isoforms are immunologically related but hold only some antigenic sites in common. Immunoblot analysis ("Western blotting") of crude spinach leaf tissue extracts probed with antibody to acyl carrier protein I reveals both isoforms. In addition, both forms of acyl carrier protein are present in dark-grown leaf tissue and in isolated chloroplasts. However, in spinach seeds and roots only acyl carrier protein II can be detected. Similar results are observed with extracts of castor oil plant leaf and seed. Therefore, the expression of the two acyl carrier protein isoforms is tissue specific.     

Two SNF1-related protein kinases from spinach leaf phosphorylate and inactivate 3-hydroxy-3-methylglutaryl-coenzyme A reductase, nitrate reductase, and sucrose phosphate synthase in vitro.

We resolved from spinach (Spinacia oleracea) leaf extracts four Ca2+-independent protein kinase activities that phosphorylate the AMARAASAAALARRR (AMARA) and HMRSAMSGLHLVKRR (SAMS) peptides, originally designed as specific substrates for mammalian AMP-activated protein kinase and its yeast homolog, SNF1. The two major activities, HRK-A and HRK-C (3-hydroxy-3-methylglutaryl-coenzyme A reductase kinase A and C) were extensively purified and shown to be members of the plant SnRK1 (SNF1-related protein kinase 1) family using the following criteria: (a) They contain 58-kD polypeptides that cross-react with an antibody against a peptide sequence characteristic of the SnRK1 family; (b) they have similar native molecular masses and specificity for peptide substrates to mammalian AMP-activated protein kinase and the cauliflower homolog; (c) they are inactivated by homogeneous protein phosphatases and can be reactivated using the mammalian upstream kinase; and (d) they phosphorylate 3-hydroxy-3-methylglutaryl-coenzyme A reductase from Arabidopsis at the inactivating site, serine (Ser)-577. We propose that HRK-A and HRK-C represent either distinct SnRK1 isoforms or the same catalytic subunit complexed with different regulatory subunits. Both kinases also rapidly phosphorylate nitrate reductase purified from spinach, which is associated with inactivation of the enzyme that is observed only in the presence of 14-3-3 protein, a characteristic of phosphorylation at Ser-543. Both kinases also inactivate spinach sucrose phosphate synthase via phosphorylation at Ser-158. The SNF1-related kinases therefore potentially regulate several major biosynthetic pathways in plants: isoprenoid synthesis, sucrose synthesis, and nitrogen assimilation for the synthesis of amino acids and nucleotides.     

Phosphorylation of a chloroplast RNA-binding protein changes its affinity to RNA.

An RNA-binding protein of 28 kDa (28RNP) was previously isolated from spinach chloroplasts and found to be required for 3' end-processing of chloroplast mRNAs. The amino acid sequence of 28RNP revealed two approximately 80 amino-acid RNA-binding domains, as well as an acidic- and glycine-rich amino terminal domain. Upon analysis of the RNA-binding properties of the 'native' 28RNP in comparison to the recombinant bacterial expressed protein, differences were detected in the affinity to some chloroplastic 3' end RNAs. It was suggested that post-translational modification can modulate the affinity of the 28RNP in the chloroplast to different RNAs. In order to determine if phosphorylation accounts for this post-translational modification, we examined if the 28RNP is a phosphoprotein and if it can serve as a substrate for protein kinases. It was found that the 28RNP was phosphorylated when intact chloroplasts were metabolically labeled with [32P] orthophosphate, and that recombinant 28RNP served as an excellent substrate in vitro for protein kinase isolated from spinach chloroplasts or recombinant alpha subunit of maize casein kinase II. The 28RNP was apparently phosphorylated at one site located in the acidic domain at the N-terminus of the protein. Site-directed mutagenesis of the serines in that region revealed that the phosphorylation of the protein was eliminated when serine number 22 from the N-terminus was changed to tryptophan. RNA-binding analysis of the phosphorylated 28RNP revealed that the affinity of the phosphorylated protein was reduced approximately 3-4-fold in comparison to the non-phosphorylated protein. Therefore, phosphorylation of the 28RNP modulates its affinity to RNA and may play a significant role in its biological function in the chloroplast.     

Lipid composition of plasma membranes from barley leaves and roots, spinach leaves and cauliflower inflorescences

Highly purified plasma membranes (PM) were obtained from barley (Hordeum vulgare L. cv. Kristina) leaves and roots, spinach (Spinacia oleracea L. cv. Viking II) leaves, and cauliflower (Brassica oleracea) inflorescences by partitioning in an aqueous polymer two-phase system. The sterol and polar lipid composition of the PM, including the fatty acid composition of the glycerolipids, was determined. Dominating lipids were free sterols, glucocerebroside, phosphatidylcholine (PC) and phosphatidylethanolamine (PE), although large variations in content were observed between the PM of the different species and organs. Thus, the spinach leaf PM contained only 7% (mol %) free sterol compared to over 30% free sterol in the other PM analysed, with the barley root PM as the other extreme (57% free sterol). On the other hand, sterol derivatives were more abundant in the spinach leaf PM, containing 13% acylated sterol glycosides. Cerebroside constituted 16% of the lipids in the barley leaf PM but only 3% in cauliflower. The phospholipids PC and PE ranged from 25 and 24%, respectively, in the spinach leaf PM to 8 and 7%, respectively, in the barley root PM. As a result of the large variations in sterol and phospholipid content, the ratio of free sterol to phospholipid varied from 2.2 in the barley root PM to only 0.1 in the spinach leaf PM. Sitosterol, campesterol and stigmasterol were the completely dominating sterols in the barley and cauliflower PM, whereas the unique sterol composition of spinach was dominated by spinasterol. Palmitic (16:0), linoleic (18:2) and linolenic (18:3) acid were the major glycerolipid fatty acids. The fatty acid composition of the barley root PM was the most saturated (44% 16:0, 13% 18:3), whereas that of the cauliflower PM was the most unsaturated (21% 16:0,42% 18:3). Thus, very large variations were observed in both total lipid and fatty acid composition of the PM investigated, which represent both mono— and dicotyledons, as well as both photosynthetic and non-photosynthetic tissue. The consequences of this large diversity in composition of the lipid bilayer for the function of integral PM proteins are discussed.     

A Pea Plasma Membrane Protein Exhibiting Blue Light-Induced Phosphorylation Retains Photosensitivity following Triton Solubilization

Phosphorylation of a polypeptide of approximately 120 kD in pea (Pisum sativum L.) plasma membranes in response to blue light has been shown to be involved in phototropic curvature, but the relationship of this protein to the kinase and photoreceptor acting upon it is uncertain. Using two-phase aqueous partitioning to isolate right-side-out plasma membrane vesicles, we have obtained evidence suggesting that the photoreceptor, kinase, and substrate are localized to the plasma membrane fraction. Latent phosphorylation accessible through Triton X-100 or freeze/thaw treatments of purified plasma membrane vesicles indicates that at least the kinase moiety is present on the internal face of the plasma membrane. Effects of solubilization of vesicles on fluence-response characteristics and on phosphorylation levels provide evidence that the receptor, kinase, and protein substrate are present together in individual mixed detergent micelles, either as a stable complex or as domains of a single polypeptide. In vivo blue-light irradiation results in a small but significant decrease in mobility of the 120-kD phosphorylated protein on sodium dodecylsulfate gel electrophoresis. This mobility shift is evident on Coomassie-stained gels and on western blots probed with polyclonal antibodies raised against the 120-kD protein. Among the plasma membrane proteins bound to the reactive nucleotide analog fluorosulfonylbenzoyladenine (FSBA), a distinct protein band at 120 kD can be detected on blots probed with anti-FSBA antibodies. This band exhibits an in vivo light-dependent mobility shift identical to that observed for the protein band and antibodies specific for the 120-kD protein, implying that the 120-kD protein has an integral nucleotide binding site and consistent with the possibility that the substrate protein is also a kinase.     

Amino acid sequence of in vivo phosphorylation sites in the main intrinsic protein (MIP) of lens membranes

The main intrinsic membrane protein of the lens fiber cell, MIP, has been previously shown to be phosphorylated in preparations of lens fragments. Phosphorylation occurred on serine residues near the cytoplasmic C-terminus of the molecule. Since MIP is thought to function as a channel protein in lens plasma membranes, possibly as a cell-to-cell channel protein, phosphorylation could regulate the assembly or gating of these channels. We sought to identify the specific serines which are phosphorylated in order to help identify the kinases involved in regulating MIP function. To this end we purified a peptide fragment from native membranes that had not been subjected to any exogenous kinases or kinase activators. Any phosphorylation detected in these fragments must be due to cellular phosphorylation and thus is termed in vivo phosphorylation. Purified membranes were also phosphorylated with cAMP-dependent protein kinase to determine the mobility of phosphorylated and unphosphorylated MIP-derived peptides on different HPLC columns and to determine possible cAMP-dependent protein kinase phosphorylation sites. Lens membranes, which contain 50-60% of the protein as MIP, were digested with lysylendopeptidase C. Peptides were released from the C-terminal region of MIP and a major product of 21-22 kDa remained membrane-associated. Separation of the lysylendopeptidase-C-released peptides on C8 reversed-phase HPLC demonstrated that one of these fragments, corresponding to residues 239-259 in MIP, was partially phosphorylated. The phosphorylated and nonphosphorylated forms of this peptide were separated on QAE HPLC. In vivo phosphorylation sites were found at residues 243 and 245 through phosphoserine modification via ethanethiol and sequence analysis. Phosphorylation was never detected on serine 240. The phosphorylation level of serine 243 could be increased by incubation of membranes with cAMP-dependent protein kinase under standard assay conditions. Other kinases that phosphorylate serines found near acidic amino acids must be responsible for the in vivo phosphorylation demonstrated at serine 245.     

Tyrosine phosphorylation modulates the interaction of calmodulin with its target proteins.

The activation of six target enzymes by calmodulin phosphorylated on Tyr99 (PCaM) and the binding affinities of their respective calmodulin binding domains were tested. The six enzymes were: myosin light chain kinase (MLCK), 3'-5'-cyclic nucleotide phosphodiesterase (PDE), plasma membrane (PM) Ca2+-ATPase, Ca2+-CaM dependent protein phosphatase 2B (calcineurin), neuronal nitric oxide synthase (NOS) and type II Ca2+-calmodulin dependent protein kinase (CaM kinase II). In general, tyrosine phosphorylation led to an increase in the activatory properties of calmodulin (CaM). For plasma membrane (PM) Ca2+-ATPase, PDE and CaM kinase II, the primary effect was a decrease in the concentration at which half maximal velocity was attained (Kact). In contrast, for calcineurin and NOS phosphorylation of CaM significantly increased the Vmax. For MLCK, however, neither Vmax nor Kact were affected by tyrosine phosphorylation. Direct determination by fluorescence techniques of the dissociation constants with synthetic peptides corresponding to the CaM-binding domain of the six analysed enzymes revealed that phosphorylation of Tyr99 on CaM generally increased its affinity for the peptides.     

Location of sites in human lipocortin I that are phosphorylated by protein tyrosine kinases and protein kinases A and C

Lipocortin I is a 39-kilodalton membrane-associated protein that in A431 cells is phosphorylated on tyrosine in response to epidermal growth factor (EGF). We have used recombinant human lipocortin I as a substrate for several protein kinases and identified phosphorylated residues by a combination of peptide mapping and sequence analysis. Lipocortin I was phosphorylated near the amino terminus at Tyr-21 by recombinant pp60c-src. The same tyrosine residue was phosphorylated by polyoma middle T/pp60c-src complex, by recombinant pp50v-abl, and with A431 cell membranes by the EGF receptor/kinase. The primary site of phosphorylation by protein kinase C was also near the amino terminus at Ser-27. The major site of phosphorylation by adenosine cyclic 3',5'-phosphate dependent protein kinase was on the carboxy-terminal half of the molecule at Thr-216. These sites are compared to the phosphorylation sites previously located in the structurally related protein lipocortin II.     

Protein phosphorylation as a mechanism for osmotic-stress activation of sucrose-phosphate synthase in spinach leaves.

Experiments were performed to investigated the mechanism of sucrose-phosphate synthase (SPS) activation by osmotic stress in darkened spinach (Spinacia oleracea L.) leaves. The activation was stable through immunopurification and was not the result of an increased SPS protein level. The previously described Ca(2+)-independent peak III kinase, obtained by ion-exchange chromatography, is confirmed to be the predominant enzyme catalyzing phosphorylation and inactivation of dephosphoserine-158-SPS. A new, Ca(2+)-dependent SPS-protein kinase activity (peak IV kinase) was also resolved and shown to phosphorylate and activate phosphoserine-158-SPS in vitro. The peak IV kinase also phosphorylated a synthetic peptide (SP29) based on the amino acid sequence surrounding serine-424, which also contains the motif described for the serine-158 regulatory phosphorylation site; i.e. basic residues at P-3 and P-6 and a hydrophobic residue at P-5. Peak IV kinase had a native molecular weight of approximately 150,000 as shown by gel filtration. The SP29 peptide was not phosphorylated by the inactivating peak III kinase. Osmotically stressed leaves showed increased peak IV kinase activity with the SP29 peptide as a substrate. Tryptic 32P-phosphopeptide analysis of SPS from excised spinach leaves fed [32P]inorganic P showed increased phosphorylation of the tryptic peptide containing serine-424. Therefore, at least part of the osmotic stress activation of SPS in dark leaves results from phosphorylation of serine-424 catalyzed by a Ca(2+)-dependent, 150-kD protein kinase.     

Phosphorylation modulates the voltage dependence of channels reconstituted from the major intrinsic protein of lens fiber membranes

Summary Major intrinsic polypeptide (MIP), a 28-kDa protein isolated from lens fiber cell membranes, forms large, nonselective channels when reconstituted into lipid bilayers. MIP channels are regulated by voltage, such that these channels close when the potential across the membrane is greater than 30 mV. We have investigated the modulation of the voltage-dependent closure of MIP channels by phosphorylation. In this report, we describe the isolation of two isomers of MIP from lens fiber cell membranes. These isomers differ by a single phosphate at a protein kinase A phosphorylation site. The phosphorylated isomer produces channels that close in response to applied voltages when reconstituted into bilayers. The nonphosphorylated isomer produces voltage-independent hannels. Direct phosphorylation with protein kinase A converts voltage-independent channels to voltage-dependent channels in situ. Analyses of macroscopic and single channel currents suggest that phosphorylation increases the voltage-dependent closure of MIP channels by increasing closed channel lifetimes and the rate of channel closure following the application of voltage.The authors gratefully acknowledge the gift of the monoclonal antibody to MIP from Drs. David Paul and Dan Goodenough. We thank Dr. Irwin Levitan for the kind gift of purified protein kinase A catalytic subunit. We also thank Ms. Mary Hawley for invaluable technical support and Mr. Paul Ross for help in generating Fig. 10. This work was supported, in part, by NIH grants EY04110 and EY05661 and a NEI postdoctoral fellowship to GRE.     

The amino acid sequence of nucleoside diphosphate kinase I from spinach leaves, as deduced from the cDNA sequence.

The primary structure of nucleoside diphosphate (NDP) kinase from spinach leaves has been deduced from its cDNA sequence. A lambda gt 11 cDNA library derived from spinach leaves was screened using an antibody against NDP kinase I, which we previously purified to electrophoretic homogeneity (T. Nomura, T. Fukui, and A. Ichikawa, 1991, Biochim. Biophys. Acta 1077, 47-55). The cDNA sequences of positive clones contained the amino acid coding region (444 base pairs) for NDP kinase I as well as 5' and 3' noncoding regions of 33 and 361 base pairs, respectively. The cDNAs hybridized to a 1.1-kb mRNA. NDP kinase I contains 148 amino acid residues with a molecular mass of 16,305, which is in excellent agreement with that of the purified enzyme (16 kDa). Homology was found between the sequence of spinach NDP kinase I and those of the rat, Myxococcus xanthus, and Dictyostelium discoideum NDP kinases, as well as the human Nm23-gene product and the awd protein of Drosophila melanogaster.     

Protein PSII-G. An additional component of photosystem II identified through its plastid gene in maize

An unidentified open reading frame, 248 or 255 amino acids in length, on the maize chloroplast DNA fragment Bam5 was sequenced. It encodes a protein which contains a high proportion of hydrophilic amino acids, of which 22% are hydroxylated, interrupted by hydrophobic domains. A synthetic peptide corresponding to a hydrophilic sequence was used to generate antibodies. Western blots of photosystem I and II complexes prepared from maize and spinach thylakoids indicate that the psbG gene product is a membrane-associated protein of the photosystem II complex that migrates as a 24-kDa species on polyacrylamide gel electrophoresis.     

Purification of a nitrate reductase kinase from Spinacea oleracea leaves, and its identification as a calmodulin-domain protein kinase

Spinach (Spinacea oleracea L.) nitrate reductase (NR) is inactivated by phosphorylation on serine-543, followed by binding of the phosphorylated enzyme to 14-3-3 proteins. We purified one of several chromatographically distinct NR_serine-543 kinases from spinach leaf extracts, and established by Edman sequencing of 80 amino acid residues that it is a calcium-dependent (calmodulin-domain) protein kinase (CDPK), with peptide sequences very similar to Arabidopsis CDPK6 (accession no. U20623; also known as CPK3). The spinach CDPK was recognized by antibodies raised against Arabidopsis CDPK. Nitrate reductase was phosphorylated at serine-543 by bacterially expressed His-tagged CDPK6, and the phosphorylated NR was inhibited by 14-3-3 proteins. However, the bacterially expressed CDPK6 had a specific activity approx. 200-fold lower than that of the purified spinach enzyme. The physiological control of NR by CDPK is discussed, and the regulatory properties of the purified CDPK are considered with reference to current models for reversible intramolecular binding of the calmodulin-like domain to the autoinhibitory junction of CDPKs. Received: 12 February 1998 / Accepted: 28 May 1998     

Protein phosphorylation as a mechanism for regulation of spinach leaf sucrose-phosphate synthase activity

Studies were conducted to determine whether protein phosphorylation may be a mechanism for regulation of spinach (Spinacia oleracea L.) leaf sucrose-phosphate synthase (SPS), shown previously to be light-dark regulated by some type of covalent modification. Radioactive phosphate was incorporated into the 120-kDa subunit of SPS during labeling of excised leaves with [32P]Pi, as shown by immunoprecipitation and denaturing gel electrophoresis of the enzyme. Conditions which activated the enzyme (illumination of leaves or mannose treatment of leaf discs in darkness) reduced the incorporation of radiolabel into SPS in the in vivo system. The partially purified SPS protein could also be phosphorylated in vitro using [gamma-32P]ATP. In the in vitro system, the incorporation of radiolabel into the 120-kDa subunit of SPS was dependent on time and magnesium concentration, and was closely paralleled by inactivation of the enzyme. These results provide the first evidence to establish protein phosphorylation as a mechanism for the covalent regulation of SPS activity.