Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals*

We report that protein phosphorylation is involved in the control of starch metabolism in Arabidopsis leaves at night. sex4 (starch excess 4) mutants, which have strongly reduced rates of starch metabolism, lack a protein predicted to be a dual specificity protein phosphatase. We have shown that this protein is chloroplastic and can bind to glucans and have presented evidence that it acts to regulate the initial steps of starch degradation at the granule surface. Remarkably, the most closely related protein to SEX4 outside the plant kingdom is laforin, a glucan-binding protein phosphatase required for the metabolism of the mammalian storage carbohydrate glycogen and implicated in a severe form of epilepsy (Lafora disease) in humans.

Starch, the main storage carbohydrate of plants, accumulates as a product of photosynthesis in leaves during the day and is converted to sucrose for export from the leaves at night. This conversion of starch to sucrose is one of the largest daily carbon fluxes on the planet, but nothing is known about how the process is initiated and controlled. The amounts of enzymes on the pathway change very little through the diurnal cycle in leaves of the model plant Arabidopsis thaliana, hence flux must be controlled by modulation of their activities (1).
Much progress in understanding the pathway has been made through the selection of Arabidopsis mutants impaired in starch degradation at night. All such mutations identified thus far are in genes encoding enzymes of the pathway, rather than proteins likely to be involved in modulation of the activities of these enzymes (2)(3)(4)(5)(6)(7)(8)(9)(10)(11). However, a mutation at a locus not yet identified, the starch excess 4 (or SEX4) locus, gives rise to a phenotype indicative of a regulatory defect rather than a defect in a structural enzyme. Mature sex4 leaves contain three to four times more starch than those of wild-type plants, apparently because a reduced capacity for starch degrada-tion at night leads to progressive accumulation of starch over the life of the leaf (12,13). Starch granules in leaves of the sex4 mutant are much larger and more rounded than those of wild-type plants (14). Measurements of activity and protein of enzymes known to be involved in starch degradation revealed only one significant reduction in the sex4 mutant in the chloroplastic ␣-amylase AMY3 (12,15). However, although both the activity and amount of protein of AMY3 are strongly reduced, this is not the cause of the deficiency in starch degradation in the sex4 mutant. T-DNA insertion lines lacking AMY3 protein have normal rates of starch degradation (15). The aim of the work described in this paper was to discover the nature of the gene at the SEX4 locus and thus shed light on the regulation of starch degradation.

EXPERIMENTAL PROCEDURES
Positional Identification of the SEX4 Locus-F2 plants from a cross between sex4-2 (Col-0 background) and Landsberg erecta showing the mutant phenotype were used for mapping. The mapping population (562 plants) was genotyped using SSLP and SNP markers available on the Arabidopsis Information Resource data base. This shows that the SEX4 gene was located within an 800-kb region between markers ATEM1 and SGCSNP42 on chromosome 3.
Plant Growth and Transformation-Plants were grown in 12-h light/ 12-h dark conditions (20°C, 60 -70% relative humidity, 175 mol of photons m Ϫ2 s Ϫ1 ), unless otherwise stated. The SEX4 cDNA (U14967 from the Arabidopsis Stock Center) was cloned into the binary vector 53AS with a 35 S cauliflower mosaic virus promoter and introduced into the sex4-1 and sex4-2 mutants via Agrobacterium-mediated transformation (by floral infiltration). Transgenic plants were selected by glufosinate resistance and confirmed by PCR and immunoblot analyses. Additionally, a C-terminal fusion construct of SEX4 cDNA and enhanced yellow fluorescent protein (Clontech TM ) was cloned into a vector with a double 35 S cauliflower mosaic virus promoter and introduced into Arabidopsis via Agrobacterium as described previously (4).
Gels, Antisera, and Immunoblotting-For the renaturation of amylolytic activity, extracts were subjected to electrophoresis on SDS-polyacrylamide gels containing starch. After washing and incubation in SDS-free medium, the gels were stained with iodine solution (15). For preparation of an antiserum to SEX4, a construct encoding a fusion between the full-length SEX4 protein and glutathione S-transferase (GST) 5 in the pGEX-4T-2 vector (Amersham Biosciences) was * This work was supported by funding from the Biotechnology and Biological Sciences Research Council of the United Kingdom (to A. M. S. and J. A. G.), from the Swiss National Science Foundation (National Centre of Competence in Research-Plant Survival) and the Roche Research Foundation (to S. C. Z.), and from the National Science Council, Taiwan (to J. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. □ S The on-line version of this article (available at http://www.jbc.org) contains supplemental material. 1 These authors contributed equally to this work. 2  expressed in Escherichia coli (BL21DE3) (15). The fusion protein was purified from inclusion bodies and used to immunize rabbits. Antiserum for AMY3 was prepared and used as described previously (15) Starch Analysis-Iodine staining of leaves and quantitative analyses of starch contents were performed as described previously (15).
Preparation of Chloroplasts-Chloroplasts were isolated from protoplasts and purified on a Percoll gradient and by treatment with protease (15,16). The purity of the chloroplast preparation was confirmed by the absence of activity of cytosolic marker enzymes. Chloroplast extracts from wild-type plants and leaf extracts from wild-type and mutant plants were loaded on a 10% SDS-polyacrylamide gel for the immunoblot analysis. Loading was adjusted so that each lane contained the same activity of chloroplastic phosphoglucose isomerase. A 1:1000 dilution of crude antiserum was used to detect the SEX4 protein.
Production of GST Fusion Protein-A fusion construct of the putative carbohydrate binding module of the SEX4 protein and GST was prepared and expressed in E. coli as described previously (17; the carbohydrate binding module is referred to as the kinase interaction sequence (KIS) domain in this reference).
Measurement of Maltose-Plants were grown in 8-h light/16-h dark conditions. Relative levels of maltose were determined by gas chromatography linked to mass spectrometry using methods described previously (18).

RESULTS
The SEX4 locus was mapped to a region of 800 kb on chromosome 3. Gene discovery was facilitated by the observation that one gene in this region (At3g52180) displays the same distinctive pattern of diurnal change in transcript abundance in the leaf as genes encoding enzymes known to be involved in starch degradation (1). Sequencing revealed mutations likely to prevent or impair function in this gene in plants carrying three independent sex4 alleles (Fig. 1). The sex4-1 allele contains a deletion that overlaps the open reading frames of both At3g52180 and At3g52190. The sex4-2 allele contains a point mutation in the seventh exon. This is predicted to change the arginine residue of the signature motif of a protein phosphatase (see last paragraph under "Results") to a lysine; hence this change is highly likely to affect protein function. The sex4-4 allele contains a point mutation that gives rise to a stop codon and results in a truncated protein ( Fig. 1 and data not  shown).
To provide further evidence about the identity of the SEX4 gene, we isolated two T-DNA insertion mutants in At3g52180 (Fig. 1, sex4-3 and sex4-5) and showed that they have starch excess phenotypes ( Fig. 2A). Levels of starch are similar to those in plants carrying the previously characterized mutant alleles (Fig. 2B). We also transformed the sex4-1 and sex4-2 mutants with a cDNA encoding the wild-type SEX4 protein.
The SEX4 protein has a predicted N-terminal chloroplast transit peptide. To discover whether the protein is in fact chloroplastic, the sex4-1 mutant was transformed with a construct encoding the wild-type SEX4 protein fused at the C terminus to yellow fluorescent protein (YFP). The resulting transgenic plants no longer displayed a starch excess phenotype and exhibited YFP fluorescence specifically in the chloroplasts (Fig.  3A). Furthermore, protein gel blots probed with an antiserum raised against the SEX4 protein revealed that chloroplasts isolated from the leaves of wild-type plants contained a protein with a similar apparent mass to that of the predicted SEX4 protein. This protein was missing from leaves of sex4-1 mutant plants (Fig. 3B).
Previously, we have shown that sex4 mutants have lower levels of sugars (sucrose, glucose, and fructose) in their leaves at night (13). To investigate this further, we measured maltose, the major product of starch breakdown (4 -6, 19), 1 h prior to the end of the dark period. In sex4-1, the relative maltose content was statistically significantly reduced (55% that of the wild-type plants). This suggests that the reduced availability of starch catabolites limits sucrose synthesis at night. Second, we crossed sex4-5 (a T-DNA insertion mutant) with a sex1 mutant. SEX1 encodes a glucan water dikinase (GWD1), which phosphorylates glucosyl residues within the amylopectin moiety of  starch (3,20). Its action is necessary for normal rates of starch degradation; in its absence, starch accumulates to levels approximately twice those observed in sex4 mutants (3,13). The phosphate groups are believed to facilitate access to the starch granule surface by the enzymes that catalyze the initial attack on the granule (20); hence GWD1 can be regarded as an initial step on the pathway of starch degradation. The starch content of leaves of the double mutant sex4/sex1 closely resembled that of sex1 and was different from that of sex4 (Fig. 2D). At the end of the light period, the starch content of sex4/sex1 was 1.7-fold higher than that of sex4 and not statistically different from that of sex1. At the end of the dark period, the starch content of sex4/sex1 was almost 2-fold higher than that of sex4 and 80% of that of sex1. The simplest explanation for these data is that SEX4 affects GWD or a step immediately downstream of it but upstream of maltose production.
SEX4 encodes a putative dual specificity protein phosphatase, PTP-KIS1 (17). Genes encoding highly similar proteins are found in other species of plants, including tomato, rice, and maize. The N-terminal part of the protein contains the phosphatase domain, and the 63% identical tomato orthologue has been shown to have phosphatase activity both on a generic phosphatase substrate and on the phosphotyrosine residues of synthetic peptides (17). In addition, PTPKIS1 possesses a C-terminal domain containing motifs characteristic of a carbohydrate binding module (CBM_20; Refs. 21 and 22) (see supplemental Fig. S2). To test whether the Arabidopsis protein can bind to carbohydrate, the heterologously expressed C-terminal domain was incubated with glycogen in vitro. The protein bound to glycogen in a saturating manner, and binding was inhibited by increasing concentrations of ␤-cyclodextrin. The protein also bound to amylose and to starch (Fig. 4 and data not shown).

DISCUSSION
Taken together, the localization of SEX4 protein in chloroplasts, its affinity for glucans, the phenotype of the sex4 mutant, and the diurnal regulation of SEX4 transcript levels (1) suggest that SEX4 interacts with starch in vivo and is directly necessary for its metabolism. SEX4 may dephosphorylate and thus modulate the activity of an enzyme or enzymes that directly exercises control over flux through the pathway of starch degradation. Alternatively, SEX4 may act indirectly on starch metabolism. In general, dual specificity protein phosphatases act on protein kinases (23). SEX4 may thus modulate the activity of a protein kinase, which in turn modulates the activity of enzyme(s) of starch degradation.
The enzymes involved in starch degradation are not fully understood, and there is little evidence thus far that they have regulatory properties of importance in the control of flux through the pathway (1,24). The extent and importance of phosphorylation in modulating their activities has not been investigated. However, phosphorylation has recently been shown to be important in modulating the activity of enzymes of starch synthesis; isoforms of starch-branching enzyme are activated by phosphorylation in chloroplasts and endosperm amyloplasts of wheat (25). Our fractionation experiments and genetic analyses indicate that targets for modulation via SEX4 lie within the chloroplast and upstream of maltose production in the pathway of starch degradation. Thus, possible targets include one or more of the following: glucan water dikinase (SEX1 or GWD1) or phosphoglucan water dikinase (GWD3 or PWD, thought to act after GWD) (7,8), isoamylase 3 (10, 11), chloroplastic ␤-amylases (9), and possibly disproportionating enzyme (2). Mutant plants lacking any one of these proteins have starch excess phenotypes, and several of these proteins have been shown to be necessary for normal rates of starch granule degradation at night. The reason why the chloroplastic ␣-amylase AMY3 is reduced in abundance in the absence of SEX4 remains to be investigated.
Remarkably, the proteins most closely related to the SEX4-like proteins in plants are mammalian laforins (17,26). These are also dual specificity protein phosphatases with CBM_20 domains, although the CBM is N-terminal in laforins (21,27). Human and mouse laforins have affinity for both glycogen and starch (27)(28)(29)(30). Similar to SEX4, laforins are necessary for normal metabolism of storage glucans. Humans and Several independently transformed lines with these characteristics were obtained. B, immunoblot of extracts of leaves of wild-type (Col) and sex4 mutant plants, and of chloroplasts (Chl) isolated from wild-type plants probed with an antiserum to SEX4. Masses of molecular markers are shown in kDa; SEX4 is marked with an arrow. The SEX4 protein is present in purified chloroplasts. It is absent from sex4-1 leaves as expected. SEX4 protein is present in sex4-2 leaves but is expected to be inactive because of the substitution of an amino acid that is strictly conserved in the active sites of all dual specificity protein phosphatases (see Fig. 1). The CBM of SEX4 was expressed as a fusion protein with glutathione S-transferase (GST), incubated with or without glycogen in the presence of BSA, and then subjected to ultracentrifugation. Control incubations contained GST or GST fused with the kinase interaction (KIS) domain of the protein kinase ZmAKIN ␤␥, which bears sequence similarities to the CBM of SEX4 (17). The SDS-polyacrylamide gel shows proteins in the pellets. Incubations contained GST and BSA (lanes 1 and 2), CBM-GST and BSA (lanes 3 and 4), or KIS-GST and BSA (lanes 5 and 6). Incubations shown in lanes 1, 3, and 5 contained glycogen; those shown in lanes 2, 4, and 6 did not. The putative CBM domain of SEX4 binds glycogen (lane 3), whereas the KIS domain of ZmAKIN ␤␥ does not (lane 5). This experiment was repeated three times with the same result. Further experiments (not shown) revealed that CBM-GST fusion protein binds glycogen in a saturating manner and that binding is inhibited by increasing concentrations of ␤-cyclodextrin, as is the case for starch-binding proteins (37). The CBM-GST fusion protein also binds amylose and soluble starch (not shown). mice carrying mutations that affect laforin function accumulate polyglucosan inclusions, putatively arising from abnormal glycogen metabolism. These are composed of glucan polymers with branching patterns thought to be more similar to those of the amylopectin component of plant starch than those of glycogen (31). Polyglucosan inclusions are implicated in neuronal death and consequent progressive myoclonus epilepsy in humans and mice (32)(33)(34). Recently, laforin was shown to dephosphorylate (and thereby activate) glycogen synthase kinase 3 at an amino-terminal serine residue (Ser-9) (35). Active glycogen synthase kinase 3 phosphorylates and inactivates glycogen synthase. Thus, loss of laforin may allow glycogen synthase activity to proceed unchecked. Arabidopsis has 10 glycogen synthase kinase 3 homologues (36), one of which (AtK-1/ASK, At1g09840) is predicted to be localized within the chloroplast. This may represent a target for SEX4, although it is worth noting, first, that the Ser-9 residue is not conserved in any known plant glycogen synthase kinase 3 homologues (36) and, second, that the existing biochemical data in this and previous studies (12,13) point toward a deficiency in starch breakdown rather than activation of the starch biosynthetic pathway.
In conclusion, despite the appreciable differences in the enzymes directly involved in starch metabolism in plants and glycogen metabolism in mammals, the striking similarities between SEX4 and laforins indicate a previously unsuspected degree of convergence or conservation in the regulation of glucan metabolism.