Similar Protein Phosphatases Control Starch Metabolism in Plants and Glycogen Metabolism in Mammals
2006; Elsevier BV; Volume: 281; Issue: 17 Linguagem: Inglês
10.1074/jbc.m600519200
ISSN1083-351X
AutoresTotte Niittylä, Sylviane Comparot-Moss, Wei‐Ling Lue, Gaëlle Messerli, Martine Trévisan, Michael David John Seymour, John A. Gatehouse, Dorthe Villadsen, Steven M. Smith, Jychian Chen, Samuel C. Zeeman, Alison M. Smith,
Tópico(s)Protein Tyrosine Phosphatases
ResumoWe 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. 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 (1Smith S.M. Fulton D.C. Chia T. Thorneycroft D. Chapple A. Dunstan H. Hylton C. Zeeman S.C. Smith A.M. Plant Physiol. 2004; 136: 2687-2699Crossref PubMed Scopus (322) Google Scholar). 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 (2Critchley J.H. Zeeman S.C. Takaha T. Smith A.M. Smith S.M. Plant J. 2001; 26: 89-100Crossref PubMed Google Scholar, 3Yu T.-S. Kofler H. Häusler R. Hille D. Flügge U.-I. Zeeman S.C. Smith A.M. Kossmann J. Lloyd J. Ritte G. Steup M. Lue W.-L. Chen J. Weber A. Plant Cell. 2001; 13: 1907-1918Crossref PubMed Scopus (255) Google Scholar, 4Niittylä T. Messerli G. Trevisan M. Chen J. Smith A.M. Zeeman S.C. Science. 2004; 303: 87-89Crossref PubMed Scopus (365) Google Scholar, 5Chia T. Thorneycroft D. Chapple A. Messerli G. Chen J. Zeeman S.C. Smith S.M. Smith A.M. Plant J. 2004; 37: 853-863Crossref PubMed Scopus (216) Google Scholar, 6Lu Y. Sharkey T.D. Planta. 2004; 218: 468-473Crossref Scopus (149) Google Scholar, 7Kötting O. Pusch P. Tiessen A. Geigenberger P. Steup M. Ritte G. Plant Physiol. 2005; 137: 242-252Crossref PubMed Scopus (212) Google Scholar, 8Baunsgaard L. Lütken H. Mikkelsen R. Glaring M.A. Pham T.T. Blennow A. Plant J. 2005; 41: 595-605Crossref PubMed Scopus (162) Google Scholar, 9Kaplan F. Guy C.L. Plant J. 2005; 44: 730-743Crossref PubMed Scopus (191) Google Scholar, 10Wattebled F. Dong Y. Dumez S. Delvallé D. Planchot V. Berbezy P. Vyas D. Colonna P. Chatterjee M. Ball S. D'Hulst C. Plant Physiol. 2005; 138: 184-195Crossref PubMed Scopus (144) Google Scholar, 11Delatte T. Umhang M. Trevisan M. Eicke S. Thorneycroft D. Smith S.M. Zeeman S.C. J. Biol. Chem. February 22, 2006; 10.1074/jbc.M513661200PubMed Google Scholar). 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 degradation at night leads to progressive accumulation of starch over the life of the leaf (12Zeeman S.C. Northrop F. Smith A.M. ap Rees T. Plant J. 1998; 15: 357-365Crossref PubMed Scopus (181) Google Scholar, 13Zeeman S.C. ap Rees T. Plant Cell Environ. 1999; 22: 1445-1453Crossref Scopus (102) Google Scholar). Starch granules in leaves of the sex4 mutant are much larger and more rounded than those of wild-type plants (14Zeeman S.C. Tiessen A. Pilling E. Kato L. Donald A.M. Smith A.M. Plant Physiol. 2002; 129: 516-529Crossref PubMed Scopus (138) Google Scholar). 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 α-amylase AMY3 (12Zeeman S.C. Northrop F. Smith A.M. ap Rees T. Plant J. 1998; 15: 357-365Crossref PubMed Scopus (181) Google Scholar, 15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). 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 (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). 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. 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 μmol of photons m-2 s-1), 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™) was cloned into a vector with a double 35 S cauliflower mosaic virus promoter and introduced into Arabidopsis via Agrobacterium as described previously (4Niittylä T. Messerli G. Trevisan M. Chen J. Smith A.M. Zeeman S.C. Science. 2004; 303: 87-89Crossref PubMed Scopus (365) Google Scholar). 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 (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). For preparation of an antiserum to SEX4, a construct encoding a fusion between the full-length SEX4 protein and glutathione S-transferase (GST) 5The abbreviations used are: GST, glutathione S-transferase; BSA, bovine serum albumin; YFP, yellow fluorescent protein; GWD, glucan water dikinase; CBM, carbohydrate binding module; KIS, kinase interaction sequence. in the pGEX-4T-2 vector (Amersham Biosciences) was expressed in Escherichia coli (BL21DE3) (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). The fusion protein was purified from inclusion bodies and used to immunize rabbits. Antiserum for AMY3 was prepared and used as described previously (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar) Starch Analysis—Iodine staining of leaves and quantitative analyses of starch contents were performed as described previously (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). Preparation of Chloroplasts—Chloroplasts were isolated from protoplasts and purified on a Percoll gradient and by treatment with protease (15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar, 16Fitzpatrick L.M. Keegstra K. Plant J. 2001; 27: 59-65Crossref PubMed Scopus (91) Google Scholar). 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 (17Fordham-Skelton A.P. Chilley P. Lumbreras V. Reignoux S. Fenton T.R. Dahm C.C. Pages M. Gatehouse J.A. Plant J. 2002; 29: 705-715Crossref PubMed Scopus (76) Google Scholar; the carbohydrate binding module is referred to as the kinase interaction sequence (KIS) domain in this reference). Glycogen Binding Assays—Protein-free glycogen (5 mg ml-1) in 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 0.1% (v/v) 2-mercaptoethanol, 0.02% (w/v) Brij-35, 0.1 mg ml-1 bovine serum albumin (BSA) was mixed with GST fusion protein. Samples were incubated at 0 °C for 30 min and then centrifuged 90 min at 100,000 × g at 4 °C to sediment the glycogen. Pellets were washed in 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, resuspended in 4× SDS sample buffer, and run on 12.5% SDS gels. The gels were stained with Coomassie Brilliant Blue R. 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 (18Roessner U. Luedemann A. Brust D. Fiehn O. Linke T. Willmitzer L. Fernie A.R. Plant Cell. 2001; 13: 11-29Crossref PubMed Scopus (849) Google Scholar). 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 (1Smith S.M. Fulton D.C. Chia T. Thorneycroft D. Chapple A. Dunstan H. Hylton C. Zeeman S.C. Smith A.M. Plant Physiol. 2004; 136: 2687-2699Crossref PubMed Scopus (322) Google Scholar). 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. Transformants no longer displayed a starch excess phenotype (Fig. 2C). All of the new sex4 mutant alleles had reduced levels of the chloroplastic α-amylase AMY3 (see supplemental Fig. S1), as is the case for sex4-1 and sex4-2 (12Zeeman S.C. Northrop F. Smith A.M. ap Rees T. Plant J. 1998; 15: 357-365Crossref PubMed Scopus (181) Google Scholar, 15Yu T.-S. Zeeman S.C. Thorneycroft D. Fulton D.C. Dunstan H. Lue W.-L. Hegemann B. Tung S.-Y. Umemoto U. Chapple A. Tsai D.-L. Wang S.-M. Smith A.M. Chen J. Smith S.M. J. Biol. Chem. 2005; 280: 9773-9779Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar). 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 (13Zeeman S.C. ap Rees T. Plant Cell Environ. 1999; 22: 1445-1453Crossref Scopus (102) Google Scholar). To investigate this further, we measured maltose, the major product of starch breakdown (4Niittylä T. Messerli G. Trevisan M. Chen J. Smith A.M. Zeeman S.C. Science. 2004; 303: 87-89Crossref PubMed Scopus (365) Google Scholar, 5Chia T. Thorneycroft D. Chapple A. Messerli G. Chen J. Zeeman S.C. Smith S.M. Smith A.M. Plant J. 2004; 37: 853-863Crossref PubMed Scopus (216) Google Scholar, 6Lu Y. Sharkey T.D. Planta. 2004; 218: 468-473Crossref Scopus (149) Google Scholar, 19Weise S.E. Weber A.P.M. Sharkey T.D. Planta. 2004; 218: 474-482Crossref PubMed Scopus (184) Google Scholar), 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 (3Yu T.-S. Kofler H. Häusler R. Hille D. Flügge U.-I. Zeeman S.C. Smith A.M. Kossmann J. Lloyd J. Ritte G. Steup M. Lue W.-L. Chen J. Weber A. Plant Cell. 2001; 13: 1907-1918Crossref PubMed Scopus (255) Google Scholar, 20Ritte G. Lloyd J.R. Eckermann N. Rottmann A. Kossmann J. Steup M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7166-7171Crossref PubMed Scopus (260) Google Scholar). Its action is necessary for normal rates of starch degradation; in its absence, starch accumulates to levels approximately twice those observed in sex4 mutants (3Yu T.-S. Kofler H. Häusler R. Hille D. Flügge U.-I. Zeeman S.C. Smith A.M. Kossmann J. Lloyd J. Ritte G. Steup M. Lue W.-L. Chen J. Weber A. Plant Cell. 2001; 13: 1907-1918Crossref PubMed Scopus (255) Google Scholar, 13Zeeman S.C. ap Rees T. Plant Cell Environ. 1999; 22: 1445-1453Crossref Scopus (102) Google Scholar). The phosphate groups are believed to facilitate access to the starch granule surface by the enzymes that catalyze the initial attack on the granule (20Ritte G. Lloyd J.R. Eckermann N. Rottmann A. Kossmann J. Steup M. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7166-7171Crossref PubMed Scopus (260) Google Scholar); 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 (17Fordham-Skelton A.P. Chilley P. Lumbreras V. Reignoux S. Fenton T.R. Dahm C.C. Pages M. Gatehouse J.A. Plant J. 2002; 29: 705-715Crossref PubMed Scopus (76) Google Scholar). 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 (17Fordham-Skelton A.P. Chilley P. Lumbreras V. Reignoux S. Fenton T.R. Dahm C.C. Pages M. Gatehouse J.A. Plant J. 2002; 29: 705-715Crossref PubMed Scopus (76) Google Scholar). In addition, PTPKIS1 possesses a C-terminal domain containing motifs characteristic of a carbohydrate binding module (CBM_20; Refs. 21Machovič M. Svensson B. MacGregor E.A. Janeček Š. FEBS J. 2005; 272: 5497-5513Crossref PubMed Scopus (59) Google Scholar and 22Coutinho P.M. Henrissat B. Gilbert H.J. Davies G. Henrissat B. Svensson B. Recent Advances in Carbohydrate Bioengineering. The Royal Society of Chemistry, Cambridge, UK1999: 3-12Google Scholar) (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 β-cyclodextrin. The protein also bound to amylose and to starch (Fig. 4 and data not shown). 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 (1Smith S.M. Fulton D.C. Chia T. Thorneycroft D. Chapple A. Dunstan H. Hylton C. Zeeman S.C. Smith A.M. Plant Physiol. 2004; 136: 2687-2699Crossref PubMed Scopus (322) Google Scholar) 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 (23Luan S. Annu. Rev. Plant Biol. 2003; 54: 63-92Crossref PubMed Scopus (218) Google Scholar). 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 (1Smith S.M. Fulton D.C. Chia T. Thorneycroft D. Chapple A. Dunstan H. Hylton C. Zeeman S.C. Smith A.M. Plant Physiol. 2004; 136: 2687-2699Crossref PubMed Scopus (322) Google Scholar, 24Smith A.M. Zeeman S.C. Smith S.M. Annu. Rev. Plant Biol. 2005; 56: 73-97Crossref PubMed Scopus (436) Google Scholar). 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 (25Tetlow I.J. Wait R. Lu Z. Akkasaeng R. Bowsher C.G. Esposito S. Kosar-Hashemi B. Morell M.K. Emes M.J. Plant Cell. 2004; 16: 694-708Crossref PubMed Scopus (288) Google Scholar). 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) (7Kötting O. Pusch P. Tiessen A. Geigenberger P. Steup M. Ritte G. Plant Physiol. 2005; 137: 242-252Crossref PubMed Scopus (212) Google Scholar, 8Baunsgaard L. Lütken H. Mikkelsen R. Glaring M.A. Pham T.T. Blennow A. Plant J. 2005; 41: 595-605Crossref PubMed Scopus (162) Google Scholar), isoamylase 3 (10Wattebled F. Dong Y. Dumez S. Delvallé D. Planchot V. Berbezy P. Vyas D. Colonna P. Chatterjee M. Ball S. D'Hulst C. Plant Physiol. 2005; 138: 184-195Crossref PubMed Scopus (144) Google Scholar, 11Delatte T. Umhang M. Trevisan M. Eicke S. Thorneycroft D. Smith S.M. Zeeman S.C. J. Biol. Chem. February 22, 2006; 10.1074/jbc.M513661200PubMed Google Scholar), chloroplastic β-amylases (9Kaplan F. Guy C.L. Plant J. 2005; 44: 730-743Crossref PubMed Scopus (191) Google Scholar), and possibly disproportionating enzyme (2Critchley J.H. Zeeman S.C. Takaha T. Smith A.M. Smith S.M. Plant J. 2001; 26: 89-100Crossref PubMed Google Scholar). 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 α-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 (17Fordham-Skelton A.P. Chilley P. Lumbreras V. Reignoux S. Fenton T.R. Dahm C.C. Pages M. Gatehouse J.A. Plant J. 2002; 29: 705-715Crossref PubMed Scopus (76) Google Scholar, 26Kerk D. Bulgrien J. Smith D.W. Barsam B. Veretnik S. Gribskov M. Plant Physiol. 2002; 129: 908-925Crossref PubMed Scopus (214) Google Scholar). These are also dual specificity protein phosphatases with CBM_20 domains, although the CBM is N-terminal in laforins (21Machovič M. Svensson B. MacGregor E.A. Janeček Š. FEBS J. 2005; 272: 5497-5513Crossref PubMed Scopus (59) Google Scholar, 27Wang J. Stuckey J.A. Wishart M.J. Dixon J.E. J. Biol. Chem. 2002; 277: 2377-2380Abstract Full Text Full Text PDF PubMed Scopus (144) Google Scholar). 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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 (31Minassian B.A. Pediatr. Neurol. 2001; 25: 21-29Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). Polyglucosan inclusions are implicated in neuronal death and consequent progressive myoclonus epilepsy in humans and mice (32Minassian B.A. Lee J.R. Herbrick J.-A. Huizenga J. Soder S. Mungall A.J. Dunham I. Gardner R. Fong C.-Y.G. Carpenter S. Jardim L. Satishchandra P. Andermann E. Snead O.C. Lopes-Cendes I. Tsui L.-C. Delgado-Escueta A.V. Rouleau G.A. Scherer S.W. Nat. Genet. 1998; 20: 171-174Crossref PubMed Scopus (413) Google Scholar, 33Serratosa J.M. Gomez-Garre P. Gallardo M.E. Anta B. de Bernabé D.B. Lindhout D. Augustijn P.B. Tassinari C.A. Malafosse R.M. Topcu M. Gris D. Dravet C. Berkovic S.F. de Cordoba S.R. Hum. Mol. Genet. 1999; 2: 345-352Crossref Scopus (197) Google Scholar, 34Ganesh S. Delgado-Escueta A.V. Sakamoto T. Avila M.R. Machado-Salas J. Hoshii Y. Akagi T. Gomi H. Suzuki T. Amano K. Agarwala K.L. Hasegawa Y. Bai D.-S. Ishihara T. Hashikawa T. Itohara S. Cornford E.M. Niki H. Yamakawa K. Hum. Mol. Genet. 2002; 11: 1251-1262Crossref PubMed Scopus (188) Google Scholar). Recently, laforin was shown to dephosphorylate (and thereby activate) glycogen synthase kinase 3 at an amino-terminal serine residue (Ser-9) (35Lohi H. Ianzano L. Zhao X.-C. Chan E.M. Turnbull J. Scherer S.W. Ackerley C.A. Minassian B.A. Hum. Mol. Genet. 2005; 14: 2727-2736Crossref PubMed Scopus (132) Google Scholar). 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 (36Jonak C. Hirt H. Trends Plant Sci. 2002; 7: 457-461Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar), one of which (AtK-1/ASKκ, 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 (36Jonak C. Hirt H. Trends Plant Sci. 2002; 7: 457-461Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar) and, second, that the existing biochemical data in this and previous studies (12Zeeman S.C. Northrop F. Smith A.M. ap Rees T. Plant J. 1998; 15: 357-365Crossref PubMed Scopus (181) Google Scholar, 13Zeeman S.C. ap Rees T. Plant Cell Environ. 1999; 22: 1445-1453Crossref Scopus (102) Google Scholar) 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. We thank Dr. Alisdair Fernie and Nicolas Schauer for assistance with the gas chromatography-mass spectrometry analysis and Therese Mandel for access to ethane methyl sulfonate-mutagenized Arabidopsis population. Download .pdf (.17 MB) Help with pdf files
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