Skip to main content
SpringerLink
Log in

Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots

  • Original Article
  • Published:
Planta Aims and scope Submit manuscript

Abstract

A 78-amino acid insertion (L78) is found in the low-affinity type (L-form) of starch phosphorylase (L-SP, EC 2.4.1.1). This insertion blocks the starch-binding site on the L-SP molecule, and it decreases the binding affinity of L-SP toward starch. The computational analysis of the amino acid sequence on L78 predicts several phosphorylation sites at its Ser residues. Indeed, from the immunoblotting results using antibodies against phosphoamino acids, we observed that the purified L-SP from mature sweet potato (Ipomoea batatas) roots is phosphorylated. This observation led us to the detection of a protein kinase activity in the protein fraction of the crude extract from the sweet potato roots. The kinase was partially purified by liquid chromatography, and its native molecular mass was estimated as 338 kDa. An expressed peptide (L78P) containing the essential part of L78 was intensively phosphorylated by the kinase. However, H-SP (the high-affinity isomer of SP lacking the L78 insertion) and the proteolytic modified L-SP, which lost its L78 fragment, could not be phosphorylated. Furthermore, using L78P mutants by site-directed mutagenesis at Ser residues on L78, we demonstrate that only one Ser residue on L78 is phosphorylated by the kinase. These results imply that this kinase is specific to L-SP, or more precisely, to the L78 insertion. The in vitro phosphorylated L-SP shows higher sensitivity to proteolytic modification, but has no change in its kinetic parameters.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

CDPK:

Calcium-dependent protein kinase

Glc-1-P:

Glucose 1-phosphate

GP:

Glycogen phosphorylase

H-SP:

High-affinity type SP (Pho2)

L-SP:

Low-affinity type SP (Pho1)

MBP:

myelin basic protein

PVDF:

Polyvinylidene fluoride

SP:

Starch phosphorylase

References

  • Albrecht T, Greve B, Pusch K, Kossmann J, Buchner P, Wobus U, Steup M (1998) Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L as revealed by sequence-specific antibodies. Eur J Biochem 251:343–352

    Article  PubMed  CAS  Google Scholar 

  • Albrecht T, Koch A, Lode A, Greve B, Schneider-Mergener J, Steup M (2001) Plastidic (Pho1-type) phosphorylase isoforms in potato (Solanum tuberosum L.) plants: expression analysis and immunochemical characterization. Planta 213:602–613

    Article  PubMed  CAS  Google Scholar 

  • Ball S, Guan HP, James M, Myers A, Keeling P, Mouille G, Buleon A, Colonna P, Preiss J (1996) From glycogen to amylopectin—a model for the biogenesis of the plant starch granule. Cell 86:349–352

    Article  PubMed  CAS  Google Scholar 

  • Baun CC, Palmiano EP, Perez CM, Juliano BO (1970) Enzymes of starch metabolism in the developing rice grain. Plant Physiol 46:429–434

    PubMed  CAS  Google Scholar 

  • Boyle WJ, Van der Geer P, Hunter T (1991) Phosphopeptide mapping and phosphoamino acid analysis by two-dimensional separation on thin-layer cellulose plates. Methods Enzymol 201:110–149

    PubMed  CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  PubMed  CAS  Google Scholar 

  • Brisson N, Giroux H, Zollinger M, Camirand A, Simard C (1989) Maturation and subcellular compartmentation of potato starch phosphorylase. Plant Cell 1:559–566

    Article  PubMed  CAS  Google Scholar 

  • Camirand A, St-Pierre B, Marineau C, Brisson N (1990) Occurrence of a copia-like transposable element in one of the introns of the potato starch phosphorylase gene. Mol Gen Gene 224:33–39

    CAS  Google Scholar 

  • Chan KF, Hurst MO, Graves DJ (1982) Phosphorylase kinase specificity. A comparative study with cAMP-dependent protein kinase on synthetic peptides and peptide analogs of glycogen synthase and phosphorylase. J Biol Chem 257:3655–3659

    PubMed  CAS  Google Scholar 

  • Chang SC, Lin PC, Chen HM, Wu JS, Juang RH (2000) The isolation and characterization of chaperonin 60 from sweet potato roots—involvement of the chaperonins in starch biosynthesis. Bot Bull Academia Sin 41:105–111

    CAS  Google Scholar 

  • Chen ZJ, Parent L, Maniatis T (1996) Site-specific phosphorylation of IκBα by a novel ubiquitin-dependent protein kinase activity. Cell 84:853–862

    Article  PubMed  CAS  Google Scholar 

  • Chen HM, Chang SC, Wu CC, Cuo TS, Wu JS, Juang RH (2002) Regulation of the catalytic behaviour of L-form starch phosphorylase from sweet potato roots by proteolysis. Physiol Plant 114:506–515

    Article  PubMed  CAS  Google Scholar 

  • Chiang C-L, Lu Y-L, Juang RH, Lee P-D, Su JC (1991) Native and degraded forms of sweet potato starch phosphorylase. Agric Biol Chem 55:641–646

    CAS  Google Scholar 

  • Duwenig E, Steup M, Kossmann J (1997) Induction of genes encoding plastidic phosphorylase from spinach (Spinacia oleracea L.) and potato (Solanum tuberosum L) by exogenously supplied carbohydrates in excised leaf discs. Planta 203:111–120

    Article  PubMed  CAS  Google Scholar 

  • Glotzer M, Murray AW, Kirschner MW (1991) Cyclin is degraded by the ubiquitin pathway. Nature 349:132–138

    Article  PubMed  CAS  Google Scholar 

  • Hanes CS (1940a) The breakdown and synthesis of starch by an enzyme system from pea seeds. Proc R Soc (Lond) B 128:421–450

    CAS  Google Scholar 

  • Hanes CS (1940b) The reversible formation of starch from glucose-1-phosphate catalyzed by potato phosphorylase. Proc R Soc (Lond) B 129:174–208

    Article  CAS  Google Scholar 

  • Hardie DG (1999) Plant protein serine/threonine kinases: classification and functions. Annu Rev Plant Physiol Plant Mol Biol 50:97–131

    Article  PubMed  CAS  Google Scholar 

  • Huber SC, Huber JL, Liao PC, Gage DA, McMichael RW Jr, Chourey PS, Hannah LC, Koch K (1996) Phosphorylation of serine-15 of maize leaf sucrose synthase. Occurrence in vivo and possible regulatory significance. Plant Physiol 112:793–802

    CAS  Google Scholar 

  • Juang RH, Chang YD, Sung HY, Su JC (1984) Oven-drying method for polyacrylamide gel slab packed in cellophane sandwich. Anal Biochem 141:348–350

    Article  PubMed  CAS  Google Scholar 

  • Kruger NJ, Ap Rees T (1983) Properties of α-glucan phosphorylase from pea-chloroplasts. Phytochemistry 22:1891–1898

    Article  CAS  Google Scholar 

  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–685

    Article  PubMed  CAS  Google Scholar 

  • Lin CT, Yeh KW, Lee PD, Su JC (1991) Primary structure of sweet potato starch phosphorylase deduced from its cDNA sequence. Plant Physiol 95:1250–1253

    PubMed  CAS  Google Scholar 

  • Liu TTY, Shannon JC (1981) A nonaqueous procedure for isolating starch granules with associated metabolites from maize endosperm. Plant Physiol 67:518–524

    PubMed  CAS  Google Scholar 

  • Mingo-Castel AM, Young RE, Smith OE (1976) Kinetin-induced tuberization of potato in vitro: on the mode of action of kinetin. Plant Cell Physiol 17:557–570

    CAS  Google Scholar 

  • Mori H, Tanizawa K, Fukui T (1991) Potato tuber type H phosphorylase isoenzyme. Molecular cloning, nucleotide sequence, and expression of a full-length cDNA in Escherichia coli. J Biol Chem 266:18446–18453

    PubMed  CAS  Google Scholar 

  • Mori H, Tanizawa K, Fukui T (1993) A chimeric α-glucan phosphorylase of plant type L and H isozymes: functional role of 78-residue insertion in type L isozyme. J Biol Chem 268:5574–5581

    PubMed  CAS  Google Scholar 

  • Nakano K, Fukui T (1986) The complete amino acid sequence of potato α-glucan phosphorylase. J Biol Chem 261:8230–8236

    PubMed  CAS  Google Scholar 

  • Nakano K, Mori H, Fukui T (1989) Molecular cloning of cDNA encoding potato amyloplast α-glucan phosphorylase and the structure of its transit peptide. J Biochem (Tokyo) 106:691–695

    CAS  Google Scholar 

  • Nelson O, Pan D (1995) Starch synthesis in maize endosperms. Annu Rev Plant Physiol Plant Mol Biol 46:475–496

    Article  CAS  Google Scholar 

  • Rogers S, Wells R, Rechsteiner M (1986) Amino acid sequences common to rapidly degraded proteins: the PEST hypothesis. Science 234:364–368

    Article  PubMed  CAS  Google Scholar 

  • Schupp N, Ziegler P (2004) The relation of starch phosphorylases to starch metabolism in wheat. Plant Cell Physiol 45:1471–1484

    Article  PubMed  CAS  Google Scholar 

  • Sprang SR, Acharya KR, Goldsmith EJ, Stuart DI, Varvill K, Fletterick RJ, Madsen NB, Johnson LN (1988) Structural changes in glycogen phosphorylase induced by phosphorylation. Nature 336:215–221

    Article  PubMed  CAS  Google Scholar 

  • St-Pierre B, Brisson N (1995) Induction of the plastidic starch-phosphorylase gene in potato storage sink tissue. Planta 195:339–344

    Article  CAS  Google Scholar 

  • Tetlow IJ, Wait R, Lu Z, Akkasaeng R, Bowsher CG, Esposito S, Kosar-Hashemi B, Morell MK, Emes MJ (2004) Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions. Plant Cell 16:694–708

    Article  PubMed  CAS  Google Scholar 

  • Van Berkel J, Conrads Strauch J, Steup M (1991) Glucan-phosphorylase forms in cotyledons of Pisum sativm L.: localization, developmental change, in vitro translation, and processing. Planta 185:432–439

    Article  CAS  Google Scholar 

  • Zeeman SC, Thorneycroft D, Schupp N, Chapple A, Weck M, Dunstan H, Haldimann P, Bechtold N, Smith AM, Smith SM (2004) Plastidial α-glucan phosphorylase is not required for starch degradation in Arabidopsis leaves but has a role in the tolerance of abiotic stress. Plant Physiol 135:849–858

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

We thank Drs. D.S. Bendall and B.G. Schlarb-Ridley (University of Cambridge); Dr. S.C. Chang (Harvard Medical School); Drs. Y.Y. Charng and C.S. Chen (Academia Sinica, ROC); Dr. C.M. Chiang (Case Western Reserve University); Drs. P.L. Huang, A.Y. Wang, and C.C. Yang (National Taiwan University); and Dr. A.B. Tobin (University of Leicester) for their helpful suggestions and discussions. We also thank Dr. J. Hook for reading the article. Part of this work was supported by the National Science Council, ROC.

Author information

Authors and Affiliations

  1. Department of Biochemical Science and Technology, Institute of Microbiology and Biochemistry, National Taiwan University, AC 2-520, Taipei, 106, Taiwan

    Guang-Huar Young, Kuang-Ching Tseng, Jiann-Shing Wu & Rong-Huay Juang

  2. Department of Life Science, Catholic Fu-Jen University, Taipei, 242, Taiwan

    Han-Min Chen

  3. Institute of Bioscience and Biotechnology, National Taiwan Ocean University, Keelung, 202, Taiwan

    Chi-Tsai Lin

Authors
  1. Guang-Huar Young
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Han-Min Chen
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Chi-Tsai Lin
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Kuang-Ching Tseng
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jiann-Shing Wu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Rong-Huay Juang
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Rong-Huay Juang.

Additional information

H.M. Chen and C.T. Lin contributed equally to this work.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Young, GH., Chen, HM., Lin, CT. et al. Site-specific phosphorylation of L-form starch phosphorylase by the protein kinase activity from sweet potato roots. Planta 223, 468–478 (2006). https://doi.org/10.1007/s00425-005-0103-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00425-005-0103-1

Keywords

Search

Navigation