Homodimers and heterodimers of Pho1-type phosphorylase isoforms in Solanum tuberosum L. as revealed by sequence-specific antibodies.
Higher plants possess two types of glucan phosphorylase (EC 2.4.1.1). One isozyme type, designated as Pho1, is located in the plastid whereas the other type, Pho2, is restricted to the cytosol. For Solanum tuberosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basner, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Both proteins (referred to as Pho1a and Pho1b, respectively) are highly similar (81-84% amino acid identity over most parts of the two sequences) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this communication antibodies that bind specifically to either Pho1a or Pho1b were used to study both isoforms at the protein level. The antibodies were applied to both potato tuber and leaf extracts following either denaturing or non-denaturing electrophoresis. Pho1a but not Pho1b was immunochemically detectable in tuber extracts whereas leaf extracts contained both the Pho1a and Pho1b protein. During denaturing electrophoresis the two antigens comigrated. When the leaf Pho1 isoforms were separated by affinity electrophoresis three bands of activity were resolved; all of them were recognized by the anti-Pho1a antibodies, but only two of these reacted with the anti-Pho1b antibodies. The isoform binding exclusively to the anti-Pho1a antibodies comigrated with the Pho1 isozyme from potato tubers. Immunoprecipitation experiments performed with anti-Pho1a antibodies removed the entire Pho1 phosphorylase activity from both tuber and leaf extracts. Addition of anti-Pho1b antibodies to tuber extracts did not affect the enzyme pattern, whereas in leaf extracts one isoform remained unchanged but the two other bands were strongly retarded. This indicates that the Pho1a protein is present in all three forms and Pho1b is associated with Pho1a. Association of Pho1a and Pho1b was further demonstrated by cross-linking experiments using bis(sulfosuccinimidyl)suberate as linker. Immunoprecipitation experiments were also performed using extracts of transformed Escherichia coli cells that expressed either Pho1a or Pho1b or both simultaneously. Under these conditions a homodimeric Pho1b phosphorylase was observed that had a lower electrophoretic mobility than the heterodimer from leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a)2 more strongly than that of the heterodimer. Thus, in leaves, Pho1a exists both as a homodimer, (Pho1a)2 and as heterodimer, (Pho1a-Pho1b); a part of it appears to be covalently modified. Pho1b, in the homodimeric form, is often below the limit of detection. In tubers the homodimer, (Pho1a)2, is the only detectable Pho1-type enzyme. To our knowledge this is the first report on a heterodimeric structure of plant phosphorylase.
Albrecht
T
T
Institut für Biochemie und Molekulare Physiologie, Universität Potsdam, Germany.
Greve
B
B
Pusch
K
K
Kossmann
J
J
Buchner
P
P
Wobus
U
U
Steup
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D015153:Blotting, Western; D003429:Cross Reactions; D003432:Cross-Linking Reagents; D019281:Dimerization; D004586:Electrophoresis / Q000379:methods; D006433:Hemocyanins / Q000235:genetics / Q000276:immunology; D051379:Mice; D008969:Molecular Sequence Data; D006005:Phosphorylases / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D018515:Plant Leaves / Q000201:enzymology; D010940:Plant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000201:enzymology; D011817:Rabbits; D011994:Recombinant Proteins / Q000235:genetics / Q000276:immunology; D011198:Solanum tuberosum / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D003432:Cross-Linking Reagents; D010940:Plant Proteins; D011994:Recombinant Proteins; D006433:Hemocyanins; D006005:Phosphorylases