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 wherea s the other type, Pho2, is restricted to the cytosol. For Solanum tube rosum L. two Pho1 type phosphorylases have been sequenced [Nakano, K. & Fukui, T. (1986) J. Biol. Chem. 261, 8230-8236; Sonnewald, U., Basne r, A., Greve, B. & Steup, M. (1995) Plant Mol. Biol. 27, 567-576]. Bot h proteins (referred to as Pho1a and Pho1b, respectively) are highly s imilar (81-84% amino acid identity over most parts of the two sequence s) with the exception of the N-terminal transit peptide and the large insertion located between the N- and the C-terminal domains. In this c ommunication antibodies that bind specifically to either Pho1a or Pho1 b were used to study both isoforms at the protein level. The antibodie s 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 c ontained both the Pho1a and Pho1b protein. During denaturing electroph oresis the two antigens comigrated. When the leaf Pho1 isoforms were s eparated by affinity electrophoresis three bands of activity were reso lved; all of them were recognized by the anti-Pho1a antibodies, but on ly two of these reacted with the anti-Pho1b antibodies. The isoform bi nding exclusively to the anti-Pho1a antibodies comigrated with the Pho 1 isozyme from potato tubers. Immunoprecipitation experiments performe d with anti-Pho1a antibodies removed the entire Pho1 phosphorylase act ivity from both tuber and leaf extracts. Addition of anti-Pho1b antibo dies to tuber extracts did not affect the enzyme pattern, whereas in l eaf extracts one isoform remained unchanged but the two other bands we re 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 e xperiments were also performed using extracts of transformed Escherich ia coli cells that expressed either Pho1a or Pho1b or both simultaneou sly. Under these conditions a homodimeric Pho1b phosphorylase was obse rved that had a lower electrophoretic mobility than the heterodimer fr om leaves. In leaves of transgenic potato plants antisense inhibition of the Pho1a gene affected the formation of (Pho1a), more strongly tha n that of the heterodimer. Thus, in leaves, Pho1a exists both as a hom odimer, (Pho1a), and as heterodimer, (Pho1a-Pho1b); a part of it appea rs 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 fi rst report on a heterodimeric structure of plant phosphorylase.