First published online April 9, 2002; 10.1104/pp.010720
Plant Physiol, May 2002, Vol. 129, pp. 278-289
Glucosylation Activity and Complex Formation of Two Classes of
Reversibly Glycosylated Polypeptides1
Sandra M.J.
Langeveld,*
Marco
Vennik,
Marijke
Kottenhagen,
Ringo
van Wijk,
Ankie
Buijk,
Jan W.
Kijne, and
Sylvia
de Pater
Department of Applied Plant Sciences of the Netherlands
Organisation for Applied Scientific Research (S.M.J.L., M.V., M.K.,
R.v.W., A.B., S.d.P.) and Institute of Molecular Plant Sciences
(J.W.K.), Center for Phytotechnology, Leiden University,
Wassenaarseweg 64, 2333 AL Leiden, The Netherlands
Reversibly glycosylated polypeptides (RGPs) have been
implicated in polysaccharide biosynthesis. In plants, these proteins may function, for example, in cell wall synthesis and/or in synthesis of starch. We have isolated wheat (Triticum aestivum)
and rice (Oryza sativa) Rgp cDNA clones
to study the function of RGPs. Sequence comparisons showed the
existence of two classes of RGP proteins, designated RGP1 and RGP2.
Glucosylation activity of RGP1 and RGP2 from wheat and rice was
studied. After separate expression of Rgp1 and
Rgp2 in Escherichia coli or yeast
(Saccharomyces cerevisiae), only RGP1 showed
self-glucosylation. In Superose 12 fractions from wheat endosperm
extract, a polypeptide with a molecular mass of about 40 kD is
glucosylated by UDP-glucose. Transgenic tobacco (Nicotiana
tabacum) plants, overexpressing either wheat
Rgp1 or Rgp2, were generated. Subsequent
glucosylation assays revealed that in RGP1-containing tobacco extracts
as well as in RGP2-containing tobacco extracts UDP-glucose is
incorporated, indicating that an RGP2-containing complex is active. Gel
filtration experiments with wheat endosperm extracts and extracts from
transgenic tobacco plants, overexpressing either wheat
Rgp1 or Rgp2, showed the presence of RGP1
and RGP2 in high-molecular mass complexes. Yeast two-hybrid
studies indicated that RGP1 and RGP2 form homo- and heterodimers.
Screening of a cDNA library using the yeast two-hybrid system and
purification of the complex by an antibody affinity column did not
reveal the presence of other proteins in the RGP complexes. Taken
together, these results suggest the presence of active RGP1 and RGP2
homo- and heteromultimers in wheat endosperm.
1
This work was partly financially supported by
the European Union Eureka Program (grant no.
EU-169311).
*
Corresponding author; e-mail Langeveld{at}rulbim.leidenuniv.nl;
fax 31-71-5274863.
© 2002 American Society of Plant Physiologists
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