In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase

Olga Komina, You Zhou, Gautam Sarath, Raymond Chollet

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Sucrose synthase (SS) is a known phosphoserine (SerP)-containing enzyme in a variety of plant "sink" organs, including legume root nodules, where it is phosphorylated primarily at Ser-11. Using immunofluorescence confocal microscopy, we documented that part of the total SS (nodulin-100) pool in mature soybean (Glycine max) nodules is apparently associated with the plasma membrane in situ, and we report that this association is very "tight," as evidenced by a variety of chemical and enzymatic pretreatments of the isolated microsomal fraction. To investigate the in situ and in planta phosphorylation state of the membrane (m) and soluble (s) forms of nodule SS, three complementary approaches were used. First, excised nodules were radiolabeled in situ with [ 32 P]Pi for subsequent analysis of phosphorylated m- and s-SS; second, immunopurified s- and m-SS were used as substrate in "on-bead" assays of phosphorylation by nodule Ca 2+ -dependent protein kinase; and third, SS-Ser-11(P) phosphopeptide-specific antibodies were developed and used. The collective results provide convincing evidence that microsomal nodulin-100 is phosphorylated in mature nodules, and that it is hypophosphorylated relative to s-SS (on an equivalent SS protein basis) in attached, unstressed nodules. Moreover, the immunological data and related phosphopeptide mapping analyses indicate that a homologous N-terminal seryl-phosphorylation domain and site reside in microsomal nodulin-100. We also observed that mild, short-term inorganic nitrogen and salt stresses have a significant negative impact on the content and N-terminal phosphorylation state of nodule m- and s-SS, with the former being the more sensitive of the two SS forms.

Original languageEnglish (US)
Pages (from-to)1664-1673
Number of pages10
JournalPlant Physiology
Volume129
Issue number4
DOIs
StatePublished - Aug 2 2002

Fingerprint

sucrose synthase
Soybeans
phosphorylation
Phosphorylation
soybeans
Membranes
nodulins
Phospho-Specific Antibodies
In Vitro Techniques
Phosphoserine
Phosphopeptides
root nodules
Plantae
fluorescence microscopy
Fluorescence Microscopy
Confocal Microscopy
Fabaceae
protein kinases
Protein Kinases
salt stress

ASJC Scopus subject areas

  • Physiology
  • Genetics
  • Plant Science

Cite this

In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase. / Komina, Olga; Zhou, You; Sarath, Gautam; Chollet, Raymond.

In: Plant Physiology, Vol. 129, No. 4, 02.08.2002, p. 1664-1673.

Research output: Contribution to journalArticle

Komina, Olga ; Zhou, You ; Sarath, Gautam ; Chollet, Raymond. / In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase. In: Plant Physiology. 2002 ; Vol. 129, No. 4. pp. 1664-1673.
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