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Site-directed mutagenesis of serine 158 demonstrates its role in spinach leaf sucrose-phosphate synthase modulationSite-directed mutagenesis of spinach sucrose-phosphate synthase (SPS) was performed to investigate the role of Ser158 in the modulation of spinach leaf SPS. Tobacco plants expressing the spinach wild-type (WT), S158A, S158T and S157F/S158E SPS transgenes were produced. Expression of transgenes appeared not to reduce expression of the tobacco host SPS. SPS activity in the WT and the S158T SPS transgenics showed light/dark modulation, whereas the S158A and S157F/S158E mutants were not similarly light/dark modulated: the S158A mutant enzyme was not inactivated in the dark, and the S157F/S158E was not activated in the light. The inability to modulate the activity of the S158A mutant enzyme by protein phosphorylation was demonstrated in vitro. The WT spinach enzyme immunopurified from dark transgenic tobacco leaves had a low initial activation state, and could be activated by PP2A and subsequently inactivated by SPS-kinase plus ATP. Rapid purification of the S158A mutant enzyme from dark leaves of transgenic plants using spinach-specific monoclonal antibodies yielded enzyme that had a high initial activation state, and pre-incubation with leaf PP2A or ATP plus SPS-kinase (the PKIII enzyme) caused little modulation of activity. The results demonstrate the regulatory significance of Ser158 as the major site responsible for dark inactivation of spinach SPS in vivo, and indicate that the significance of phosphorylation is the introduction of a negative charge at the Ser158 position.
Document ID
20040141990
Document Type
Reprint (Version printed in journal)
Authors
Toroser, D.
(US Department of Agriculture, Agricultural Research Service Raleigh, NC 27607, United States)
McMichael, R. Jr
Krause, K. P.
Kurreck, J.
Sonnewald, U.
Stitt, M.
Huber, S. C.
Davies, E.
Date Acquired
August 22, 2013
Publication Date
February 1, 1999
Publication Information
Publication: The Plant journal : for cell and molecular biology
Volume: 17
Issue: 4
ISSN: 0960-7412
Subject Category
Life Sciences (General)
Distribution Limits
Public
Copyright
Other
Keywords
Non-NASA Center
NASA Discipline Plant Biology
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