Biochemical and Molecular Characterization of RcSUS1, a Cytosolic Sucrose Synthase Phosphorylated in Vivo at Serine 11 in Developing Castor Oil Seeds

Eric T Fedosejevs,Sheng Ying,Joonho Park,Erin M Anderson,Robert T Mullen,Yi-Min She,William C Plaxton

doi:10.1074/jbc.m114.585554

Abstract

Sucrose synthase (SUS) catalyzes the UDP-dependent cleavage of sucrose into UDP-glucose and fructose and has become an important target for improving seed crops via metabolic engineering. A UDP-specific SUS homotetramer composed of 93-kDa subunits was purified to homogeneity from the triacylglyceride-rich endosperm of developing castor oil seeds (COS) and identified as RcSUS1 by mass spectrometry. RcSUS1 transcripts peaked during early development, whereas levels of SUS activity and immunoreactive 93-kDa SUS polypeptides maximized during mid-development, becoming undetectable in fully mature COS. The cytosolic location of the enzyme was established following transient expression of RcSUS1-enhanced YFP in tobacco suspension cells and fluorescence microscopy. Immunological studies using anti-phosphosite-specific antibodies revealed dynamic and high stoichiometric in vivo phosphorylation of RcSUS1 at its conserved Ser-11 residue during COS development. Incorporation of (32)P(i) from [γ-(32)P]ATP into a RcSUS1 peptide substrate, alongside a phosphosite-specific ELISA assay, established the presence of calcium-dependent RcSUS1 (Ser-11) kinase activity. Approximately 10% of RcSUS1 was associated with COS microsomal membranes and was hypophosphorylated relative to the remainder of RcSUS1 that partitioned into the soluble, cytosolic fraction. Elimination of sucrose supply caused by excision of intact pods of developing COS abolished RcSUS1 transcription while triggering the progressive dephosphorylation of RcSUS1 in planta. This did not influence the proportion of RcSUS1 associated with microsomal membranes but instead correlated with a subsequent marked decline in SUS activity and immunoreactive RcSUS1 polypeptides. Phosphorylation at Ser-11 appears to protect RcSUS1 from proteolysis, rather than influence its kinetic properties or partitioning between the soluble cytosol and microsomal membranes.

Highlights

The pathways and control of oil seed sugar unloading and metabolism are not well understood
Purification of RcSUS1 from 75 g of endosperm harvested from stage V–VII developing castor oil seeds (COS)
The castor Sucrose synthase (SUS) family comprises five genes that exhibited distinctive expression profiles with RcSUS1 being the dominant isozyme of developing COS (Fig. 2, A–C) [40, 41]

Summary

Introduction

The pathways and control of oil seed sugar unloading and metabolism are not well understood. Results: The UDP-specific sucrose synthase isozyme RcSUS1 is the dominant sucrolytic enzyme of developing castor oil seeds. A UDP-specific SUS homotetramer composed of 93-kDa subunits was purified to homogeneity from the triacylglyceride-rich endosperm of developing castor oil seeds (COS) and identified as RcSUS1 by mass spectrometry. Elimination of sucrose supply caused by excision of intact pods of developing COS abolished RcSUS1 transcription while triggering the progressive dephosphorylation of RcSUS1 in planta. This did not influence the proportion of RcSUS1 associated with microsomal membranes but instead correlated with a subsequent marked decline in SUS activity and immunoreactive RcSUS1 polypeptides.

Methods

Results

Conclusion