Plastidial Starch Phosphorylase in Sweet Potato Roots Is Proteolytically Modified by Protein-Protein Interaction with the 20S Proteasome

Yi-Chen Lin,I-Min Chou,An-Na Chen,Chi-Tsai Lin,Guang-Huar Young,Han-Min Chen,Chiung-Hsiang Cheng,Shih-Chung Chang,Rong-Huay Juang,Chia-Pei Chen,Maria G Masucci

doi:10.1371/journal.pone.0035336

Abstract

Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidial starch phosphorylase (Pho1) may be regulated by proteolytic modification. During the purification of Pho1 from sweet potato roots, we observed an unknown high molecular weight complex (HX) showing Pho1 activity. The two-dimensional gel electrophoresis, mass spectrometry, and reverse immunoprecipitation analyses showed that HX is composed of Pho1 and the 20S proteasome. Incubating sweet potato roots at 45°C triggers a stepwise degradation of Pho1; however, the degradation process can be partially inhibited by specific proteasome inhibitor MG132. The proteolytically modified Pho1 displays a lower binding affinity toward glucose 1-phosphate and a reduced starch-synthesizing activity. This study suggests that the 20S proteasome interacts with Pho1 and is involved in the regulation of the catalytic activity of Pho1 in sweet potato roots under heat stress conditions.

Highlights

Starch is the main storage polysaccharide in plants
Several essential enzymes are involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, starch synthase, branching enzyme, and debranching enzyme [1]
During the purification of Pho1 from sweet potato roots by size-exclusion chromatography (Fig. 1A), we found an unknown high molecular weight complex (HX) on native PAGE showing Pho1 activity after in-gel activity staining (Fig. 1B)

Summary

Introduction

Starch is the main storage polysaccharide in plants. Several essential enzymes are involved in starch biosynthesis, including ADP-glucose pyrophosphorylase, starch synthase, branching enzyme, and debranching enzyme [1]. Starch phosphorylase (Pho, or SP, EC 2.4.1.1) plays a key role in starch metabolism [2,3,4,5]. Pho catalyzes the reversible phosphorolysis of starch and produces glucose 1-phosphate (Glc-1-P) as one of its products [6,7]. An insert sequence containing 78 amino acids (L78) was found uniquely in the middle of the Pho molecule, though not in Pho. An insert sequence containing 78 amino acids (L78) was found uniquely in the middle of the Pho molecule, though not in Pho2 This insertion, located near the glucan binding site, is believed to cause a steric hindrance and prevents Pho from binding to polyglucan substrates effectively [11]

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Results

Conclusion