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

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

Plastidial α-glucan phosphorylase 1 complexes with disproportionating enzyme 1 in Ipomoea batatas storage roots for elevating malto-oligosaccharide metabolism.

It has been proposed that malto-oligosaccharides (MOSs) are possibly recycled back into amylopectin biosynthesis via the sequential reactions catalyzed by plastidial α-glucan phosphorylase 1 (Pho1) and disproportionating enzyme 1 (Dpe1). In the present study, the reciprocal co-immunoprecipitation experiments using specific antibodies against Pho1 and Dpe1 demonstrated that these two enzymes can form a complex (the PD complex) in Ipomoea batatas storage roots. The immunohistochemistry analyses also revealed the co-localization of Pho1 and Dpe1 in the amyloplasts, and the protein levels of Pho1 and Dpe1 increased gradually throughout sweet potato storage root development. A high molecular weight PD complex was co-purified from sweet potato storage root lysates by size exclusion chromatography. Enzyme kinetic analyses showed that the PD complex can catalyze maltotriose and maltotetraose to generate glucose-1-phosphate in the presence of inorganic phosphate, and it also performs greater Dpe1 activity toward MOSs than does free form Dpe1. These data suggest that Pho1 and Dpe1 may form a metabolon complex, which provides elevated metabolic fluxes for MOS metabolism via a direct transfer of sugar intermediates, resulting in recycling of glucosyl units back into amylopectin biosynthesis more efficiently