Identification and Functional Study of Plastidial Starch Phosphorylase Interacting Protein Complexes in Sweet Potato Roots
Date Issued
2012
Date
2012
Author(s)
Lin, Yi-Chen
Abstract
Post-translational regulation plays an important role in cellular metabolism. Earlier studies showed that the activity of plastidal starch phosphorylase (L-SP) may be regulated by proteolytic modification. During the purification of L-SP from sweet potato roots, an unknown high molecular weight complex (HX) showing L-SP activity was constantly observed. Its mobility was significantly slower than the typical L-SP on native PAGE. We utilized mass spectrometry, coimmunoprecipitation, Ouchterlony double immunodiffusion, two-dimensional gel electrophoresis, and confocal microscopy as tools to demonstrate that HX was composed of L-SP and the 20S proteasome. Furthermore, we found that the amount of HX decreased immediately after 45°C heat treatment, which caused stepwise degradation of L-SP in a time-dependent mode. This degradation process was strongly inhibited by MG132, suggesting that the 20S proteasome might be involved in L-SP degradation. In addition, kinetic studies indicated that the proteolytic modification of L-SP caused it to decrease the binding affinity toward Glc-1-P and subsequently reduced its starch-synthesizing activity. This work demonstrates the role of the 20S proteasome as a regulator of L-SP activity, which may be controlled by stressful condition.
On the other hand, immunoprecipitation experiments with L-SP mAbs showed that another protein might associate with L-SP. This protein was identified as DPE1 (D-enzyme, disproportionating enzyme, 4-alpha-glucanotransferase; EC 2.4.1.25) by LC/MS/MS. DPE1 catalyses the cleavage and transfer reactions involving alpha-1,4 linked glucans and alters the chain length distribution of oligosaccharides. Previous studies suggested that DPE1 might work in conjunction with L-SP. Furthermore, we utilized 2-DE (native PAGE/SDS-PAGE), GST pull-down assay, and confocal microscopy as tools to demonstrate that L-SP might interact with DPE1, suggesting that these enzymes may form protein complexes (SP-DPE complexes). The results from gel-filtration chromatography and 2-DE (native PAGE/SDS-PAGE) indicated that SP-DPE complexes might be composed of four L-SP subunits and four DPE1 subunits with molecular weight around 700 kDa. In addition, protein complex forms of DPE1 showed a higher affinity toward maltotriose and a higher catalytic activity toward maltotetraose than DPE1 monomers. The efficient passage of the product of one enzyme to the next enzyme in SP-DPE complexes was also be observed. Moreover, the protein levels of SP-DPE complexes were shown to become higher in the middle stage of sweet potato root development where starch accumulated fast. These results suggest that SP-DPE complexes may either efficiently recycle short chain malto-oligosaccharides to produce Glc-1-P for starch synthesis, or may specifically edit short-chain amylopectin, thus resulting in the formation of correct starch structure.
Subjects
Starch Phosphorylase
Protein Complexes
Sweet Potato Roots
Type
thesis
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