Bioinformatic and in vitro Analyses of Arabidopsis Starch Synthase 2 Reveal Post-translational Regulatory Mechanisms

Abstract

Starch synthase 2 (SS2) is an important enzyme in leaf starch synthesis, elongating intermediate-length glucan chains. Loss of SS2 results in a distorted starch granule phenotype and altered physiochemical properties, highlighting its importance in starch biosynthesis, however, the post-translational regulation of SS2 is poorly understood. In this study, a combination of bioinformatic and in vitro analysis of recombinant SS2 was used to identify and characterize SS2 post-translational regulatory mechanisms. The SS2 N-terminal region, comprising the first 185 amino acids of the mature protein sequence, was shown to be highly variable between species, and was predicted to be intrinsically disordered. Intrinsic disorder in proteins is often correlated with protein phosphorylation and protein-protein interactions. Recombinant Arabidopsis thaliana SS2 formed homodimers that required the N-terminal region, but N-terminal peptides could not form stable homodimers alone. Recombinant SS2 was shown to be phosphorylated by chloroplast protein kinases and recombinant casein kinase II at two N-terminal serine residues (S63, S65), but mutation of these phosphorylation sites (Ser>Ala) revealed that they are not required for homo-dimerization. Heteromeric enzyme complex (HEC) formation between SS2 and SBE2.2 was shown to be ATP-dependent. However, SS2 homo-dimerization and protein phosphorylation are not required for its interaction with SBE2.2, as truncation of the SS2 N-terminus did not disrupt ATP-dependent HEC assembly. SS2 phosphorylation had no affect on its catalytic activity. Intriguingly, the removal of the N-terminal region of SS2 resulted in a 47-fold increase in its activity. As N-terminal truncation disrupted dimerization, this suggests that SS2 is more active when monomeric, and that transitions between oligomeric state may be a mechanism for SS2 regulation.

Protein phosphorylation regulates maize endosperm starch synthase IIa activity and protein−protein interactions

“…However, until now it has not been clear whether SSIIa within this HEC is also phosphorylated or whether phosphorylation affects the catalytic activity of SSIIa or its interaction with other proteins. Phosphorylation of SSIIa in wheat endosperm amyloplasts and SS2 in Arabidopsis chloroplasts has been observed previously by Tetlow (2004) and Patterson et al (2018). In the present study, unlike previous investigations, SSIIa was immunopurified and shown to be substantially free from contamination by other starch synthases (Figure 1).…”

Abscisic acid‐controlled redox proteome of Arabidopsis and its regulation by heterotrimeric Gβ protein

“…Despite lacking glycosyltransferase activity, the noncanonical starch synthase isoform AtSS5 promotes the initiation of chloroplast‐localized starch granules by providing a binding site for both glucans and myosine‐resembling chloroplast proteins (Abt et al ., 2020). By contrast, AtSS2 enzymatically elongates intermediate‐length glucan chains during the formation of starch granules and is regulated by phosphorylation from chloroplast casein kinases (CKII) (Commuri & Keeling, 2001; Patterson et al ., 2018). In response to oxidative stress, maltose is released from starch and metabolized into sucrose, which can then scavenge ROS in the cytosol (Thalmann et al ., 2016).…”

Multi-omic analysis of the Arabidopsis clock activator mutantrve 4 6 8reveals connections to carbohydrate metabolism and proteasome regulation

“…leaves), where starch is produced and degraded on a daily basis (Smith and Zeeman, 2020). Recent biochemical characterization of SS2 phosphorylation using phospho-ablative SS2 mutants (S63A and S65A) found no enzymatic or protein complex consequences to the loss of SS2 phosphorylation despite being phosphorylated by CKII; (Patterson et al, 2018)). Our data finds SS2 to be phosphorylated at Ser65; part of a canonical CKII phosphorylation motif ( pS [D/E][D/E][D/E]) (Purzner et al, 2018), exclusively at ZT0 in the rve 4 6 8 mutant.…”