Structural and functional basis for starch binding in the SnRK1 subunits AKINβ2 and AKINβγ

Abstract

Specialized carbohydrate-binding domains, the Starch-Binding Domain (SBD) and the Glycogen Binding Domain (GBD), are motifs of approximately 100 amino acids directly or indirectly associated with starch or glycogen metabolism. Members of the regulatory β subunit of the heterotrimeric complex AMPK/SNF1/SnRK1 contain an SBD or GBD. In Arabidopsis thaliana, the β regulatory subunit AKINβ2 and a γ-type subunit, AKINβγ, also have an SBD. In this work, we compared the SBD of AKINβ2 and AKINβγ with the GBD present in rat AMPKβ1 and demonstrated that they conserved the same overall topology. The majority of the amino acids identified in the protein-carbohydrate interactions in the rat AMPKβ1 are conserved in the two plant proteins. In AKINβγ, there is an insertion of three amino acids that creates a loop adjacent to one of the conserved tryptophan residues. Functionally, the SBD from AKINβγ and AKINβ2 could bind starch, but there was an important difference in the association when an amylose/amylopectin (A/A) mixture was used. The physiological relevance of binding to starch was clear for AKINβγ, because immunolocalization experiments identified this protein inside the chloroplast. SnRK1 activity was not affected by the addition of A/A to the reaction mixture. However, addition of starch inhibited the activity 85%. Furthermore, proteins associated with A/A and starch in an in vitro-binding assay accounted for 10-20% of total SnRK1 kinase activity. Interestingly, the identification of the SnRK1 subunits associated to the protein-carbohydrate complex indicated that only the catalytic subunits, AKIN10 and AKIN11, and the regulatory subunit AKINβγ were present. These results suggest that a dimer formed between either catalytic subunit and AKINβγ could be associated with the A/A mixture in its active form but the same subunits are inactivated when binding to starch.

Keywords: AKINβ2; AKINβγ; SnRK1; Starch Binding Domain (SBD); chloroplast proteins.

Figure 1

Sequence and structural alignment. (A) Amino acids 92–178 from AKINβ1, 93–180 from AKINβ2, 12–102 from AKINβγ and 70–154 from rat AMPKβ1 were included for the sequence alignment. The sequence identity with the Glycogen Binding Domain (GBD) of rat used to construct the structural model was 42%. (B) Lateral view of the structural alignment for the models generated with AKINβ2 (light blue) and AKINβγ (red) where the insertion of the three amino acids (VPM) is indicated. The structure of the β-ciclodextrin is showed in dark blue and the conserved tryptophans in orange. ^*Indicates the conserved critical residues for protein-carbohydrate interaction.

Figure 2

Binding of AKINβγ and AKINβ2 to starch and A/A. Samples of each protein were incubated with starch and A/A as described in the Materials and Methods section. The carbohydrate-protein complexes were collected by centrifugation and samples of the soluble fraction (S) and bound fraction (P) were separated by SDS-PAGE, and Western blot analysis was performed using protein-specific antibodies. (A) Protein-starch complexes. (B) Protein-A/A complexes.

Figure 3

Immunolocalization of AKINβγ and AKINβ2. (A) Leaf sections incubated with AKINβγ- and AKINβ2-specific antibodies showing goat anti-rabbit Alexa 568-fluorochrome staining (red signal, 40×), chlorophyll (green signal, 40×) and overlaid images of the Alexa 568 fluorochrome and chlorophyll (40×). (B) The proteins extracted from the chloroplasts treated with (+T) or without (−T) thermolysin were analyzed by Western blot using AKINβγ- and AKINβ2-specific antibodies. The large subunit of Rubisco was used as a loading control.

Figure 4

Effect of A/A and starch on SnRK1 activity. (A) SnRK1 kinase activity measured under normal conditions (CE), with 0.4 mg of A/A in the reaction mixture (A/A), with 0.5 mg of starch (STARCH) and using the protein-A/A complex (A/A bound) and the protein-starch complex (STARCH bound) as protein source. Specific activity for the control was 2.04 ± 0.08 nmol/min/mg. (B) Western blot analysis of the proteins extracted from the starch (starch) and from the A/A complexes (A/A).