Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1

Authors: HARDIN, SHANE - UNIVERSITY OF ILLINOIS; DUNCAN, KATERI - UNIVERSITY OF ILLINOIS; Huber, Steven

Submitted to: Plant Physiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/2/2006
Publication Date: 7/1/2006
Citation: Hardin, S.C., Duncan, K.A., Huber, S.C. 2006. Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1. Plant Physiology. 141:1106-1119.

Interpretive Summary: Sucrose synthase is an important enzyme of sucrose metabolism in growing plant organs, such as developing seeds, and channels carbon from sucrose into various metabolic and biosynthetic pathways. The activity of sucrose synthase in a growing organ is thought to be a marker for, or possible determinant of, growth. Interestingly, this soluble protein is also found to be partially associated with cellular membranes such as the plasma membrane, where it is thought to provide C for cell wall glucans synthesis. Hence, there is great interest in identifying factors that control the activity and also the intracellular localization of this critical plant enzyme. In the present study, we demonstrate that maize sucrose synthase (the SUS1 protein) has an intrinsic ability to interact directly with membranes, and that this ability is due, in part, to sequences contained within the catalytic domain of the enzyme. In addition, we demonstrate that the N-terminal non-catalytic region also contributes to membrane binding. The presence of sucrose and low pH were identified as factors that promote partitioning of SUS1 to membranes. These results add substantially to our understanding of the ability of sucrose synthase to associate with cellular membranes, and identify physiological factors that may influence the distribution of the enzyme in vivo. These results also provide a foundation for guiding the rational creation of mutant SUS transgenics that possess altered membrane localization, and to potentially control carbon allocation to structural compounds in plants.

Technical Abstract: Sucrose synthase (SUS) cleaves sucrose to form UDP-glucose and fructose, and exists in soluble (s-SUS) and membrane-associated (m-SUS) forms, with the latter proposed to channel UDP-glucose to the cellulose synthase complex on the plasma membrane of plant cells during synthesis of cellulose. However, the structural features responsible for membrane localization and the mechanisms regulating its dual intracellular localization are unknown. The Zea mays (L.) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (i) partial membrane localization when expressed in Escherichia coli, and (ii) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and Ala substitutions) and in vitro microsome binding assays with the SUS1 protein to define intrinsic membrane-binding regions and potential regulatory factors that could be provided by cellular microenvironment. The results suggest that two regions of SUS1 contribute to membrane affinity: (i) the amino-terminal non-catalytic domain and (ii) a region with sequence similarity to the C-terminal PH (pleckstrin homology) domain of human pleckstrin. Ala substitutions within the PH-like domain of SUS1 reduced membrane association in E. coli and with plant microsomes in vitro without reducing enzymatic activity. Microsomal association of wild type SUS1 displayed cooperativity with SUS1 protein concentration and was stimulated by both lowering the pH and adding sucrose. These studies offer insight into the molecular level regulation of SUS1 localization and its participation in carbon partitioning in plants. Moreover, transgenics with active SUS mutants altered in membrane affinity may be of technological utility.