title: Determination of structural requirements and probable regulatory effectors for membrane association of maize sucrose synthase 1.
abstract: Sucrose (Suc) synthase (SUS) cleaves Suc to form UDP glucose and fructose, and exists in soluble and membrane-associated 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 maize (Zea mays) SUS1 isoform is likely to have the intrinsic ability to interact directly with membranes because we show: (1) partial membrane localization when expressed in Escherichia coli, and (2) binding to carbonate-stripped plant microsomes in vitro. We have undertaken mutational analyses (truncations and alanine 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: (1) the amino-terminal noncatalytic domain, and (2) a region with sequence similarity to the C-terminal pleckstrin homology domain of human pleckstrin. Alanine substitutions within the pleckstrin homology-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 Suc. 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.
authors.affiliation: United States Department of Agriculture, Agricultural Research Service, Photosynthesis Research Unit, Urbana, Illinois 61801, USA.
authors.forename: Shane C
authors.initials: SC
authors.lastname: Hardin
authors.forename: Kateri A
authors.initials: KA
authors.lastname: Duncan
authors.forename: Steven C
authors.initials: SC
authors.lastname: Huber
mesh_terms: D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000737:chemistry; D002462:Cell Membrane / Q000201:enzymology*; D005964:Glucosyltransferases / Q000737:chemistry / Q000378:metabolism*; D006004:Glycogen Debranching Enzyme System / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010750:Phosphoproteins / Q000737:chemistry; D017434:Protein Structure, Tertiary; D017386:Sequence Homology, Amino Acid; D013395:Sucrose / Q000378:metabolism; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics
chemical_list: D001798:Blood Proteins; D006004:Glycogen Debranching Enzyme System; D010750:Phosphoproteins; C039077:platelet protein P47; D013395:Sucrose; D005964:Glucosyltransferases; C022699:sucrose synthase; C020569:4 alpha-glucanotransferase