Characterization and identification of a galacturonosyltransferase involved in pectin biosynthesis
Sterling, Jason Dwight
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Pectins are a class of acidic, plant cell wall polysaccharides that contain (1,4)-linked .-D-galactopyranosyluronic acid (D-GalpA) as part of their backbone structure. Pectin is involved in the regulation of plant growth and development, the elucidation of plant defense responses, and the maintenance of plant cell adhesion. While a significant amount of published information is available on the structure and function of pectin in the plant cell wall, relatively little is known about pectin biosynthesis. This study centers around the characterization and identification of an .-1,4-galacturonosyltransferase (GalAT) involved in the biosynthesis of the backbone structures of pectin. The subcellular localization of a GalAT was determined by sucrose density gradient centrifugation of pea epicotyl membranes. GalAT activity was found to co-fractionate with Golgi-resident latent UDPase activity. Treatment of Golgi vesicles with Proteinase K in the absence of detergent showed that intact Golgi membranes from pea protected GalAT activity from proteolytic degradation. These results show that pectin biosynthesis occurs in the Golgi apparatus and that the pea GalAT has its catalytic site facing the Golgi lumen. A radioactive assay was developed to assay GalAT activity. The assay uses cetylpyridinium chloride-treated 14filters to separate unincorporated UDP-D-[C]GalpA from radioactive products formed during the GalAT reaction. The versatility of this assay was demonstrated by its ability to rapidly and accurately measure GalAT activity in multiple chromatography fractions during the partial purification of a GalAT from tobacco (Nicotiana tabacum L. cv. Samsun) and Arabidopsis thaliana (cv. Columbia). Using a new detergent solubilization technique and a combination of SP-Sepharose, Reactive Yellow 3, and UDP-agarose chromatography, GalAT activity was purified 17-fold from solubilized Arabidopsis membranes. The partially purified fraction was digested with sequencing-grade trypsin and the released peptides were analyzed by liquid chromatography-tandem mass spectrometry. Peptide analysis revealed the presence of two proteins (JS33 and JS36) with sequence similarity to other glycosyltransferases. Truncated versions of these genes lacking their N-terminal transmembrane domain were cloned into a vector containing an N-terminal signal sequence designed for secretion of the recombinant proteins. Both gene constructs were transiently and stably expressed in human embryonic kidney (HEK) 293 cells. Preliminary transient transfection experiments indicated that GalAT activity was detectable in media from HEK293 cells transiently expressing the JS36 gene construct providing evidence that JS36 encoded a putative GalAT. Inactive, recombinant proteins were detected in cell lysates from stable HEK293 lines expressing either JS33 or JS36. Database analysis indicates that JS33 and JS36 are part of a 25 member gene family in Arabidopsis. Mutation of two members of this putative GalAT gene family gives phenotypes that are similar to previously characterized pectin mutants. Further research needs to be conducted to determine what role(s) these genes play in pectin biosynthesis.