Co- and/or post-translational modifications are critical for TCH4 XET activity.
TCH4 encodes a xyloglucan endotransglycosylase (XET) of Arabidopsis thaliana. XETs endolytically cleave and religate xyloglucan polymers; xyloglucan is one of the primary structural components of the plant cell wall. Therefore, XET function may affect cell shape and plant morphogenesis. To gain insight into the biochemical function of TCH4, we defined structural requirements for optimal XET activity. Recombinant baculoviruses were designed to produce distinct forms of TCH4. TCH4 protein engineered to be synthesized in the cytosol and thus lack normal co- and post-translational modifications is virtually inactive. TCH4 proteins, with and without a polyhistidine tag, that harbor an intact N-terminus are directed to the secretory pathway. Thus, as predicted, the N-terminal region of TCH4 functions as a signal peptide. TCH4 is shown to have at least one disulfide bond as monitored by a mobility shift in SDS-PAGE in the presence of dithiothreitol (DTT). This disulfide bond(s) is essential for full XET activity. TCH4 is glycosylated in vivo; glycosidases that remove N-linked glycosylation eliminated 98% of the XET activity. Thus, co- and/or post-translational modifications are critical for optimal TCH4 XET activity. Furthermore, using site-specific mutagenesis, we demonstrated that the first glutamate residue of the conserved DEIDFEFL motif (E97) is essential for activity. A change to glutamine at this position resulted in an inactive protein; a change to aspartic acid caused protein mislocalization. These data support the hypothesis that, in analogy to Bacillus beta-glucanases, this region may be the active site of XET enzymes.
Campbell
P
P
Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892, USA.
Braam
J
J
D000818:Animals; D017360:Arabidopsis / Q000201:enzymology / Q000235:genetics*; D029681:Arabidopsis Proteins; D016367:Baculoviridae / Q000235:genetics; D002460:Cell Line; D004220:Disulfides; D004229:Dithiothreitol; D005822:Genetic Vectors / Q000235:genetics; D005936:Glucans; D006031:Glycosylation; D016695:Glycosyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011134:Polysaccharides / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D018411:Spodoptera; D013439:Sulfhydryl Reagents; D014990:Xylans
D029681:Arabidopsis Proteins; D004220:Disulfides; D005936:Glucans; D011134:Polysaccharides; D021382:Protein Sorting Signals; D011993:Recombinant Fusion Proteins; D013439:Sulfhydryl Reagents; D014990:Xylans; C029353:xyloglucan; D016695:Glycosyltransferases; C110295:TCH4 protein, Arabidopsis; D004229:Dithiothreitol