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Co- and/or post-translational modifications are critical for TCH4 XET activity
1Department of Biochemistry and Cell Biology, Rice University, Houston, TX 77005-1892, USA.
The Plant Journal : for Cell and Molecular Biology
|October 1, 1998
Summary
The TCH4 protein requires specific modifications like glycosylation and disulfide bonds for optimal xyloglucan endotransglycosylase (XET) activity. Its N-terminal signal peptide and active site glutamate are crucial for function in Arabidopsis thaliana.
Area of Science:
- Plant Biology
- Molecular Biology
- Biochemistry
Background:
- Xyloglucan endotransglycosylase (XET) enzymes modify xyloglucan, a key component of plant cell walls.
- TCH4 is an XET in Arabidopsis thaliana, and its function impacts plant cell shape and morphogenesis.
Purpose of the Study:
- To elucidate the biochemical function of TCH4 by defining its structural requirements for optimal activity.
- To investigate the role of post-translational modifications and specific amino acid residues in TCH4 function.
Main Methods:
- Engineered recombinant baculoviruses to produce distinct forms of TCH4.
- Utilized SDS-PAGE with and without dithiothreitol (DTT) to detect disulfide bonds.
- Employed glycosidases to assess the impact of N-linked glycosylation.
- Performed site-specific mutagenesis on the conserved DEIDFEFL motif.
Main Results:
- TCH4 synthesized in the cytosol (lacking modifications) is inactive, indicating the importance of co- and post-translational modifications.
- An intact N-terminus functions as a signal peptide, directing TCH4 to the secretory pathway.
- Disulfide bonds are essential for full XET activity.
- Removal of N-linked glycosylation reduced XET activity by 98%, highlighting its critical role.
- Mutating the conserved glutamate residue (E97) in the DEIDFEFL motif to glutamine resulted in an inactive protein, while mutation to aspartic acid caused mislocalization.
Conclusions:
- Co- and post-translational modifications, including glycosylation and disulfide bond formation, are critical for TCH4's xyloglucan endotransglycosylase activity.
- The N-terminal region acts as a signal peptide.
- The conserved glutamate residue (E97) within the DEIDFEFL motif is essential for TCH4 activity, likely forming part of the active site.