Abstract: Xyloglucans (XyGs) are critical components of the plant cell wall. XyG-synthesizing glycosyltransferases (GTs) have been identified to localize in the Golgi. Their protein–protein interactions suggest the formation of multiprotein complexes; however, the mechanism of complex formation and their stoichiometry is unclear. The transient co-expression of all XyG-synthesizing enzymes and Golgi/ER markers in Arabidopsis (Arabidopsis thaliana) protoplasts prepared from various mutants of XyG glycosyltransferases demonstrated that protein interactions are not required for delivery to the Golgi. The stability of the proteins can affect the protein–protein interactions and the co-existence of the protein in multiprotein complexes. To investigate the stability of all XyG-synthesizing GTs in the Golgi, we performed cycloheximide (CHI) treatments of Arabidopsis seedlings expressing these enzymes fused with cyan fluorescent protein (CFP) in the background of corresponding mutants. The results showed cellulose synthase-like C4 (CSLC4), galactosyltransferases (MUR3), and fucosyltransferase (FUT1) have similar longer stability: the half-life of CSLC4 was 3.5 h, the half-life of MUR3 was 5 h, and the half-life of FUT1 was about 4 h. The XyG xylosyltransferases XXT1, XXT2, and XXT5 showed a short-term half-life of about 30 min. The half-life of galactosyltransferases (XLT2) was about 45 min. These results demonstrate that the XyG GTs complex is, most likely, compositionally dynamic in the Golgi, with glucan synthase, galactosyltransferase, and fuctosyltransferase being the longest-living proteins in the complex. This study advances our understanding of protein–protein interactions among polysaccharide synthesizing enzymes and their co-existence in the compositionally dynamic complexes in the Golgi. 

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