Diverse Stability and Independent Trafficking of XyG-Synthesizing Glycosyltransferases in Distinct Golgi cisternae.

Xyloglucan (XyGs), a plant-derived polysaccharide, supports gut health, is widely used as a food additive with stabilizing and gelling properties, and is increasingly being explored for drug delivery applications. XyG-synthesizing glycosyltransferases (GTs) are localized to Golgi, and their protein-protein interactions suggest the formation of multiprotein complexes; however, the mechanisms underlying protein complex assembly and transport, protein stability and degradation remain unknown. By employing the transient expression of YFP-fused GTs along with a cis-Golgi marker and generating fluorescence intensity profiles, we demonstrated the differential distribution of GTs in Golgi apparatus. The GTs half-lives were estimated using treatments with the protein synthesis inhibitor CHX. It was observed that the GTs exhibit distinct half-lives and, based on their turnover rates, display an apparent trend toward two groups. Our findings revealed that cellulose synthase-like C4 (CSLC4), galactosyltransferase (MUR3), and fucosyltransferase (FUT1) exhibit longer stability. In contrast, XyG xylosyltransferases XXT1, XXT2, XXT5, and galactosyltransferase XLT2 tend to have shorter half-lives. We also uncovered that protein-protein interactions among XyG-synthesizing GTs are not prerequisites for Golgi localization and our data are consistent with a model in which XyG-synthesizing GTs can reach the Golgi independently of their known interacting partners, exhibiting distinct sub-Golgi localization that shapes multiprotein complex assembly in specific cisternae. This spatial organization governs partner GT access and residence time for functional efficiency, while GT half-life variations regulate stability and interaction dynamics. Collectively, these factors provide a critical framework for independent operation and coordinated organization of Golgi-resident XyG-synthesizing proteins.