A Hybrid Approach Enabling Large-Scale Glycomic Analysis of Post-Golgi Vesicles Reveals a Transport Route for Polysaccharides

Abstract

The plant endomembrane system facilitates the transport of polysaccharides, associated enzymes, and glycoproteins through its dynamic pathways. Although enzymes involved in cell wall biosynthesis have been identified, little is known about the endomembrane-based transport of glycan components. This is partially attributed to technical challenges in biochemically determining polysaccharide cargo in specific vesicles. Here, we introduce a hybrid approach addressing this limitation. By combining vesicle isolation with a large-scale carbohydrate antibody arraying technique, we charted an initial large-scale map describing the glycome profile of the SYNTAXIN OF PLANTS61 (SYP61) trans-Golgi network compartment in Arabidopsis (Arabidopsis thaliana). A library of antibodies recognizing specific noncellulosic carbohydrate epitopes allowed us to identify a range of diverse glycans, including pectins, xyloglucans (XyGs), and arabinogalactan proteins in isolated vesicles. Changes in XyG-and pectin-specific epitopes in the cell wall of an Arabidopsis SYP61 mutant corroborate our findings. Our data provide evidence that SYP61 vesicles are involved in the transport and deposition of structural polysaccharides and glycoproteins. Adaptation of our methodology can enable studies characterizing the glycome profiles of various vesicle populations in plant and animal systems and their respective roles in glycan transport defined by subcellular markers, developmental stages, or environmental stimuli.

Separating Golgi Proteins from Cis to Trans Reveals Underlying Properties of Cisternal Localization

“…Given their trans-Golgi association, and the family size, it seems likely that DUF707s make an important contribution to the diversity of terminal substitutions on glycan chains, possibly relating to the cell wall. Nonfucosylated xyloglucan epitopes were not observed in the very latest Golgi cisternae ( Figures 3A to 3C) but have been recorded in post-Golgi compartments and the cell wall (Wilkop et al, 2019). This suggests that their absence from the very late Golgi was not a consequence of further substitution preventing antibody binding.…”

“…Following on from the TEM imaging, the FFE fractions (from R1) were analyzed for the same classes of polysaccharide, using carbohydrate antibody arrays immobilized on nitrocellulose membranes, which has been successfully applied to endomembrane enrichments (Okekeogbu et al, 2019) and post-Golgi compartments (Wilkop et al, 2019). Here, we were able to probe an expanded number of polysaccharide epitopes compared with TEM due to the high-throughput nature of the array assays.…”

Overexpression of trans‐Golgi network t‐SNAREs rescues vacuolar trafficking and TGN morphology defects in a putative tethering factor mutant

“…It is not clear which vesicle trafficking pathway is disrupted in tno1 mutants, although it appears to involve both SYP41 and SYP61 (Kim and Bassham, 2011). Immunoisolation of vesicles containing SYP61, which may include TGN fragments and TGN-derived vesicles, revealed that they contained secretory cargo including both extracellular proteins and cell wall polysaccharides (Drakakaki et al, 2012;Wilkop et al, 2019). However, there is no evidence to date for a role for TNO1 in secretion, only in transport to the vacuole.…”

AtTRAPPC11/ROG2: A Role for TRAPPs in Maintenance of the Plant Trans-Golgi Network/Early Endosome Organization and Function

“…The trans-Golgi network/early endosome (TGN/EE) provides a main trafficking hub for the endomembrane system and comprises a heterogeneous population of vesicles shuttling cargo between Golgi, vacuole, plasma membrane (PM), and late endosomal compartments (Dettmer et al, 2006;Viotti et al, 2010;Wattelet-Boyer et al, 2016;Brillada and Rojas-Pierce, 2017;Rosquete et al, 2018). The plant SYNTAXIN OF PLANTS61 (SYP61) defines a TGN/EE compartment involved in endosomal recycling and transport of extracellular cargo (Robert et al, 2008;Drakakaki et al, 2012;Wilkop et al, 2019) and has been implicated in abiotic stress responses (Zhu et al, 2002).…”