title: Combined biochemical and cytological analysis of membrane trafficking using lectins.
abstract: We have tested the application of high-mannose-binding lectins as analytical reagents to identify N-glycans in the early secretory pathway of HeLa cells during subcellular fractionation and cytochemistry. Post-endoplasmic reticulum (ER) pre-Golgi intermediates were separated from the ER on Nycodenz-sucrose gradients, and the glycan composition of each gradient fraction was profiled using lectin blotting. The fractions containing the post-ER pre-Golgi intermediates are found to contain a subset of N-linked α-mannose glycans that bind the lectins Galanthus nivalis agglutinin (GNA), Pisum sativum agglutinin (PSA), and Lens culinaris agglutinin (LCA) but not lectins binding Golgi-modified glycans. Cytochemical analysis demonstrates that high-mannose-containing glycoproteins are predominantly localized to the ER and the early secretory pathway. Indirect immunofluorescence microscopy revealed that GNA colocalizes with the ER marker protein disulfide isomerase (PDI) and the COPI coat protein β-COP. In situ competition with concanavalin A (ConA), another high-mannose specific lectin, and subsequent GNA lectin histochemistry refined the localization of N-glyans containing nonreducing mannosyl groups, accentuating the GNA vesicular staining. Using GNA and treatments that perturb ER-Golgi transport, we demonstrate that lectins can be used to detect changes in membrane trafficking pathways histochemically. Overall, we find that conjugated plant lectins are effective tools for combinatory biochemical and cytological analysis of membrane trafficking of glycoproteins.
authors.affiliation: Sir William Dunn School of Pathology, University of Oxford, Oxford OX1 3RE, UK. gareth.morgan@kcl.ac.uk
authors.forename: Gareth W
authors.initials: GW
authors.lastname: Morgan
authors.forename: Mark
authors.initials: M
authors.lastname: Kail
authors.forename: Michael
authors.initials: M
authors.lastname: Hollinshead
authors.forename: David J
authors.initials: DJ
authors.lastname: Vaux
mesh_terms: D003584:Cytological Techniques; D004721:Endoplasmic Reticulum / Q000737:chemistry / Q000378:metabolism; D027801:Galanthus / Q000737:chemistry; D006023:Glycoproteins / Q000032:analysis / Q000737:chemistry* / Q000378:metabolism*; D006056:Golgi Apparatus / Q000737:chemistry / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007425:Intracellular Membranes / Q000737:chemistry / Q000378:metabolism*; D007472:Iohexol; D027806:Lens Plant / Q000737:chemistry; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism*; D018532:Pisum sativum / Q000737:chemistry; D037121:Plant Lectins / Q000737:chemistry*; D021381:Protein Transport; D013395:Sucrose
chemical_list: D006023:Glycoproteins; D009844:Oligosaccharides; D037121:Plant Lectins; D007472:Iohexol; D013395:Sucrose
keywords: Confocal microscopy; ER; Glycosylation; Golgi complex; Lectin; Subcellular fractionation