title: Rice SCAMP1 defines clathrin-coated, trans-golgi-located tubular-vesicular structures as an early endosome in tobacco BY-2 cells.
abstract: We recently identified multivesicular bodies (MVBs) as prevacuolar compartments (PVCs) in the secretory and endocytic pathways to the lytic vacuole in tobacco (Nicotiana tabacum) BY-2 cells. Secretory carrier membrane proteins (SCAMPs) are post-Golgi, integral membrane proteins mediating endocytosis in animal cells. To define the endocytic pathway in plants, we cloned the rice (Oryza sativa) homolog of animal SCAMP1 and generated transgenic tobacco BY-2 cells expressing yellow fluorescent protein (YFP)-SCAMP1 or SCAMP1-YFP fusions. Confocal immunofluorescence and immunogold electron microscopy studies demonstrated that YFP-SCAMP1 fusions and native SCAMP1 localize to the plasma membrane and mobile structures in the cytoplasm of transgenic BY-2 cells. Drug treatments and confocal immunofluorescence studies demonstrated that the punctate cytosolic organelles labeled by YFP-SCAMP1 or SCAMP1 were distinct from the Golgi apparatus and PVCs. SCAMP1-labeled organelles may represent an early endosome because the internalized endocytic markers FM4-64 and AM4-64 reached these organelles before PVCs. In addition, wortmannin caused the redistribution of SCAMP1 from the early endosomes to PVCs, probably as a result of fusions between the two compartments. Immunogold electron microscopy with high-pressure frozen/freeze-substituted samples identified the SCAMP1-positive organelles as tubular-vesicular structures at the trans-Golgi with clathrin coats. These early endosomal compartments resemble the previously described partially coated reticulum and trans-Golgi network in plant cells.
authors.affiliation: Department of Biology and Molecular Biotechnology Program, Chinese University of Hong Kong, Shatin, New Territories, Hong Kong, China.
authors.forename: Sheung Kwan
authors.initials: SK
authors.lastname: Lam
authors.forename: Ching Lung
authors.initials: CL
authors.lastname: Siu
authors.forename: Stefan
authors.initials: S
authors.lastname: Hillmer
authors.forename: Seonghoe
authors.initials: S
authors.lastname: Jang
authors.forename: Gynheung
authors.initials: G
authors.lastname: An
authors.forename: David G
authors.initials: DG
authors.lastname: Robinson
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
mesh_terms: D000595:Amino Acid Sequence; D000730:Androstadienes / Q000494:pharmacology; D015415:Biomarkers; D020126:Brefeldin A / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism* / Q000648:ultrastructure; D006056:Golgi Apparatus / Q000378:metabolism; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D015388:Organelles / Q000145:classification / Q000648:ultrastructure; D012275:Oryza / Q000235:genetics*; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D011726:Pyridinium Compounds / Q000032:analysis; D000644:Quaternary Ammonium Compounds / Q000032:analysis; D011993:Recombinant Fusion Proteins / Q000032:analysis; D016415:Sequence Alignment; D014026:Nicotiana / Q000235:genetics* / Q000648:ultrastructure; D033921:Vesicular Transport Proteins / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D015415:Biomarkers; C092350:FM 4-64; D010940:Plant Proteins; D011726:Pyridinium Compounds; D000644:Quaternary Ammonium Compounds; D011993:Recombinant Fusion Proteins; D033921:Vesicular Transport Proteins; D020126:Brefeldin A; D000077191:Wortmannin