title: Golgi apparatus-localized synaptotagmin 2 is required for unconventional secretion in Arabidopsis.
abstract: BACKGROUND
  Most secretory proteins contain signal peptides that direct their sorting to the ER and secreted via the conventional ER/Golgi transport pathway, while some signal-peptide-lacking proteins have been shown to export through ER/Golgi independent secretory pathways. Hygromycin B is an aminoglycoside antibiotic produced by Streptomyces hygroscopicus that is active against both prokaryotic and eukaryotic cells. The hygromycin phosphotransferase (HYG(R)) can phosphorylate and inactivate the hygromycin B, and has been widely used as a positive selective marker in the construction of transgenic plants. However, the localization and trafficking of HYG(R) in plant cells remain unknown. Synaptotagmins (SYTs) are involved in controlling vesicle endocytosis and exocytosis as calcium sensors in animal cells, while their functions in plant cells are largely unclear.
  
  
  RESULTS
  We found Arabidopsis synaptotagmin SYT2 was localized on the Golgi apparatus by immunofluorescence and immunogold labeling. Surprisingly, co-expression of SYT2 and HYG(R) caused hypersensitivity of the transgenic Arabidopsis plants to hygromycin B. HYG(R), which lacks a signal sequence, was present in the cytoplasm as well as in the extracellular space in HYG(R)-GFP transgenic Arabidopsis plants and its secretion is not sensitive to brefeldin A treatment, suggesting it is not secreted via the conventional secretory pathway. Furthermore, we found that HYG(R)-GFP was truncated at carboxyl terminus of HYG(R) shortly after its synthesis, and the cells deficient SYT2 failed to efficiently truncate HYG(R)-GFP,resulting in HYG(R)-GFP accumulated in prevacuoles/vacuoles, indicating that SYT2 was involved in HYG(R)-GFP trafficking and secretion.
  
  
  CONCLUSIONS
  These findings reveal for the first time that SYT2 is localized on the Golgi apparatus and regulates HYG(R)-GFP secretion via the unconventional protein transport from the cytosol to the extracelluar matrix in plant cells.
authors.affiliation: Key Laboratory of Photosynthesis and Molecular Environmental Physiology, Institute of Botany, Chinese Academy of Sciences, Beijing, China.
authors.forename: Haiyan
authors.initials: H
authors.lastname: Zhang
authors.forename: Liang
authors.initials: L
authors.lastname: Zhang
authors.forename: Bin
authors.initials: B
authors.lastname: Gao
authors.forename: Hai
authors.initials: H
authors.lastname: Fan
authors.forename: Jingbo
authors.initials: J
authors.lastname: Jin
authors.forename: Miguel A
authors.initials: MA
authors.lastname: Botella
authors.forename: Liwen
authors.initials: L
authors.lastname: Jiang
authors.forename: Jinxing
authors.initials: J
authors.lastname: Lin
mesh_terms: D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020126:Brefeldin A / Q000494:pharmacology; D002451:Cell Compartmentation / Q000187:drug effects; D017343:Genes, Plant / Q000235:genetics; D006056:Golgi Apparatus / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006921:Hygromycin B / Q000494:pharmacology; D008954:Models, Biological; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D021381:Protein Transport / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D055571:Secretory Pathway / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism; D050861:Synaptotagmin II / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000187:drug effects
chemical_list: D029681:Arabidopsis Proteins; D011993:Recombinant Fusion Proteins; D050861:Synaptotagmin II; D049452:Green Fluorescent Proteins; D020126:Brefeldin A; D006921:Hygromycin B