title: O-glycosylation is essential for intracellular targeting of synaptotagmins I and II in non-neuronal specialized secretory cells.
abstract: We have examined the trafficking of synaptotagmin (Syt) I and II in the mast cell line rat basophilic leukemia (RBL-2H3). We demonstrate that both Syt I and Syt II travel through the plasma membrane and require endocytosis to reach their final intracellular localization. However, N- or C-terminal tagging of Syt II, but not of Syt I, prevents its internalization, trapping the tagged protein at the plasma membrane. Furthermore, a chimeric protein comprising a tagged luminal domain of Syt II fused with the remaining domains of Syt I also localizes to the plasma membrane, whereas a chimera consisting of tagged luminal domain of Syt I fused with Syt II colocalizes with Syt I on secretory granules. We also show that endocytosis of both Syt I and Syt II is strictly dependent on O-glycosylation processing, whereby O-glycosylation mutants of either protein fail to internalize and remain at the plasma membrane. Our results indicate that the luminal domains of Syt I and Syt II govern their internalization capacity from the plasma membrane and identify O-glycosylation as playing a crucial role in Syt trafficking in non-neuronal secretory cells.
authors.affiliation: Department of Cell and Developmental Biology, Sackler School of Medicine, Tel Aviv University, Tel Aviv 69978, Israel.
authors.forename: Yafit
authors.initials: Y
authors.lastname: Atiya-Nasagi
authors.forename: Hila
authors.initials: H
authors.lastname: Cohen
authors.forename: Ora
authors.initials: O
authors.lastname: Medalia
authors.forename: Mitsunori
authors.initials: M
authors.lastname: Fukudan
authors.forename: Ronit
authors.initials: R
authors.lastname: Sagi-Eisenberg
mesh_terms: D000818:Animals; D002135:Calcium-Binding Proteins / Q000096:biosynthesis / Q000378:metabolism*; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism / Q000648:ultrastructure; D004705:Endocytosis / Q000502:physiology; D006031:Glycosylation; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000378:metabolism; D008407:Mast Cells / Q000378:metabolism* / Q000648:ultrastructure; D008562:Membrane Glycoproteins / Q000096:biosynthesis / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000502:physiology; D021381:Protein Transport / Q000502:physiology; D051381:Rats; D022142:Secretory Vesicles / Q000378:metabolism / Q000648:ultrastructure; D050861:Synaptotagmin II; D050857:Synaptotagmins
chemical_list: D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050861:Synaptotagmin II; C494264:Syt2 protein, rat; D050857:Synaptotagmins; D049452:Green Fluorescent Proteins