Vesicle-associated membrane protein-2/synaptobrevin binding to synaptotagmin I promotes O-glycosylation of synaptotagmin I.
Synaptotagmin I (Syt I), an evolutionarily conserved integral membrane protein of synaptic vesicles, is now known to regulate Ca2+-dependent neurotransmitter release. Syt I protein should undergo several post-translational modifications before maturation and subsequent functioning on synaptic vesicles (e.g. N-glycosylation and fatty acylation in vertebrate Syt I), because the apparent molecular weight of Syt I on synaptic vesicles (mature form, 65,000) was much higher than the calculated molecular weight (47,400) predicted from the cDNA sequences both in vertebrates and invertebrates. Common post-translational modification(s) of Syt I conserved across phylogeny, however, have never been elucidated. In the present study, I discovered that dithreonine residues (Thr-15 and Thr-16) at the intravesicular domain of mouse Syt I are post-translationally modified by a complex form of O-linked sugar (i.e. the addition of sialic acids) in PC12 cells and that the O-glycosylation of Syt I in COS-7 cells depends on the coexpression of vesicle-associated membrane protein-2 (VAMP-2)/synaptobrevin. I also showed that a transmembrane domain of Syt I directly interacts with isolated VAMP-2, but not VAMP-2, in the heterotrimeric SNARE (SNAP receptor) complex (vesicle SNARE, VAMP-2, and two target SNAREs, syntaxin IA and SNAP-25). Since di-Thr or di-Ser residues are often found at the intravesicular domain of invertebrate Syt I, and VAMP-dependent O-glycosylation was also observed in squid Syt expressed in COS-7 cells, I propose that VAMP-dependent O-glycosylation of Syt I is a common modification during evolution and may have important role(s) in synaptic vesicle trafficking.
Fukuda
Mitsunori
M
Fukuda Initiative Research Unit, RIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan. mnfukuda@brain.riken.go.jp
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002135:Calcium-Binding Proteins; D017931:DNA Primers; D006031:Glycosylation; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011499:Protein Processing, Post-Translational; D021381:Protein Transport; D050683:R-SNARE Proteins; D017386:Sequence Homology, Amino Acid; D050863:Synaptotagmin I; D050857:Synaptotagmins
D002135:Calcium-Binding Proteins; D017931:DNA Primers; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D009419:Nerve Tissue Proteins; D050683:R-SNARE Proteins; D050863:Synaptotagmin I; D050857:Synaptotagmins