Increased plasma membrane localization of O-glycosylation-deficient mutant of synaptotagmin I in PC12 cells.

Synaptotagmin I (Syt I) is a Ca2+-binding protein on synaptic vesicles and presumably functions as a Ca2+ sensor for neurotransmitter release. Native Syt I protein in neuroendocrine PC12 cells undergoes several posttranslational modifications, such as O-glycosylation, N-glycosylation, and fatty acylation, and the latter two modifications have been shown to be required for the proper function of murine Syt I in PC12 cells. However, nothing is known about the physiological significance of the O-glycosylation of Syt I in dense-core vesicle exocytosis in PC12 cells. In this study, we created an O-glycosylation-deficient mutant (named TA = T15A/T16A) and an N-glycosylation-deficient mutant of Syt I (named T26A) and investigated their subcellular distribution in Syt I-deficient PC12 cells, where other Syt isoforms (e.g., IV and IX) and other membrane trafficking proteins (e.g., Rab27A, SNAP-25, syntaxin-1, and VAMP-2) are normally expressed. We found that some cells expressing high level of recombinant wild-type (WT) Syt I protein show mistargeting of Syt I(WT) protein to the plasma membrane, whereas most of the cells show normal dense-core vesicle localization of Syt I(WT) protein. Similar mistargeting was also observed in cells expressing high levels of the Syt I(T26A) and Syt I(TA) mutants, but the mistargeting of the Syt I(TA) mutant to the plasma membrane was much more evident than with the Syt I(WT) or (T26A) mutant. The results indicate that O-glycosylation, not N-glycosylation, is partially involved in efficient targeting of Syt I protein to dense-core vesicles in PC12 cells.