Targeting of synaptotagmin to neurite terminals in neuronally differentiated PC12 cells.
We have investigated structural elements that determine the accumulation of synaptotagmin, a major synaptic vesicle protein, in neurite terminals of neuronally differentiated neuroendocrine pheochromocytoma PC12 cells. We performed extensive deletion and point mutagenesis of rat synaptotagmin II, expressed mutant proteins in PC12 cells differentiated by nerve growth factor (NGF) and monitored their intracellular distribution by immunofluorescence. We found a structural element located at the carboxy-terminal domain of the synaptotagmin molecule, which is necessary for its accumulation at the terminal. Using alanine-scanning mutagenesis, we have identified two amino acids in this element, tryptophan W405 and leucine L408, that are critical for correct targeting of synaptotagmin II to neurite terminals. Changing either one of them to alanine prevents the accumulation of the protein at the terminals. These amino acids are evolutionarily conserved throughout the entire synaptotagmin family and also among synaptotagmin-related proteins, suggesting that different synaptotagmins may have similar mechanisms of targeting to neuronal cell terminals.
Krasnov
P A
PA
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Enikolopov
G
G
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport / Q000235:genetics; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D002454:Cell Differentiation; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D016501:Neurites / Q000378:metabolism* / Q000648:ultrastructure; D009474:Neurons / Q000166:cytology / Q000378:metabolism* / Q000648:ultrastructure; D016716:PC12 Cells; D017354:Point Mutation; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017384:Sequence Deletion; D050857:Synaptotagmins; D014162:Transfection
D002135:Calcium-Binding Proteins; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D011994:Recombinant Proteins; D050857:Synaptotagmins