Although the extravesicular p40 domain of the transmembrane protein, p65 synaptotagmin (Syt) 1, is essential for the nonclassical export of the signal peptide-less structure, FGF1, it was not possible to identify a specific intracellular protease responsible for the processing of p65 Syt1. Surprisingly, analysis of the p65 Syt1 coding sequence revealed the presence of two potential alternative ATG codons corresponding to Met103 and Met113 both of which were flanked by Kozak sequences. Indeed, in vitro translation of a Met103Ile but not a Met113Ile p65 Syt 1 point mutant exhibited reduced expression of p40 Syt 1 and the double p65 Syt1 Met103Ile and Met113Ile point mutant was unable to translate the p40 Syt1 isoform. Since the expression of the p65 Syt1 double point mutant inhibited the stress-induced release of FGH, it is likely that the alternative translation of the p65 Syt1 transcript at Met 103 may be involved in the generation of intracellular p40 Syt 1, a critical component of the FGF1 release pathway. (C) 2003 Elsevier Inc. All rights reserved.