Cloning and sequence analysis of cDNA encoding p38, a major synaptic vesicle protein.

Abstract

We have isolated from a lambda gt11 rat brain cDNA library cDNA clones encoding greater than 95% of the open reading frame and untranslated regions of the mRNA for p38, the most abundant of the integral membrane proteins of the synaptic vesicle. Phage containing cDNA that encoded vesicle proteins were identified by screening fusion proteins with a polyclonal serum to rat brain synaptic vesicles. To identify phage carrying p38 sequences, fusion proteins were used to affinity purify monospecific antibodies from the original heterogeneous serum; antibodies to a 38,000-D protein were then identified by Western blotting. Inserts carrying DNA-encoding p38 sequences were subcloned into plasmid vectors and used to generate cDNA probes for Northern blot analysis. A major transcript of 2.4 kb was expressed specifically in brain and endocrine tissue but not in liver, consistent with the tissue-specific expression of the protein detected by antibody techniques. Using three overlapping clones that encoded fusion proteins, we identified and sequenced approximately 85% of the cDNA. Two additional Eco RI fragments at the 5' end of the mRNA were obtained from a fourth clone identified by screening a second lambda gt11 library with a 5' cDNA probe. The cDNA encoded an open reading frame of 298 amino acids with a 3' untranslated region of 1.4 kb. The protein shares no sequence homology with other Ca2+-binding proteins. The availability of a cDNA clone for an integral synaptic vesicle protein should facilitate studies of its function in transmitter release, its intracellular targeting, and regulation of synaptic vesicle biogenesis during development and regeneration of nerve terminals.