Identification of a 24 kDa phosphoprotein associated with an intermediate stage of memory in Hermissenda

A requirement for protein synthesis is a critical feature in dissociating different phases of memory. However, in examples of cellular and synaptic plasticity in which an early or intermediate requirement for protein synthesis has been implicated, specific proteins have not been identified. Here we report the identification of a 24 kDa phosphoprotein (CSP24) associated with an intermediate stage of memory, distinct from short-term memory, detected after one-trial conditioning of Hermissenda. CSP24, initially identified from (32)PO(4)-labeled proteins resolved by two dimensional (2-D) PAGE, was excised from multiple Coomassie blue-stained 2-D gels and subjected to reverse phase HPLC and automated sequence analysis. The sequenced peptides exhibited a homology to the beta-thymosin family of actin-binding protein. Anti-CSP24 antibody recognized CSP24 on 1- and 2-D gels by Western blot analysis. Labeled CSP24 immunoprecipitated with anti-CSP24 antibody revealed that significantly more (32)PO(4) was incorporated in preparations that received one-trial conditioning compared with unpaired controls. In contrast, labeled CSP24 immunoprecipitated with anti-CSP24 from conditioned and unpaired control preparations receiving a procedure that only produced short-term enhanced excitability did not exhibit differences in (32)PO(4) incorporation into the immunoprecipitates. These results show that a specific identified phosphoprotein is associated with an intermediate stage of memory for one-trial conditioning in Hermissenda.