Confocal microscopy reveals thimet oligopeptidase (EC 3.4.24.15) and neurolysin (EC 3.4.24.16) in the classical secretory pathway

Thimet oligopeptidase (EC 3.4.24.15; EP24.15) and neurolysin (EC 3.4.24.16; EP24.16) are closely related enzymes involved in the metabolic inactivatio n of bioactive peptides, Both of these enzymes were previously shown to be secreted from a variety of cell types, although their primary sequence lack s a signal peptide, To investigate the mechanisms responsible for this secr etion, we examined by confocal microscopy the subcellular localization of t hese two enzymes in the neuroendocrine cell line AtT20, Both EP24.15 and EP 24.16 were found by immunohistochemistry to be abundantly expressed in AtT2 0 cells, Western blotting experiments confirmed that the immunoreactivity d etected in the soma of these cells corresponded to previously cloned isofor ms of the enzymes. At the subcellular level, both enzymes colocalized exten sively with the integral trans-Golgi network protein, syntaxin-6, in the ju xtanuclear region. In addition, both EP24.15 and EP24.16 were found within small vesicular organelles distributed throughout the cell body. Some, but not all, of these organelles also stained positively for ACTH, These result s demonstrate that both EP24.15 and EP24.16 are present within the classica l secretory pathway. Their colocalization with ACTH further suggests that t hey may be targeted to the regulated secretory pathway, even in the absence of a signal peptide.