SECRETION OF TETRAIN, A TETRAHYMENA CYSTEINE PROTEASE, AS A MATURE ENZYME AND ITS IDENTIFICATION AS A MEMBER OF THE CATHEPSIN-L SUBFAMILY

A protease in the culture medium of Tetrahymena pyriformis was purifie d to homogeneity, The purified protease had an apparent molecular mass of 28 kDa on SDS/PAGE. The amino acid sequences of the N-terminal and internal peptides of the protease showed complete identity with those of tetrain, an enzyme previously reported as a Tetrahymena cysteine p rotease but not characterized in detail. Two overlapping cDNA clones f or terrain were sequenced, and the nucleotide sequence predicts that t hese clones encode a 330-amino acid protein composed of a 16-residue N -terminal signal sequence followed by a 103-residue propeptide and a 2 11-residue mature protease. The primary structure and enzymatic proper ties support the conclusion that terrain belongs to the cathepsin L su bfamily. Immunoblotting analyses showed that mature tetrain was found exclusively in the culture medium. Immunofluorescence microscopy demon strated that retrain was concentrated in or around the food vacuoles o f cells in the late logarithmic phase, but the staining of food vacuol es was not obvious in the stationary phase. These results suggest that terrain is synthesized at the logarithmic phase and is secreted into the culture medium as a mature form.