Localization and Purification of Phagosome‐Associated Proteases in Tetrahymena thermophila

Phagosome maturation involves sequential introduction of oxidases, hydrolases and proteases that degrade encapsulated materials. In the ciliate Tetrahymena thermophila , we found that protease activity is associated with small vesicle‐like structures at the phagosome surface and occasionally in the lumen. Our current focus is to localize and purify phagosome associated cysteine proteases from T. thermophila using fluorescent and affinity‐tagged conjugates of an irreversible inhibitor DCG‐04 (lys(Boc)‐6‐aminohexanoic acid‐tyr‐leu‐ethyl (2S, 3S)‐oxirane‐2, 3‐dicarboxylate after Greenbaum et al, 2000). Two irreversible active‐site directed fluorescent cysteine protease inhibitors (6 FAM SE DCG‐04 and BODIPY 588/616‐DCG‐04) were synthesized using a combination of solution phase and standard F‐moc solid phase peptide chemistry. The products were confirmed by MALDI‐TOF mass spectrometry and purified by reverse phase HPLC. The binding of 6 FAM SE DCG‐04 to formaldehyde fixed, detergent permeabilized cells was assessed by fluorescence microscopy in the presence and absence of a 100‐fold excess of E‐64, a competing irreversible cysteine protease inhibitor. There was extensive punctate binding of the fluorescent inhibitor only in the absence of E‐64. 6 FAM SE DCG‐04 was taken up by live cells and following fixation, the fluor was observed to co‐localize with a fluorogenic protease substrate. Biotinylated DCG‐04 was used to isolate inhibited cysteine proteases by affinity chromatography. One‐dimensional electrophoresis revealed at least 21 distinct proteins ranging in size from about 18 ‐ 200 kDa. Future efforts will focus on proteomic analysis of these isolated proteins.