MATURATION OF PHAGOSOMES IS ACCOMPANIED BY SPECIFIC PATTERNS OF SMALLGTPASES

In this study we purified phagosomes of the ciliated protozoan Tetrahy mena thermophila to analyze aspects of the maturation pathway of phago cytotic vesicles. Phagosomes were labeled with magnetic microparticles and then purified in high amounts with the help of a permanent magnet . By combining a pulse-chase labeling protocol with the magnetic separ ation procedure we were able to isolate phagosomes of defined ages, wh ich represent distinct stages of their maturation pathway. GTP-overlay assays showed that a set of small GTPases of the ras superfamily is a ssociated with these phagosomes. Phagosomes isolated at different stag es of maturation revealed a change in the pattern of the small GTPases . Some small GTPases identified by the GTPase overlay assays could be aligned to India ink stained protein spots in two-dimensional gels of isolated phagosomes. The results presented here are a first step to id entify the members of small GTPases associated with phagosomes during their maturation pathway. Microsequencing of pooled polypeptides by ma ss-spectrometric techniques will identify the primary structure of the se small GTPases.