Annexins inParamecium cells

Abstract

Annexins were isolated from Paramecium cell homogenates by standard ethylene glycol tetraacetic acid (EGTA) extraction and 100 000-g centrifugation. Two different antibodies (Abs) against synthetic peptides were used, Call-15 and B15, which in mammalian cells recognize a sequence of annexin II or a common sequence occurring in several annexins (except for annexin II), respectively. With anti-Call-15 Abs, western blots from EGTA extracts showed strongly reactive bands of 44.5 and 46 kDa and of higher values. Some of these bands bound to the 100 000-g pellet fraction when Ca(2+) was added. Immuno- and affinity labelling revealed selective, Ca(2+)-dependent labelling of the cell cortex, with enrichment around trichocyst docking sites (facing subplasmalemmal Ca(2+) stores). Cortical fluorescence labelling decreased in wild-type (7S) cells when trichocyst ghosts were detached after synchronous exocytosis. Similarly, cortical labelling was reduced when intact trichocysts were detached from the cell surface of non-discharge mutant cells (nd9-28 degrees C, showing identical bands on blots), which then contained numerous heavily labelled phagolysosomes. This strongly suggests annexin downregulation. All together, the dynamic labelling of cortical structures we observed strongly supports involvement of calpactin-like annexins in trichocyst docking. Anti-B15 Abs recognized a band of 51 kDa and some of higher values. These Abs selectively labelled the outlines of the cytoproct, the site of spent phagolysosome exocytosis. In conclusion, our data indicate involvement of specific sets of annexins in site-specific positioning and attachment of widely different secretory organelles at the cell surface in Paramecium cells.

The Copines, a Novel Class of C2 Domain-containing, Calciumdependent, Phospholipid-binding Proteins Conserved from Paramecium to Humans

“…Paramecium is thus unusual in that the major protein, copine, falls into a different class. Although no annexin has been definitively characterized from Paramecium, there are reports of Paramecium proteins that cross-react with anti-annexin antisera (38). 3 It is possible that some of the additional proteins obtained in the lipid-binding fraction in this study are annexins; however, we detected no immunological cross-reaction between these proteins and antisera raised against mammalian annexin I, II, IV, VI, or VII or nematode Nex-1 annexin (data not shown).…”