During phagocytosis cells internalize large particles that are subsequently degraded in lysosomes. Digestion capability of phagosomes is acquired in the process of maturation that culminates in the fusion of phagosomes with lysosomes leading to formation of the phagolysosomes. In single celled eukaryote Paramecium we identified the components indispensable for this process: homologues of lysosomal membrane protein 2 (LAMP-2) and a7 subunit of the 26S proteasome - the interacting partner of Rab7 (Dong et al. J. Biol. Chem. 279:21334) that was cloned by us in this cell (Surmacz et al. Acta Biochim Pol 53:149).

LAMP-2 cross-reacting polypeptide of ~106 kDa was glycosylated as shown by Pro-Q Emerald fluorescent staining and Western analysis of the same blot preceded by PNGase F digestion. During phagocytosis LAMP-2 expression was 2.5-fold higher than in the control cells when P2 protein fractions (100 000 x g) of equal load were quantified by immunoblotting.
LAMP-2 homologue was localized by confocal microscopy and ultrastructural studies. It was detected in lysosomes and in the phagolysosomes where it colocalized with Rab7 during internalization of latex beads. The presence of proteasome-related polypeptides of ~22 and ~24 kDa was revealed by immunoblotting with the antibody against human a7 of 20S proteasome. By electron microscopic analysis a7 subunit of 26S proteasome was detected in the vicinity of phagosomal membrane in the tiny vesicles. In some of them it colocalized with Rab7. The proteasomal antigen was not observed in LAMP-2-positive primary lysosomes. These results indicate that LAMP-2, Rab7 and a7 subunit of 26S are localized to phagolysosomal compartment and may be involved in the process of phagosome maturation in Paramecium.

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