Phagosome maturation in unicellular eukaryote Paramecium: the presence of RILP, Rab7 and LAMP-2 homologues.
Phagosome maturation is a complex process enabling degradation of internalised particles. Our data obtained at the gene, protein and cellular level indicate that the set of components involved in this process and known up to now in mammalian cells is functioning in unicellular eukaryote. Rab7-interacting partners: homologues of its effector RILP (Rab-interacting lysosomal protein) and LAMP-2 (lysosomal membrane protein 2) as well as alpha7 subunit of the 26S proteasome were revealed in Paramecium phagolysosomal compartment. We identified the gene/transcript fragments encoding RILP-related proteins (RILP1 and RILP2) in Paramecium by PCR/RT-PCR and sequencing. The deduced amino acid sequences of RILP1 and RILP2 show 60.5% and 58.3% similarity, respectively, to the region involved in regulating of lysosomal morphology and dynein-dynactin recruitment of human RILP. RILP colocalised with Rab7 in Paramecium lysosomes and at phagolysosomal membrane during phagocytosis of both the latex beads and bacteria. In the same compartment LAMP-2 was present and its expression during latex internalisation was 2.5-fold higher than in the control when P2 protein fractions (100,000 x g) of equal load were quantified by immunoblotting. LAMP-2 cross-reacting polypeptide of approximately106 kDa was glycosylated as shown by fluorescent and Western analysis of the same blot preceded by PNGase F treatment. The alpha7 subunit of 26S proteasome was detected close to the phagosomal membrane in the small vesicles, in some of which it colocalised with Rab7. Immunoblotting confirmed presence of RILP-related polypeptide and a7 subunit of 26S proteasome in Paramecium protein fractions. These results suggest that Rab7, RILP and LAMP-2 may be involved in phagosome maturation in Paramecium.
Wyroba
E
E
Department of Cell Biology, Nencki Institute of Experimental Biology, 3 Pasteur Street, 02-093 Warsaw, Poland. e.wyroba@nencki.gov.pl
Surmacz
L
L
Osinska
M
M
Wiejak
J
J
D048868:Adaptor Proteins, Signal Transducing / Q000235:genetics*; D000595:Amino Acid Sequence; D000818:Animals; D006023:Glycoproteins / Q000378:metabolism; D052119:Lysosomal-Associated Membrane Protein 2 / Q000235:genetics / Q000378:metabolism*; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D010247:Paramecium / Q000378:metabolism / Q000502:physiology* / Q000648:ultrastructure; D010588:Phagosomes / Q000378:metabolism / Q000502:physiology*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D020691:rab GTP-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D000090802:rab7 GTP-Binding Proteins
D048868:Adaptor Proteins, Signal Transducing; D006023:Glycoproteins; D052119:Lysosomal-Associated Membrane Protein 2; C423317:RILP protein, human; D000090802:rab7 GTP-Binding Proteins; C000716463:rab7 GTP-binding proteins, human; D020691:rab GTP-Binding Proteins