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A Giant Phosphoprotein Localized at the Spongiome Region of Crithidia luciliae thermophila
Author(s) -
BAQUI MUNIRA M. A.,
MORAES NADIA,
MILDER REGINA V.,
PUDLES JULIO
Publication year - 2000
Publication title -
journal of eukaryotic microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.067
H-Index - 77
eISSN - 1550-7408
pISSN - 1066-5234
DOI - 10.1111/j.1550-7408.2000.tb00086.x
Subject(s) - phosphoprotein , biology , crithidia , crithidia fasciculata , immunogold labelling , polyclonal antibodies , biochemistry , immunofluorescence , molecular mass , vacuole , microbiology and biotechnology , cytoplasm , ultrastructure , phosphorylation , antibody , protozoa , enzyme , anatomy , immunology , genetics
. A giant protein with an apparent molecular mass of 2,300‐kDa was identified in the Triton X‐100 soluble fraction of Crithidia luciliae thermophila. Polyclonal antibody raised against this protein reacted by immunoblot analysis with proteins of similar molecular mass in Crithidia fasciculata and Crithidia oncopelti. In addition, the antibody immunoprecipitates the protein either after in vivo phosphorylation with [32P]orthophosphoric acid or after metabolically labeling with [35S]methionine. Indirect immunofluorescence microscopy analysis performed either with fixed or with live parasites showed a single fluorescent spot at the level of the flagellar pocket region. Immunogold electron microscopy of thin sections of the parasite revealed that the antigen is localized at a restricted area of the spongiome, between the contractile vacuole and the flagellar pocket. Furthermore, Triton X‐114 phase separation of whole cell membrane proteins, metabolically labeled with [35S]methionine, demonstrated that the giant protein remains in the aqueous phase. These results indicate that this phosphoprotein behaves as a peripheral membrane protein localized at the spongiome region, suggesting that it might be involved in the osmoregulatory process.