Heterologous expression of pyruvate dehydrogenase E1 subunits of the microsporidium Paranosema (Antonospora) locustae and immunolocalization of the mitochondrial protein in amitochondrial cells.
Microsporidia, a large group of fungi-related intracellular parasites, are characterized by drastically reduced metabolism. They possess genes encoding glycolysis components, and the glycerol-phosphate shuttle, but lack mitochondria, Krebs cycle, respiratory chain and pyruvate-converting enzymes, except alpha and beta subunits of E(1) enzyme of pyruvate dehydrogenase (PDH) complex. Here, we have expressed PDH subunits from the microsporidum Paranosema (Antonospora) locustae in Escherichia coli. Western blot analysis with antibodies raised against recombinant proteins has revealed their specific accumulation in mature spores of P. locustae but not in the intracellular development stages. Two subunits were coprecipitated as a single heterooligomeric complex by anti-alpha or anti-beta PDH antibodies. Ultracentrifugation of spore homogenate has shown the presence of PDH in the soluble fraction. Relocalization of the mitochondrial protein in microsporidial spore cytoplasm was confirmed by immunoelectron microscopy of ultrathin cryosections with affinity-purified anti-alpha PDH antibodies. On cryosections, parasite enzyme was found partly associated with the cytoplasmic side of ER and other intraspore membranes, suggesting that electrons might be transferred to any membrane acceptor and finally to oxygen in the parasite cell.
Dolgikh
Viacheslav V
VV
Laboratory of Microbiological Control, All-Russian Institute for Plant Protection, St Petersburg, Pushkin, Russia. dol1slav@yahoo.com
Seliverstova
Elena V
EV
Naumov
Anton M
AM
Senderskiy
Igor V
IV
Pavlova
Olga A
OA
Beznoussenko
Galina V
GV
D016817:Apansporoblastina / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics / Q000648:ultrastructure; D020285:Cryoelectron Microscopy; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000096:biosynthesis / Q000235:genetics; D047468:Immunoprecipitation; D016253:Microscopy, Immunoelectron; D024101:Mitochondrial Proteins / Q000096:biosynthesis / Q000235:genetics; D011485:Protein Binding; D021122:Protein Subunits / Q000096:biosynthesis* / Q000235:genetics; D030481:Pyruvate Dehydrogenase (Lipoamide) / Q000096:biosynthesis* / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D013172:Spores, Fungal / Q000737:chemistry / Q000648:ultrastructure
D005656:Fungal Proteins; D024101:Mitochondrial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D030481:Pyruvate Dehydrogenase (Lipoamide)