Molecular characterization and intracellular distribution of the alpha 5 subunit of Trypanosoma cruzi 20S proteasome.
Three different monoclonal antibodies were produced against Trypanosona cruzi proteasomes. These antibodies were shown to react with a single 27-kDa band on immunoblots of purified proteasomes. Using a 7E5 monoclonal antibody (IgG1) that recognized the alpha5 subunit of protozoan protease we have studied the intracellular distribution of the T. cruzi 20S proteasome. Contrary to all cell types described to date, T. cruzi 20S proteasome was found not only in the cytoplasm and nucleus but also in the kinetoplast. As revealed by confocal microscopy, the reactivity of monoclonal antibody 7E5 was highly specific for protozoan proteasome because the antibody recognized only the proteasomes from parasites and not those from the mammalian host in T. cruzi infected cells. These findings were confirmed by immunoblots or immunoprecipitations, followed by chymotrypsin-like activity detection in kinetoplasts isolated by differential centrifugation and sucrose density gradients. Proteasome 20S was present in all T. cruzi stages and only slight differences in terms of relative abundance were found. The potential role of the proteasome in kinetoplast remodeling remains to be determined.
Gutiérrez
Bessy
B
Health Sciences Faculty, University of Antofagasta, Antofagasta, Chile.
Osorio
Luis
L
Motta
María Cristina M
MC
Huima-Byron
Telervo
T
Erdjument-Bromage
Heydeie
H
Muñoz
Christian
C
Sagua
Hernán
H
Mortara
Renato A
RA
Echeverría
Alex
A
Araya
Jorge E
JE
González
Jorge
J
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D003593:Cytoplasm / Q000201:enzymology; D005455:Fluorescent Antibody Technique; D007150:Immunohistochemistry; D008018:Life Cycle Stages; D018613:Microscopy, Confocal; D046988:Proteasome Endopeptidase Complex / Q000235:genetics / Q000378:metabolism; D021122:Protein Subunits / Q000235:genetics / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology* / Q000648:ultrastructure; D014349:Trypanosoma cruzi / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D021122:Protein Subunits; D046988:Proteasome Endopeptidase Complex