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Yolk
Granules of Rhodnius prolixus have Several Similarities with Protozoan
Acidocalcisomes
Motta, L.S.1; da-Silva, W.S.2;
Miranda, K3; Gomes, F.M.1; Docampo, R.4; de
Souza, W.3; Machado, E.A1.
1 Laboratório
de Entomologia Médica, Instituto de Biofísica Carlos Chagas Filho, Universidade
Federal do Rio de Janeiro.
2 Laboratório de Bioenergética, Departamento
de Bioquímica Médica, Universidade Federal do Rio de Janeiro.
3 Laboratório de Ultraestrutura Celular Hertha
Meyer, Instituto de Biofísica Carlos Chagas Filho, Universidade Federal do Rio
de Janeiro.
4 Department of Cellular Biology and
Center for Tropical and Global Emerging Diseases, The University of Georgia.
Introduction: Intracellular compartments that
store ions, molecules and macromolecules are usual in several organisms. In
insects, during oogenesis, a large content of yolk proteins is incorporated by
oocytes and accumulated in structures called yolk granules (YG). The
acidification of this organelle is essential to activate hydrolases that are
responsible to make the yolk available during embryogenesis. In protozoan
there are organelles named acidocalcisomes that have an acidic nature, high
electron density, and high content of polyphosphates (polyPs), calcium,
magnesium, and other elements. Also, proton-pyrophosphatases (H+-PPase) has been found
as a marker for acidocalcisomes in unicellular eukaryotes. H+-PPases
catalyze the hydrolysis of PPi and the eletrogenic translocation of protons
into the vacuoles. Acidocalcisomes have been shown to be conserved during evolution
from prokaryotes to eukaryotes. Rhodnius prolixus is an insect
involved with Chagas’s disease transmission and lately we have observed some
similarities within Rhodnius’ YGs and protozoan’s acidocalcisomes. Objectives:
Determination of elemental composition, polyphosphate content and H+-PPase
characterization in Rhodnius’ YGs. Methodology and Results: In
this work we have measured the H+-PPase activity in membrane fractions prepared by
ultracentrifugation from fresh Rhodnius’ eggs. In these preparations the enzyme was
dependent of Mg+2, inhibited by NaF, IDP and Ca+2. The
membrane proteins of ovary and YG were analyzed by Western blot using anti
plant-H+-PPase, showing an immunostaining associated to a single 55
KDa polypeptide, which was immunolocalized in membrane of yolk granules. We
also described the yolk granules acidification by PPi and ATP using acridine
orange uptake. The elemental composition
was determined using X-ray microanalysis performed at an Electron
Microscope in YG fractions applied to Formvar-coated copper or nickel grids. In small YG (less than 3µm) we
observed a high concentration of phosphate, calcium and other elements also
found in acidocalcisomes. In addition we measure the
polyphosphate content in R. prolixus fresh eggs and found higher
concentrations of polyP short chain. Conclusion: Our results showed that only some YG
were acidified after PPi treatments and that some YG have several structural
similarities with acidocalcisomes,
indicating that small YG may have the same functions as acidocalcisomes.
Supported
by CNPq, PRONEX, CAPES and FAPERJ.
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