Immunocytochemical mapping of the hemoglobin biosynthesis site in amphibian erythroid cells

During the past 25 years, several studies have attempted to determine the s ite of integration of the heme and the four globin chains in vertebrate ery throid cells that is important in the formation of the hemoglobin molecule. Mitochondrion-like organelles or hemosomes were pointed out as responsible for this task. We performed several experiments to investigate this hypoth esis. The intracellular distribution of hemoglobin in amphibian erythroid c ells was detected by post-embedding immune-electron microscopy, using a pol yclonal anti-human hemoglobin-proteinA-gold complex. Hemoglobin mapping sho wed an intense labeling in the cell cytoplasm, but none in cytoplasmic stru ctures such as endoplasmic reticulum, mitochondria, mitochondrion-like orga nelles, Golgi complex, ribosomes or ferruginous inclusions. The mitochondri al fraction obtained according to the protocol described for some authors, showed by ultrastructural examination that this fraction has a heterogeneou s content, also composed by microvesicles rich in cytoplasmic hemoglobin, a n artifact generated by mechanical action during cell fractionation. Thus, when this fraction is lysed and its content submitted to electrophoresis, h emoglobin bands would be found inevitably, causing false-positive results, erroneously attributed to hemoglobin content of mitochondrion-like organell es. Our data do not confirm the hypothesis that the final hemoglobin biosyn thesis occurs inside mitochondrion-like organelles. They suggest that the h emoglobin molecule be assembled in the erythrocyte cytoplasm outside of mit ochondria or hemosomes.