Organization of Hemoglobin Synthesis in Chicken Erythrocytes: Protein Migration Through the Nuclear Membrane

Abstract

Abstract Avian erythrocytes retain their nuclei throughout cellular development, and it has been suggested that the nucleus is the site of hemoglobin synthesis. It was the goal of this work to re-examine the role of the nucleus in the synthesis of chicken hemoglobins, and to analyze the synthesis of hemoglobin polypeptides at the ribosomal level. The two chicken hemoglobins, Hb1 and Hb2, were studied by column chromatography, by electrophoresis, by immunodiffusion analysis, and by tryptic peptide mapping; they are very similar according to these criteria, and they appear to share many common amino acid sequences. Autoradiographic tryptic peptide maps of cytoplamic ribosomes pulse-labeled with [14C]leucine show the presence on ribosomes of nascent globin chains. Nascent growing polypeptide chains were isolated from ribosomes following pulses to erythrocytes of [3H]leucine and were fractionated according to size by gel filtration; the growing chains are heterogenous in size, ranging from complete globin polypeptides down to single amino acids. The kinetics of labeling these various-sized peptides shows that they are synthesized by sequential addition of amino acids to one growing end; the synthesis time of a globin chain at 37° was estimated from these kinetics as 1 min. Only a small fraction of the nascent polypeptides are the size of completed globin chains, which implies that the release of completed chains from ribosomes occurs in a period of time which is much shorter than the time required to synthesize the whole chain. An autoradiographic study of erythrocytes submitted to short pulses of [3H]leucine establishes conclusively that the hemoglobins are synthesized in the cytoplasm; the newly synthesized hemoglobin molecules subsequently become distributed between the nucleus and the cytoplasm, presumably passing through open pores in the nuclear membrane.