Biosynthesis of Reticulocyte Membrane Proteins by Membrane-Free Polyribosomes

Abstract

Intact rabbit reticulocyte cells synthesize two predominant species of membrane polypeptides; at least 10 reticulocyte membrane polypeptide species are not produced by the cells. Cell-free extracts of reticulocytes, free of any membranes or membrane-bound polyribosomes, synthesize large amounts of these two membrane polypeptides; one of these polypeptides appears to be modified, probably by loss of 20-40 amino acids, after it is incorporated into the membrane. Deprivation of hemin results in inhibition of synthesis by lysates of membrane proteins, globin, and all other cytoplasmic proteins.The protein composition of mammalian erythrocyte membrane is now fairly well established. There exist 7-9 main components (1-8); the major species include two polypeptides of molecular weight greater than 200,000 (4), a protein of molecular weight 100,000, and a sialoylgylcoprotein which contains the MN, A, and B blood-group activities (8). The latter two species are the only membrane proteins found on the cell surface (3, 9-12); they may also penetrate to the interior surface of the membrane (13-15). Little is known, however, about the biosynthesis of erythrocyte membrane proteins: the types of erythropoetic cells that synthesize each protein, the type of polysome (free or membrane-attached) that synthesizes the proteins, and the factors that regulate their synthesis.The reticulocyte is the penultimate cell in the erythropoetic series; it has no nucleus, and makes no RNA or DNA.About 90% of protein produced is a and ,3-globin chains (16,17). The protein composition of reticulocyte membranes is very similar to that of erythrocyte membranes (my unpublished observations). In this paper, I show that reticulocytes synthesize only 2 major and 2-3 minor membrane proteins; most of the membrane proteins are no longer being synthesized in significant amounts at this stage.In most mammalian cells a fraction of the polyribosomes are firmly attached to membranes; these are the site of synthesis of most, if not all, proteins that are eventually transported out of the cell, such as trypsinogen, chymotrypsinogen, albumin, and other serum proteins (18-22). It is not at all clear that this is the sole function of these polysomes.Burka has claimed that rabbit reticulocytes contain membrane-bound polyribosomes (23) that synthesize most of the nonglobin proteins made by the cell (24). By contrast, we showed recently that membrane-free reticulocyte polyribosomes will produce precisely the same globin and nonglobin proteins as does the intact cell (17). In this paper I show, using dodecyl sulfate-gel electrophoresis and peptide map-1526 ping procedures, that a membrane-free lysate makes large amounts of the two membrane polypeptides produced by the intact cell; one of these proteins appears to be modified, probably by removal of 20-40 amino acids when it is incorporated into the membrane. MATERIALS AND METHODSMaterials. Acetylphenylhydrazine was purchased from Sigma, and [35S]methionine (30,000 Ci/mol) from Amersham/Searle. Sources of all o...

Biogenesis of microsomal membrane glycoproteins in rat liver. III. Release of glycoproteins from the Golgi fraction and their transfer to microsomal membranes.

“…Subunits of several mitochondrial enzymes are believed to be synthesized on cytoplasmic ribosomes and to be subsequently transferred to their locations in the mitochondria (22). Cytoplasmic pools of polypeptides play an important role in the biosynthesis of plasma membranes, as has been demonstrated previously for reticulocytes (23), and in the assembly of the viral envelope (24).…”

Asynchronous synthesis of erythrocyte membrane proteins.

“…Biosynthetic studies with rabbit reticulocytes have shown that two nonglobin proteins of 33,000 and 56,000 daltons (8) are made by the free polyribosomes in the cytoplasm and then attach to the inner surface of the membrane (9). The latter protein loses about 30 amino acids with incorporation into the membrane.…”

A rabbit reticulocyte model for the role of hemin-controlled repressor in hypochromic anemias.

“…8) Furthermore, the hemin effect is not specific to the synthesis of globin. Recently, it has been shown that hemin is necessary for maximal protein synthesis of the nonglobin proteins of the reticulocyte (27,(37)(38)(39), as well as for Krebs II ascites tumor cells (40), platelets (41), and brain and liver cells (34). Thus, one may speculate that the suppression of heme synthesis and enhancement of HCR possibly might have many more complex effects rather than solely the inhibition of hemoglobin synthesis and production of hypochromic red cells.…”