Membrane proteins synthesized by rabbit reticulocytes.

Abstract

Intact rabbit reticulocyte cells synthesize two predominant species of polypeptides which are components of the cell plasma membrane. Previous work (Lodish, H. F. 1973. Proc. Natl. Acad. Sci. U. S. A. 70:1526-1530 showed that these proteins were synthesized by polyribosomes not attached to membranes. We show here that both polypeptides are confined to the cytoplasmic surface of the cell membrane. These studies utilized iodination of whole cells and of membranes with lactoperoxidase, and digestion of whole cells and membranes with chymotrypsin. One of these proteins is synthesized as a precursor, and about 20-40 amino acids are removed after it is incorporated into the membrane. We discuss the probable sites of synthesis of these and other classes of membrane proteins.Although a great deal is known of the protein composition of mammalian erythrocyte membranes, and of the asymmetric distribution of these proteins within the membrane, little is known about the biosynthesis of erythrocyte membrane proteins. The literature concerning the proteins of erythrocyte membranes has been reviewed several times (1-4). As resolved by sodium dodecyl sulfate (SDS) gel electrophoresis, all mammalian erythrocyte membranes contain about seven to nine principal protein components, although the detailed pattern of membrane proteins shows some species differences (1-11). The major polypeptides common to all mammalian erythrocytes include two polypeptides of molecular weight greater than 200,000 (7) and one of 43,000 (the three spectrin polypeptides) (1, 3, 4, 12), a protein of molecular weight 90,000, and a sialoylglycoprotein which in the human contains the MN, A, and B blood group antigens (11). The latter two species are the only membrane proteins found on the cell surface; recent work suggests that they penetrate to the interior surface of the membrane (references 6, 13-20, reviewed in reference 4). Most membrane proteins are confined to the cytoplasmic surface of the membrane; glyceraldehyde-3-phosphate dehydrogenase, for instance, is found only on the inner surface of the human erythrocyte membrane, and the isolated enzyme forms stable interactions with high affinity for a limited number of sites on the cytoplasmic face of the membrane (21).Rabbit reticulocytes contain, with possibly one or two exceptions (22), the same proteins in their membranes as do rabbit erythrocytes. We recently showed that rabbit reticulocytes synthesize only two major and two to three minor membrane proteins; most of the membrane proteins are no longer being synthesized in significant amounts at this stage (23). We also showed that these two proteins as well as all other proteins made by reticulocytes are synthesized on polyribosomes which are not attached to membranes (23,24). A small fraction of reticulocyte polysomes are apparently bound to the cell membrane. Woodward et

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.…”

“…Red cells that had reached a "steady state" of radioactive labeling about 10 days after the injection of isotope were treated with a-chymotrypsin to digest the proteins on their external surface (9 Autoradiofluorography. In order to enhance the detection of 35S in each band, we permeated the gels with a scintillant, 20% (wt/vol) 2,5-diphenyloxazole (PPO, Interex, Natick, Mass.)…”

“…Each mouse was etherized and bled by cutting off a segment of tail, allowing about 0.1 ml of blood to drip into 10 ml of heparinized cold Krebs-Ringer phosphate buffer (15 7.4 (16). After the membranes had been pelleted at 20,000 X g for 10 min, the hemolysate was saved for specific activity determination of hemoglobin using acid acetone precipitation and Lowry (17) Reticulocytes from anemic acetylphenylhydrazine-treated mice were labeled in vitro as described (9).…”

Cloning Expeditions: Risky but Rewarding

“…We showed that several proteins, now known to be cytoskeletal proteins, are made on membrane-free polysomes (15,16). One of my favorite experiments demonstrated that the major red cell membrane and cytoskeleton proteins are made at different times during development (17).…”

Molecular genetic characterization of the mRNA coding for an inducible suppressor factor specific for L-glutamic acid60-L-alanine30-L-tyrosine10.

“…Sci. USA 84 (1987) 6233 face of the plasma membrane (27)(28)(29)(30)(31). The best-characterized protein is pp60vsrc, the transforming protein of Rous sarcoma virus that is modified by the covalent attachment of the 14-carbon saturated fatty acid myristic acid to its NH2 terminus following translation on free polysomes and is then transported within 15 min to the plasma membrane (32,33).…”