Biogenesis of erythrocyte membrane proteins in vivo studies in anemic rabbits

To study the process of red cell membrane protein synthesis we have followed the time course of [³H]leucine appearance in total protein and individual peptides of the erythrocyte membrane following injection of the amino acid into phenylhydrazine-anemic rabbits. Multiple peripheral blood samples were taken from single animals over a 5-week period. Erythrocyte membrane proteins were separated by polycrylamide gel electrophoresis in sodium dodecylsulfate and dithiothreitol; incorporation of radioactivity was determined by gel slicing and liquid scintillation spectrometry. Appearance of [³H]leucine in circulating erythrocytes reached a peak at 1–3 days, with a steady decline thereafter. The radioactive amino acid appeared first in the lowest molecular weight peptides and last in the largest peptides; at the earliest time point (8 h), little radioactivity was observed in any of the four largest peptides present in the membranes (bands A, 1, 2 and 3). Certain smaller peptides (bands 4, 5 and 9) were the predominant species labeled at this time. By 24 h all peptides showed significant incorporation. With maturation of the red cells, label largely disappeared from bands A, 9 and several smaller peptides; this was confirmed by finding that the peptides are virtually absent from mature circulating erythrocytes. These data are interpreted as showing that red cell membrane proteins are synthesized asynchronously during the life cycle of the erythrocyte; the largest peptides are made predominantly in the earlier marrow stages of development, while certain of the smaller peptides are still being synthesized in the reticulocyte stage. Several membrane proteins appear to be specific to the reticulocyte and are lost during the process of cell maturation in the circulation.     

Biogenesis of erythrocyte membrane proteins. In vitro studies with rabbit reticulocytes.

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [3-H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polyacrylamide gel electrophoresis of labeled membranes on large (19 mm) gels which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [3-H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removable of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Biogenesis of erythrocyte membrane proteins in vitro studies with rabbit reticulocytes

The capability of rabbit reticulocytes to synthesize red cell membrane proteins has been tested in vitro. Reticulocyte-rich blood from phenylhydrazine-treated rabbits was incubated in vitro in a complete amino acid medium containing ferrous salts, glucose, rabbit plasma and [³H]leucine. Red cell ghost membranes were prepared by hypotonic lysis and leucine incorporation into hemoglobin and total membrane proteins determined. The pattern of incorporation into individual peptides was determined by polycrylamide gel electrophoresis of labeled membranes on large (19 mm) gel which were then sliced into 1 mm sections; radioactivity was compared with densitometric tracings of Coomassie blue stained analytical (6 mm) gels. Incorporation of [³H]leucine into both hemoglobin and membrane protein was linear over 1 h. Gel analysis of labeled membranes revealed that the amino acid was primarily incorporated into peptides with molecular weights of 90 000 or less; three peptides of molecular weights 90 000, 60 000 and 33 000 showed the highest specific activity. Synthesis of the four largest peptide species was negligible. Removal of ferrous salts inhibited synthesis of both globin and membrane protein equally (approx. 50%). However, puromycin and cycloheximide preferentially inhibited the synthesis of globin as compared to membrane proteins. Reticulocytes remain capable of synthesizing a number of membrane proteins; these results are consistent with studies of red cell membrane synthesis in anemic rabbits in vivo.     

Biogenesis of mitochondrial outer membrane proteins

Mitochondria are surrounded by two distinct membranes: the outer and the inner membrane. The mitochondrial outer membrane mediates numerous interactions between the mitochondrial metabolic and genetic systems and the rest of the eukaryotic cell. Proteins of this membrane are nuclear-encoded and synthesized as precursor proteins in the cytosol. They are targeted to the mitochondria and inserted into their target membrane via various pathways. This review summarizes our current knowledge of the sorting signals for this specific targeting and describes the mechanisms by which the mitochondrial import machineries recognize precursor proteins, mediate their membrane integration and facilitate assembly into functional complexes.     

Two-dimensional separation of erythrocyte membrane proteins.

The details of a two-dimensional separation procedure specially designed for the study of erythrocyte membranes are presented. In this highly reproducible method, the membrane proteins are dissolved in sodium dodecyl sulfate and separated first on the basis of charge by isoelectric focusing. The samples are loaded either at the cathode (CIF) or anode (AIF). The CIF samples gave better separation of the acidic proteins, while the AIF was better for the separation of the high molecular weight polypeptides of the erythrocyte. Over 90 discrete polypeptides could be detected with this method in the pH range of 5 to 8. Special attention was given to the higher molecular weight components. For example, six components could be detected within the 90,000 to 100,000 molecular weight range of protein 3, the major membrane protein. A component with the same or very nearly the same molecular weight as spectrin band 2 was detected. It is more basic than spectrin band 2, and both spectrin band 2 and the basic component are readily phosphorylated in the intact cell. However, the phosphorylation of band 2 is cAMP independent while the phosphorylation of the basic component is enhanced by cAMP. In contrast to spectrin, the basic component is not extracted from the membrane with 0.1 mm EDTA, although dilute NaOH will remove it from the membrane. The Ca²⁺-activated transferase of the erythrocyte cytoplasm will not crosslink this component. Calcium does, however, activate the conversion of this component to a lower molecular weight. This high molecular weight basic component has properties attributed to the component labeled 2.1 in Fairbanks' system of nomenclature.     

Biogenesis of inner membrane proteins in Escherichia coli

The inner membrane proteome of the model organism Escherichia coli is composed of inner membrane proteins, lipoproteins and peripherally attached soluble proteins. Our knowledge of the biogenesis of inner membrane proteins is rapidly increasing. This is in particular true for the early steps of biogenesis - protein targeting to and insertion into the membrane. However, our knowledge of inner membrane protein folding and quality control is still fragmentary. Furthering our knowledge in these areas will bring us closer to understand the biogenesis of individual inner membrane proteins in the context of the biogenesis of the inner membrane proteome of Escherichia coli as a whole. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.     

Biogenesis of inner membrane proteins in Escherichia coli

For a long time, it was generally assumed that the biogenesis of inner membrane proteins in Escherichia coli occurs spontaneously, and that only the translocation of large periplasmic domains requires the aid of a protein machinery, the Sec translocon. However, evidence obtained in recent years indicates that most, if not all, inner membrane proteins require the assistance of protein factors to reach their native conformation in the membrane. Here, we review and discuss recent advances in our understanding of the biogenesis of inner membrane proteins in E. coli.     

Biogenesis of beta-barrel membrane proteins of mitochondria

beta-Barrel membrane proteins have several important functions in outer membranes of Gram-negative bacteria and in the organelles of endosymbiotic origin, mitochondria and chloroplasts. The biogenesis of beta-barrel membrane proteins was, until recently, an unresolved process. A breakthrough was achieved when a specific pathway for the insertion of beta-barrel outer-membrane proteins was identified in both mitochondria and Gram-negative bacteria. The key component of this pathway is Tob55 (also known as Sam50) in mitochondria and Omp85 in bacteria, both beta-barrel membrane proteins themselves. Tob55 is part of the hetero-oligomeric TOB (topogenesis of mitochondrial outer-membrane beta-barrel proteins) or SAM (sorting and assembly of mitochondria) complex, which is present in the mitochondrial outer membrane. Tob55 belongs to an evolutionarily conserved protein family, the members of which are present in almost all eukaryotes and in Gram-negative bacteria and chloroplasts. Thus, is it emphasized that the insertion pathway of mitochondrial beta-barrel membrane proteins was conserved during evolution of mitochondria from endosymbiotic bacterial ancestors.     

Effect of Intralipid-infusion on erythrocyte membrane in rabbits

Through Intralipid infusion in rabbits, the phospholipids derived from Intralipid were incorporated into erythrocytes, although Intralipid is mainly composed of triglycerides. This is supported by the increase in oleic acid and the compensatory decrease in linoleic acid of the phospholipids in the erythrocyte membrane, corresponding to the content of linoleic acid in the phospholipids from Intralipid. The excess phospholipid rendered the membrane more fluid, probably by overwhelming the rigidifying effect of the increased cholesterol content. Furthermore, the shape of erythrocytes was changed from biconcave to spur, dose dependently. The morphological alterations in erythrocyte membranes could not be completely elucidated by the changes in lipid. These results suggested that the alteration in lipid metabolism in Intralipid-infused rabbits caused various effects on the erythrocyte membrane, through the elevation of triglyceride, cholesterol, and phospholipid contents in plasma.     

Aging of the erythrocyte. IV. Spin-label studies of membrane lipids, proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

Aging of the erythrocyte IV. Spin-label studies of membrane lipids,proteins and permeability

Spin-label studies demonstrated age-related alterations of the erythrocyte membrane concerning both lipid and protein components. Decrease in fluidity of membrane lipids correlated with decreased membrane permeability to a hydrophobic spin label TEMPO, permeability to a more hydrophilic TEMPOL being less affected. The rigidification of membrane lipids was much more pronounced in whole membranes than in liposomes composed of membrane lipids, suggesting changes in lipid-protein interactions as an important factor in the decrease of lipid fluidity in aged red cells. ESR spectra of membrane-bound maleimide spin label evidenced alterations in the state of membrane proteins during cell aging in vivo.     

In vivo analysis of integration of membrane proteins in Escherichia coli

The in vivo process of membrane protein integration was studied by pulse-labelling Escherichia coli cells, and assessing integral anchoring of labelled proteins to the lipid bilayer based on their resistance to alkali extraction. To conduct this experiment, conditions for extracting E. coli proteins with alkali were refined, and the immunoprecipitation procedures were improved to allow effective detection of integral membrane proteins. Examination of pulse-labelled, integral membrane proteins, including lactose permease (LacY), SecY, cytochrome omicron subunit II and leader peptidase revealed that all were in the alkali-insoluble fraction, indicating that membrane integration of these proteins takes place rapidly in wild-type cells. However, when LacY was synthesized in excess from a multicopy plasmid, significant proportions were found in the alkali-soluble fraction, indicating that the solubility in alkali is not an intrinsic property of the protein, and suggesting that LacY depends on some limited cellular factor for membrane integration. The unintegrated species of LacY sedimented slowly through an alkaline sucrose gradient. The secY24 mutant cells accumulated higher proportions of unintegrated LacY molecules at lower levels of overproduction than the sec+ cells. LacY overproduction in wild-type cells was found to inhibit processing (export) of beta-lactamase but not of OmpA and OmpF. These results are interpreted to mean that integration of LacY depends on multiple cellular components, one of which is also involved in export of beta-lactamase.     

Resolution of erythrocyte membrane proteins by two-dimensional electrophoresis.

A two-dimensional electrophoresis method has been developed which solubilizes erythrocyte membrane proteins, and which resolves the components of the band that migrates in detergent gels as if its molecular mass were 95,000 daltons. This method uses gel electrophoresis with sodium dodecyl sulfate in the first dimension and phenol, aqueous urea, and acetic acid in the second dimension. The 95,000 dalton band is known to contain several different membrane proteins, including those associated with anion transport, glucose transport, and (Na+,K+) transport. Two-dimensional electrophoresis resolved this band into one major spot and several minor ones. Pronase digestion of whole erythrocytes, followed by preparation of ghosts and two-dimensional electrophoresis, showed that only the major component of this band was digested by pronase.     

Biogenesis of peroxisomes. Topogenesis of the peroxisomal membrane and matrix proteins

Genetic and proteomic approaches have led to the identification of 32 proteins, collectively called peroxins, which are required for the biogenesis of peroxisomes. Some are responsible for the division and inheritance of peroxisomes; however, most peroxins have been implicated in the topogenesis of peroxisomal proteins. Peroxisomal membrane and matrix proteins are synthesized on free ribosomes in the cytosol and are imported post-translationally into pre-existing organelles (Lazarow PB & Fujiki Y (1985) Annu Rev Cell Biol1, 489-530). Progress has been made in the elucidation of how these proteins are targeted to the organelle. In addition, the understanding of the composition of the peroxisomal import apparatus and the order of events taking place during the cascade of peroxisomal protein import has increased significantly. However, our knowledge on the basic principles of peroxisomal membrane protein insertion or translocation of peroxisomal matrix proteins across the peroxisomal membrane is rather limited. The latter is of particular interest as the peroxisomal import machinery accommodates folded, even oligomeric, proteins, which distinguishes this apparatus from the well characterized translocons of other organelles. Furthermore, the origin of the peroxisomal membrane is still enigmatic. Recent observations suggest the existence of two classes of peroxisomal membrane proteins. Newly synthesized class I proteins are directly targeted to and inserted into the peroxisomal membrane, while class II proteins reach their final destination via the endoplasmic reticulum or a subcompartment thereof, which would be in accord with the idea that the peroxisomal membrane might be derived from the endoplasmic reticulum.