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

The presence in the Golgi fraction of glycoproteins destined to be incorporated into the microsomal membrane was investigated. When incubated in sucrose, washed Golgi vesicles released four major, weakly acidic glycoproteins, some of which could be incorporated into microsomal membranes by incubation. Double labeling with [3H]glucosamine and [14C]leucine demonstrated the incorporation of both protein and oligosaccharide moieties, and the main peak of radioactivity was associated with the 70,000 mol wt region after SDS-gel electrophoresis. The proteins that could be incorporated into microsomes were probably associated to a large extent with the outer surface of the Golgi membrane. Centrifugation of the proteins released from the Golgi in a KBr solution (p = 1.24) resulted in a separation of glycoproteins, those in the top layer most actively incorporated into microsomes. The lipoglycoproteins in the top layer that could be incorporated appeared in the 70,000 mol wt region after SDS-gel electrophoresis, as did the corresponding proteins isolated from the supernate. These results suggest that glycoproteins with completed oligosaccharide chains are released from the Golgi system to the cytosol and are subsequently transferred to microsomes as constitutive membrane components.     

Biogenesis of plasma membrane glycoproteins. Purification and properties of two rat liver plasma membrane glycoproteins

As a preliminary to a study of the biogenesis of individual plasma membrane glycoproteins, the marker enzyme nucleotide pyrophosphatase (NPPase) and a major rat liver plasma membrane sialoprotein, subsequently found to be identical with the enzyme dipeptidyl peptidase IV (DPP IV), were purified 10,000- and 2,000-fold, respectively, from rat liver. Both were amphipathic proteins which formed defined micellar complexes with detergents and aggregated in their absence. Gel filtration, sucrose density gradient centrifugation, and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the Triton X-100 complex of NPPase to contain a single 150,000-dalton peptide, while that of DPP IV was composed of two 120,000-dalton subunits; each complex also contained about 150,000-dalton Triton X-100. Trypsin cleaved the detergent complexes with release of major hydrophilic fragments which no longer bound detergent micelles; the accompanying change in peptide size was small for NPPase and undetectable for DPP IV, which also retained the dimer structure of its native form. DPP IV was the only major glycoprotein in rat liver plasma membrane which bound strongly to wheat germ agglutinin. Monospecific rabbit antibodies against NPPase and DPP IV precipitated the antigens without affecting their enzymatic activities.     

Biogenesis of microsomal membrane glycoproteins in rat liver. I. Presence of glycoproteins in microsomes and cytosol

The glycoproteins of microsomes and cytosol were studied. Various washing procedures did not release the proteins from the microsomes, and immunological tests demonstrated that the sialoproteins are not serum components. Low concentrations of deoxycholate and incubation in 0.25 M sucrose solution liberated a small amount of microsomal sialoprotein and this fraction exhibited a high degree of labeling of protein-bound N-acetylneuraminic acid. A part of the glycoprotein fraction could not be solubilized, even with a high concentration of the detergent. Thoroughly perfused rat liver contained sialoproteins in the particle-free supernate. The level of sialoprotein present could not be due to contamination with serum or broken organelles. The high in vivo incorporation of [3H]glucosamine into protein-bound sialic acid of Golgi membranes and cytosol was paralleled by a delayed and lesser rate of incorporation into the rough and smooth microsomal membranes. This incorporation pattern suggests the possibility that the glycoproteins of cytosol and Golgi may later be incorporated into the membrane of the endoplasmic reticulum.     

Biogenesis of plasma membrane glycoproteins. Tracer kinetic study of two rat liver plasma membrane glycoproteins in vivo

Antibodies to purified nucleotide pyrophosphatase (NPPase) and dipeptidyl peptidase IV (DPP IV) were used to study the biogenesis of these rat liver plasma membrane glycoproteins in vivo. Following injection of tritiated leucine, the radioactivity in NPPase and DPP IV decayed at markedly different rates in the plasma membrane, with apparent half-lives of about 1 and 5 days, respectively. In short term experiments, labeling of total plasma membrane proteins was rapid and insensitive to colchicine, while labeling of both NPPase and DPP IV showed a lag of about 15 min, followed by colchcine-sensitive/cycloheximide-insensitive increases to half-maximal and maximal values at about 1 and 2 h, respectively. A peak of labeled DPP IV in rough microsomes at 15 min showed increased mobility on polyacrylamide gels and was largely inaccessible to antibodies in intact microsomes, consistent with its being an underglycosylated precursor, exposed on the cisternal side of the rough endoplasmic reticulum. In contrast, the behavior of unlabeled DPP IV in preparations of rough microsomes and Golgi was consistent with its being contributed by contaminating right-side-out plasma membrane vesicles. This conclusion was also necessary to fit the tracer kinetic data to a simple membrane-flow model, which gave precursor pools (1 microgram/g of liver) and fluxes (1 microgram/h/g of liver) for both DPP IV and NPPase which were about 3 orders of magnitude less than those for the synthesis of rat serum albumin. Thus, unlike hepatoma tissue culture cells (Doyle, D., Baumann, H., England, B., Friedman, E., Hou, E., and Tweto, J. (1978) J. Biol. Chem. 253, 967-973), normal rat liver does not contain large amounts of preformed intracellular plasma membrane precursors.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. I. Localization of lectin-binding sites in microsomal membranes

Carbohydrate-containing structures in rat liver rough microsomes (RM) were localized and characterized using iodinated lectins of defined specificity. Binding of [125I]Con A increased six- to sevenfold in the presence of low DOC (0.04--0.05%) which opens the vesicles and allows the penetration of the lectins. On the other hand, binding of [125I]WGA and [125I]RCA increased only slightly when the microsomal vesicles were opened by DOC. Sites available in the intact microsomal fraction had an affinity for [125I]Con A 14 times higher than sites for lectin binding which were exposed by the detergent treatment. Lectin-binding sites in RM were also localized electron microscopically with lectins covalently bound to biotin, which, in turn, were visualized after their reaction with ferritin-avidin (F-Av) markers. Using this method, it was demonstrated that in untreated RM samples, binding sites for lectins are not present on the cytoplasmic face of the microsomal vesicles, even after removal of ribosomes by treatment with high salt buffer and puromycin, but are located on smooth membranes which contaminate the rough microsomal fraction. Combining this technique with procedures which render the interior of the microsomal vesicles accessible to lectins and remove luminal proteins, it was found that RM membranes contain binding sites for Con A and for Lens culinaris agglutinin (LCA) located exclusively on the cisternal face of the membrane. No sites for WGA, RCA, soybean (SBA) and Lotus tetragonobulus (LTA) agglutinins were detected on either the cytoplasmic or the luminal faces of the rough microsomes. These observations demonstrate that: (a) sugar moieties of microsomal glycoproteins are exposed only on the luminal surface of the membranes and (b) microsomal membrane glycoproteins have incomplete carbohydrate chains without the characteristic terminal trisaccharides N-acetylglucosamine comes from galactose comes from sialic acid or fucose present in most glycoproteins secreted by the liver. The orientation and composition of the carbohydrate chains in microsomal glycoproteins indicate that the passage of these glycoproteins through the Golgi apparatus, followed by their return to the endoplasmic reticulum, is not required for their biogenesis and insertion into the endoplasmic reticulum (ER) membrane.     

Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins

Rat liver microsomal glycoproteins were purified by affinity chromatography on concanavalin A Sepharose columns from membrane and content fractions, separated from rough microsomes (RM) treated with low concentrations of deoxycholate (DOC). All periodic acid-Schiff (PAS)-positive glycoproteins of RM showed affinity for concanavalin A Sepharose; even after sodium dodecyl sulfate (SDS) acrylamide gel electrophoresis, most of the microsomal glycoproteins bound [125I]concanavalin A added to the gels, as detected by autoradiography. Two distinct sets of glycoproteins are present in the membrane and content fractions derived from RM. SDS acrylamide gel electrophoresis showed that RM membranes contain 15--20 glycoproteins (15--22% of the total microsomal protein) which range in apparent mol wt from 23,000 to 240,000 daltons. A smaller set of glycoproteins (five to seven polypeptides), with apparent mol wt between 60,000 and 200,000 daltons, was present in the microsomal content fraction. The disposition of the membrane glycoproteins with respect to the membrane plane was determined by selective iodination with the lactoperoxidase (LPO) technique. Intact RM were labeled on their outer face with 131I and, after opening of the vesicles with 0.05% DOC, in both faces with 125I. An analysis of iodination ratios for individual proteins separated electrophoretically showed that in most membrane glycoproteins, tyrosine residues are predominantly exposed on the luminal face of the vesicles, which is the same face on which the carbohydrate moieties are exposed. Several membrane glycoproteins are also exposed on the cytoplasmic surface and therefore have a transmembrane disposition. In this study, ribophorins I and II, two integral membrane proteins (mol wt 65,000 and 63,000) characteristic of RM, were found to be transmembrane glycoproteins. It is suggested that the transmembrane disposition of the ribophorins may be related to their possible role in ribosome binding and in the vectorial transfer of nascent polypeptides into the microsomal lumen.     

Purification and characterization of rat liver microsomal beta-glucuronidase.

Beta-Glucuronidase (EC 3.2.1.31) has been isolated from rat-liver microsomes by a novel chromatographic method employing antibody to rat preputial gland beta-glucuronidase coupled to Sepharose. The purified enzyme, homogeneous by several methods, was purified some 1700-fold. The microsomal beta-glucuronidase has been characterized with respect to catalysis, stability, and molecular weight. The purified enzyme is a tetramer of 290 000 daltons. Comparative studies with lysosomal beta-glucuronidase indicate that while these two enzymes are electrophoretically distinct, they are catalytically and immunologically identical and have indistinguishable molecular dimensions. The results suggest that microsomal and lysosomal beta-glucuronidase are charge isomers.     

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.     

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.     

Incorporation of galactose from UDP-galactose into microsomal and golgi membranes of rat liver

Rough and smooth microsomes and Golgi membranes were incubated with UDP[¹⁴C]galactose and the incorporation of radioactivity into the lipid extract and into endogenous protein acceptors were measured. Antagonistic pyrophosphatases were inhibited with ATP and interference from β-galactosidase activity was greatly decreased by carrying out the incubation at pH 7.8. After incubation the particles were centrifuged to remove free oligosaccharide residues. Radioactivity was found in the lipid extract from Golgi membranes but not from rough and smooth microsomes. This radioactivity, however, was not associated with dolichol or retinyl phosphates. The incorporation of radioactivity into proteins of the Golgi fraction was more than double than that of the microsomal fractions. In addition, the transferases in these two types of particles exhibited different properties. Trypsin treatment of intact rough microsomal vesicles, smooth vesicles and Golgi membranes removed about 5, 15 and 50%, respectively, of newly incorporated protein-bound galactose, indicating that the proportion of the newly galactosylated proteins, which are localized at the cytoplasmic surface of the membrane, is lowest in rough microsomes, intermediate in smooth, and highest in Golgi membranes.     

Purification of phospholipid methyltransferase from rat liver microsomal fraction.

Phospholipid methyltransferase, the enzyme that converts phosphatidylethanolamine into phosphatidylcholine with S-adenosyl-L-methionine as the methyl donor, was purified to apparent homogeneity from rat liver microsomal fraction. When analysed by SDS/polyacrylamide-gel electrophoresis only one protein, with molecular mass about 50 kDa, is detected. This protein could be phosphorylated at a single site by incubation with [alpha-32P]ATP and the catalytic subunit of cyclic AMP-dependent protein kinase. A less-purified preparation of the enzyme is mainly composed of two proteins, with molecular masses about 50 kDa and 25 kDa, the 50 kDa form being phosphorylated at the same site as the homogeneous enzyme. After purification of both proteins by electro-elution, the 25 kDa protein forms a dimer and migrates on SDS/polyacrylamide-gel electrophoresis with molecular mass about 50 kDa. Peptide maps of purified 25 kDa and 50 kDa proteins are identical, indicating that both proteins are formed by the same polypeptide chain(s). It is concluded that rat liver phospholipid methyltransferase can exist in two forms, as a monomer of 25 kDa and as a dimer of 50 kDa. The dimer can be phosphorylated by cyclic AMP-dependent protein kinase.     

N-Glycosylation of membrane glycoproteins in retinol-deficient rat liver

The effect of vitamin A deficiency onN-linked oligosaccharides of membrane glycoproteins was studied in rat liver in order to evaluate the suggested role of retinol in proteinN-glycosylation. First, oligosaccharides of newly synthesized glycoproteins from rough endoplasmic reticulum of vitamin A deficient liver were compared with that of pair-fed controls. Oligosaccharides were metabolically labelled withd-[2-³H]mannose, released from the glycoproteins with endoglycosidase H, purified by reversed phase HPLC and ion exchange chromatography, and were reduced with sodium borohydride. HPLC fractionation of the oligosaccharide alditols showed that the glycoproteins carried mainly four oligosaccharide species, Glc₁Man₉GlcNAc₂, Man₉GlcNAc₂, Man₈GlcNAc₂ and Man₇GlcNAc₂, in identical relative amounts in the vitamin A deficient and the control tissue. In particular, no increase in the proportion of short chain oligosaccharides was noted in vitamin A deficient liver. Second, the number ofN-linked oligosaccharides was estimated in dipeptidylpeptidase IV (DPP IV), a major glycoprotein constituent of the hepatic plasma membrane, comparing the newly synthesized glycoprotein from rough endoplasmic reticulum and the mature form of DPP IV from the plasma membrane. No evidence was obtained that retinol deficiency caused incomplete glycosylation of this membrane glycoprotein. From these data, the suggested role of retinol as a cofactor involved in the synthesis ofN-linked oligosaccharides of glycoproteins must be questioned.Abbreviations DolP Dolichyl phosphate - DolPP dolichyl pyrophosphoryl - RetPMan retinyl phosphate mannose - DPP IV dipeptidyl peptidase IV (EC 3.4.14.5) - endo H endo--N-acetylglucosaminidase H (EC 3.2.1.96) - endo F endo--N-acetylglucosaminidase F (EC 3.2.1.96) - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis