Golgi-enriched Fractions Research Articles

Translocation of ricin A-chain into proteoliposomes reconstituted from Golgi and endoplasmic reticulum.

Translocation to the cytosol is an essential and rate-limiting step in the cytotoxicity of the potent plant toxin ricin. In an attempt to study the mechanism of ricin A-chain translocation in a cell-free assay, we have partially purified Golgi and endoplasmic reticulum from Jurkat cells by discontinuous sucrose gradient fractionation. The membranes of the organelle fractions were solubilized by the addition of sodium cholate and reconstituted into proteoliposomes by dialyzing out the detergent. The resulting vesicles supported cell-free translocation of RTA (as assessed by an enzyme protection assay) at a rate which was linearly dependent on the concentration of the vesicle preparation. Ricin B-chain (RTB) neither translocated into the vesicles, nor increased the efficiency of RTA translocation. Liposomes prepared from purified phospholipids were not capable of supporting RTA translocation. Furthermore, protease treatment of concanavalin A adsorption of proteins from lysates prior to vesicle reconstitution resulted in abrogation of the translocation process, suggesting that the protein components of organelle membranes are required for RTA translocation. Reconstitution of translocation-competent proteoliposomes from detergent-solubilized membranes of endoplasmic reticulum- and Golgi-enriched fractions provides a convenient cell-free system to study the mechanism of RTA translocation.

Retinal GlcNAc-transferases and the Glycosylation of Rhodopsin

The major oligosaccharide chains of bovine, frog and human rhodopsins are identical in structure. These molecules are unique in terms of their abridged size as compared to other asparagine-linked glycoproteins, a property which must be reflected in the activities and properties of the glycosyltransferases of the retina that are concerned with their biosynthesis. One of the unusual characteristics of the major oligosaccharide chain is its lack of branching. A key enzymatic step required for branching to occur would be the transfer of a residue of N-acetylglucosamine to the terminal unsubstituted mannose residue of the major rhodopsin oligosaccharide. We have investigated the kinetic properties of enzymes that would catalyse this process. The N-acetylglucosaminyl-transferases (GlcNAc-transferases) of Golgi-enriched fractions of bovine retina, human retina, rat liver Golgi and a partially purified GlcNAc-transferase II from rat liver were examined using as acceptors rhodopsin, opsin and the oligosaccharide isolated from rhodopsin. The identification of the substrates and products of the reactions was carried out by chromatographic means. From an evaluation of the Vmax /Km ratios it was observed that bovine and human retinas have very limited abilities compared to the rat liver enzymes to carry out the transfer of GlcNAc to these acceptors, showing from 100- to about 800-fold lower efficiency in this regard. A comparison of the activities obtained with intact rhodopsin or opsin and the rhodopsin oligosaccharide indicated that the activity of GlcNAc-transferase II was not appreciably influenced by the polypeptide portion of the molecule. It was also observed that prior galactosylation of rhodopsin blocked the addition of GlcNAc to rhodopsin. It is suggested that these properties contribute to the assembly of the abridged structures that have been observed in the oligosaccharides of rhodopsins of all species thus far examined.

Association of kinesin with the Golgi apparatus in rat hepatocytes.

The Golgi apparatus is a dynamic membranous structure, which has been observed to alter its location and morphology during the cell cycle and after microtubule disruption. These dynamics are believed to be supported by a close structural interaction of the Golgi with the microtubule cytoskeleton and associated motor enzymes. One microtubule-dependent motor enzyme, kinesin, has been implicated in Golgi movement and function although direct evidence supporting this interaction is lacking. In this study, we utilized two well-characterized kinesin antibodies in conjunction with subcellular fractionation techniques, immunoblot analysis and immunofluorescence microscopy to conduct a detailed study on the association of kinesin with the Golgi and other membranous organelles in a polarized epithelial cell, the primary rat hepatocyte. We found that kinesin represents approximately 0.3% of total protein in rat liver homogenates, with approximately 30% membrane-associated and the remainder in the cytosol. Among membrane fractions, kinesin was concentrated markedly in Golgi-enriched fractions, which were prepared using two independent techniques. Kinesin was also abundant in fractions enriched in transcytotic carriers and secretory vesicles, with lower levels detected on fractions enriched in endosomes, endoplasmic reticulum, lysosomes and mitochondria. Immunofluorescence microscopy showed that kinesin is concentrated on Golgi-like structures in both primary cultured hepatocytes and rat hepatocyte-derived clone 9 cells. Double-label immunofluorescence demonstrated that kinesin staining colocalizes with the Golgi marker, alpha-mannosidase II, in both cell types. These results provide compelling evidence showing that kinesin is associated with the Golgi complex in cells and implicate this motor enzyme in Golgi structure, function and dynamics.

3‘-Azido-3‘-deoxythymidine potently inhibits protein glycosylation. A novel mechanism for AZT cytotoxicity.

3'-Azido-3'-deoxythymidine (AZT) is one of the primary chemotherapeutic agents used in the treatment of human immunodeficiency virus (HIV) infection. Unfortunately, AZT therapy is accompanied by severe side effects. Using Golgi-enriched membrane fractions, we have determined that 3'-azido-3'-deoxythymidine monophosphate, the primary AZT metabolite in treated cells, potently inhibits protein glycosylation. This inhibition results from direct competition with several pyrimidine-sugars for transport into Golgi membranes. This potential mechanism of cytotoxicity does not involve 3'-azido-3'-deoxythymidine triphosphate, the AZT metabolite most likely responsible for its antiviral effects; thus, it may be possible to develop novel therapeutic strategies that prevent inhibition of glycosylation without affecting the anti-HIV properties of AZT.

Kinetic and spatial interrelationships between ganglioside glycosyltransferases and O-acetyltransferase(s) in human melanoma cells

The melanoma-associated disialogangliosides 9(7)-O-acetyl-GD3 and 9(7)-O-acetyl-GD2 have been structurally well characterized. However, the compartmentalization and sequence of action of the biosynthetic activities responsible for synthesizing these molecules remain obscure. Here, we have studied the spatial and temporal interrelationships among the activities responsible for the later stages of ganglioside biosynthesis and those for O-acetylation in cultured human melanoma cells. First, brefeldin A treatment was used to separate biosynthetic steps into compartments distal or proximal to the transport block imposed by the drug. In keeping with prior reports, GM2/GD2 synthase was consistently rendered inaccessible to its acceptors GM3 and GD3. In contrast, the effect on GD3 biosynthesis was cell line-specific. Synthesis of GD3 was nearly abrogated in two lines, while it accumulated in a third line. This indicates that the spatial organization of ganglioside processing activities can vary even between similar cell lines. However, in all cell lines studied, the ratio of 9(7)-O-acetyl-GD3 to GD3 was not changed by brefeldin A, indicating that the majority of ganglioside O-acetyltransferase activity is co-localized with GD3 biosynthetic activity in the same Golgi subcompartment(s). As an alternative approach, Golgi-enriched fractions from melanoma cells were incubated with radiolabeled and nonlabeled nucleotide sugars or acetyl-CoA. In these preparations, biosynthesis is dependent upon the co-localization of appropriate sugar nucleotide transporters, glycosyltransferases, and acceptors that are endogenously present within intact topologically correct compartments. Incubations with CMP-Neu5Ac and acetyl-CoA corroborated the results with brefeldin A, co-localizing ganglioside O-acetyltransferase activity in compartments where GD3 biosynthesis takes place. Analyses with CMP-Neu5Ac and UDP-GalNAc showed that GD2 and GD3 synthesis occur in partially overlapping compartments. Labeling with acetyl-CoA and UDP-GalNAc indicated that although labeled acetate can be transferred from acetyl-CoA directly to GD2, ganglioside O-acetyltransferase activity does not substantially overlap with the biosynthetic compartment(s) for GD2. Instead, O-acetyl-GD3 appears to be co-localized with the compartment of GD2 biosynthesis and serves as an acceptor for GD2 synthase. Thus, both 9-O-acetyl-GD3 and GD2 can be precursors of 9-O-acetyl-GD2, but apparently in distinct compartments.

Inhibition by brefeldin A of a Golgi membrane enzyme that catalyses exchange of guanine nucleotide bound to ARF.

A wide variety of membrane transformations important in intracellular transport are inhibited by the fungal metabolite brefeldin A (refs 1-4), implying that the target for this drug is central to the formation and maintenance of subcellular compartments. Brefeldin A added to cells causes the rapid and reversible dissociation of a Golgi-associated peripheral membrane protein (M(r) 110,000) which was found to be identical to one of the subunits of the coat of Golgi-derived (non-clathrin) coated vesicles, beta-COP, implying that brefeldin A prevents transport by blocking the assembly of coats and thus the budding of enclosed vesicles. In addition to the coatomer (a cytosol-derived complex of seven polypeptide chains, one of which is beta-COP), the non-clathrin (COP) coat of Golgi-derived vesicles contains stoichiometric amounts of a small (M(r) approximately 20,000) GTP-binding protein, the ADP-ribosylation factor (ARF). Binding of ARF to Golgi membranes is necessary before coatomer/beta-COP can bind these membranes (ref. 12; and D. J. Palmer et al., manuscript submitted), so the primary effect of brefeldin A seems to be on the reaction responsible for ARF binding. Indeed, like beta-COP, ARF is dissociated from the Golgi complex by treatment with brefeldin A and brefeldin A prevents ARF from associating in vitro, but the mechanism of this action by brefeldin A has been unclear. Here we report the discovery of an enzyme in a Golgi-enriched fraction that catalyses guanine nucleotide (GDP-GTP) exchange on ARF-1 protein, and which is inhibited by brefeldin A. We suggest that activation of ARF proteins for membrane localization by compartmentalized exchange enzymes is in general the first committed step in membrane transformation pathways.

Subcellular distribution and activity of glucose transporter isoforms GLUT1 and GLUT4 transiently expressed in COS-7 cells

In adipose and muscle cells, the glucose transporter isoform GLUT4 is mainly located in an intracellular, vesicular compartment from which it is translocated to the plasma membrane in response to insulin. In order to test the hypothesis that this preferential targeting of a glucose transporter to an intracellular storage site is conferred only by its primary sequence, we compared the subcellular distribution of the fat/muscle glucose transporter GLUT4 with that of the erythrocyte/brain-type glucose transporter GLUT1 after transient expression in COS-7 cells. Full-length cDNA was ligated into the expression vector pCMV that is driven by the cytomegalovirus promoter, and introduced into COS cells by the DEAE-dextran method. Cells were homogenized and fractionated by differential centrifugation to yield plasma membranes and a Golgi-enriched fraction of intracellular membranes (low-density microsomes). In these membrane fractions, the abundance of glucose transporters was assessed by immunoblotting with specific antibodies against GLUT1 and GLUT4, and their transport activity was assayed after solubilization and reconstitution into lecithin liposomes. Uptake rates of 2-deoxyglucose assayed in parallel samples were higher in cells expressing GLUT1 or GLUT4 as compared with control cells (transfection of pCMV without transporter cDNA). Reconstituted glucose transport activity in plasma membranes was about 5-fold higher after expression of GLUT1 and GLUT4 as compared with control cells. The relative amount of GLUT4 in the low-density microsomes as detected by reconstitution and immunoblotting exceeded that of the GLUT1, but was much lower than that observed in typical insulin-sensitive cells, e.g., rat fat cells or 3T3-L1 adipocytes. These data indicate that COS-7 cells transfected with glucose transporter cDNA express the active transport proteins and can be used for functional studies.

Characterization of GTP-binding proteins in Golgi-associated membrane vesicles from rat adipocytes.

We have previously reported that guanine nucleotides inhibit glucose transport activity reconstituted from adipocyte membrane fractions. In order to further investigate the hypothetical involvement of guanine-nucleotide-binding proteins (GTP-binding proteins) in the regulation of insulin-sensitive glucose transport activity, we studied their subcellular distribution in adipocytes treated or not with insulin. Adipocytes were homogenized and fractionated to yield plasma membranes (PM) and a Golgi-enriched fraction of intracellular membranes (low-density microsomes, LDM). In these membrane fractions, total guanosine 5'-[gamma-[35S]thio]triphosphate ([35S]GTP[S]) binding, alpha- and beta-subunits of heterotrimeric G-proteins, proto-oncogenes Ha-ras and K-ras, and 23-28 kDa GTP-binding proteins were assayed. The levels of alpha s and alpha i (the alpha-subunits of Gs and Gi) were approx. 8-fold lower in LDM than in PM; beta-subunits, Ha-ras and K-ras were not detectable in LDM. Total GTP[S]-binding sites and 23-28 kDa GTP-binding proteins were present in LDM in approximately the same concentrations as in PM. Insulin gave rise to the characteristic translocation of glucose transporters, but failed to alter the subcellular distribution of any of the GTP-binding proteins. Fractionation of the LDM on a discontinuous sucrose gradient revealed that alpha s and alpha i, as detected with antiserum against a common peptide sequence (alpha common), and the bulk of the 23-28 kDa G-proteins sedimented at different sucrose densities. None of the GTP-binding proteins co-sedimented with glucose transporters. Furthermore, the inhibitory effect of GTP[S] on the reconstituted transport activity was lost in the peak fractions of glucose transporters partially purified on the sucrose gradient. These data indicate that LDM from adipocytes contain several GTP-binding proteins in discrete vesicle populations. However, the intracellular GTP-binding proteins are not tightly associated with the vesicles containing the glucose transporter.

Tyrosylprotein sulfotransferase in rat submandibular salivary glands

1. 1. The transfer of sulfate ester group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to poly-(Glu 6, Ala 3, Tyr 1 ) (EAY; M r 47 kDa) in rat submandibular salivary gland has been investigated. The highest tyrosylprotein sulfotransferase activity was obtained in the Golgi-enriched fraction in the presence of 2mM 5'AMP, 20 mM MnCl 2 and 50 mM NaF at pH 6.2. 2. 2. The apparent K m values for EAY and PAPS were 1.6 × 10 −6 and 1.9 × 10 −6M, respectively. 3. 3. Inclusion of NaCl, EDTA, NEM and DTT was inhibitory for the enzyme activity. The enzyme was 28 times less susceptible to 2,6-dichloro-4-nitrophenol inhibition than to phenol sulfotransferase inhibition. 4. 4. This study is the first report characterizing a sulfotransferase activity specific for tyrosylprotein in rat submandibular salivary glands.

Transient hyperglycosylation of rhodopsin with galactose

Rhodopsin's oligosaccharide chains contain predominantly two types of sugar residues: mannose and N-acetylglucosamine. In the present work, bovine and rat rhodopsin were analysed biochemically for the presence of a third sugar, galactose. Treatment of bovine rod outer segments (ROS) with galactose oxidase followed by reduction with tritium-labeled sodium borohydride revealed the presence of existing molecules of galactose on rhodopsin. Rats injected intravitreally with [ 3H]galactose and [ 14C]leucine and maintained in darkness were killed 1 hr, 6 hr, 1, 3, or 5 days following the injection. Retinas were collected for subcellular fractionation and rhodopsin from each of the fractions was purified by ConA sepharose chromatography and SDS-PAGE. During the first 6 hr, galactose selectively labeled rhodopsin in the Golgi-enriched fraction resulting in increased [ 3H] [ 14C] ratios in both Golgi and ROS. The data suggested that trimming was occurring at the transition from Golgi to ROS. Furthermore, a decrease in isotope ratio in the ROS between 6 hr and 1 day suggested further trimming of rhodopsin after membrane assembly in the ROS. Additional in vivo experiments demonstrated existing molecules of galactose on rhodopsin's oligosaccharide chain using lectin affinity chromatography. Rats injected intravitreally with [ 35S]methionine were dark-adapted for 2 hr. Following subcellular fractionation of retinas, ConA purified rhodopsin from ROS was applied to one of two additional lectin columns: Ricinus communis agglutinin (RCA) or Griffonia simplicifolia I (GSA). Eight to nine per cent of the labeled rhodopsin was bound to and eluted from RCA, whereas none bound to GSA, indicating the presence of a β-galactoside. The RCA agarose eluted protein co-electrophoresed with a rhodopsin standard and was light sensitive. Galactose was shown to be the terminal sugar on this subset of rhodopsin and was not capped by neuraminic acid. Binding of rhodopsin's oligosaccharide to RCA was abolished by pre-treatment with β-galactosidase. Decreased binding of rhodopsin to RCA was observed following intravitreal injection of castanospermine but not swainsonine. Of those two inhibitors of glycoprotein trimming, only castanospermine would be expected to prevent the addition of galactose to the oligosaccharide. The association of galactose with rat rhodopsin appeared to be a transient one. At 2 hr, 8–9% of rhodopsin contained galactose, at 6 hr only 2·2% had galactose and by 24 hr less than 1% did. The galactose was trimmed from rhodopsin's oligosaccharide presumably after its role was complete. Separation of rhodopsin of the plasma membranes from rhodopsin of discs indicated that 75% of the galactose-containing rhodopsin was in the plasma membrane and only 25% was in the discs. These findings suggested a possible role for galactose in new disc formation with subsequent removal after the discs are sealed.

Novel purification of the catalytic domain of Golgi alpha-mannosidase II. Characterization and comparison with the intact enzyme.

Rat liver alpha-mannosidase II, a hydrolase involved in the processing of asparagine-linked oligosaccharides, is an integral membrane glycoprotein facing the lumen of Golgi membranes. We have previously shown (Moremen, K. W., and Touster, O. (1986) J. Biol. Chem. 261, 10945-10951) that mild chymotrypsin digestion of permeabilized or solubilized Golgi membranes will result in the cleavage of the intact 124,000-dalton alpha-mannosidase II subunit, releasing a 110,000-dalton hydrophilic polypeptide which contains the catalytic site. Consistent with the removal of a membrane binding domain, the chymotrypsin-generated 110,000-dalton peptide was found exclusively in the aqueous phase in Triton X-114 phase separation studies, whereas the intact enzyme was found in the detergent phase. Taking advantage of this conversion in phase partitioning behavior, a purification procedure was developed to isolate the 110,000-dalton proteolytic digestion product as a homogeneous polypeptide for further characterization and protein sequencing at a yield of greater than 65% from a rat liver Golgi-enriched membrane fraction. An improved purification procedure for the intact enzyme was also developed. The two forms of the enzyme were compared yielding the following results. (a) The catalytic activity of the intact and cleaved forms of alpha-mannosidase II were indistinguishable in Km, Vmax, inhibition by the alkaloid, swainsonine, and in their activity toward the natural substrate GlcNAc-Man5GlcNAc. (b) Both the intact and cleaved forms of the enzyme appear to be disulfide-linked dimers. (c) The two forms of the enzyme contain different NH2-terminal sequences suggesting that the cleaved NH2 terminus contains the membrane-spanning domain. (d) Additional peptide sequences were obtained from proteolytic fragments and cyanogen bromide digestion products in order to create a partial protein sequence map of the enzyme. These results are consistent with a model common among Golgi processing enzymes of a hydrophilic catalytic domain anchored to the lumenal face of Golgi membranes through an NH2-terminal hydrophobic membrane-anchoring domain.

Beta-VLDL increases endothelial cell plasma membrane cholesterol.

In this study, the distribution of free cholesterol in cholesterol-loaded endothelial cells was examined. For these studies, cell fractionation methods were used to assess marker enzyme activity and cholesterol distribution. Treatment of rabbit aortic endothelial cells for 3 days with 50 micrograms/ml of beta-very low density lipoprotein (beta-VLDL) or malondialdehyde-low density lipoprotein (MDA-LDL) but not LDL caused a 50-100% increase in total cell unesterified cholesterol. The accumulation of free rather than esterified cholesterol in endothelial cells may be due to the ratio of hydrolysis to esterification, which we have shown in this study to be 10-fold higher in endothelial cells than in smooth muscle cells. This free cholesterol is found in the fractions enriched in plasma membrane markers and, to a lesser extent, in the Golgi-enriched fractions. The amount of cholesterol per mg of protein was increased approximately 50% in these fractions from cells treated for 3 days with 50 micrograms/ml of beta-VLDL. These increases in cholesterol content were reversible upon incubation of cells for 3 days in medium containing 15% fetal bovine serum. Alterations in several membrane functions were also observed in cholesterol-loaded cells. The activity of alkaline phosphatase, an enzyme marker for plasma membranes, was decreased by 25% and an alteration in membrane-associated microfilaments was seen with phalloidin staining. This morphological change in microfilaments was reflected in a decrease in filament ends as shown by cytochalasin binding and occurred without a change in total actin or vinculin. These microfilament changes were reversible.(ABSTRACT TRUNCATED AT 250 WORDS)

Golgi-enriched membrane fractions from rat brain and liver contain long-chain polyisoprenyl pyrophosphate phosphatase activity.

The subcellular distribution of polyisoprenyl pyrophosphate phosphatase activity has been examined in rat brain by assaying the release of 32Pi from [beta-32P]dolichyl pyrophosphate (Dol-P-P) as described previously (Scher,M.G. and Waechter, C.J. (1984) J. Biol. Chem., 259, 14580-14585). The highest specific activities of Dol-P-P phosphatase in rat brain were found in the Golgi-enriched light microsomal, synaptic plasma membrane and heavy microsomal fractions. A comparative analysis of the distribution of galactosyltransferase and dolichol kinase reveals that Dol-P-P phosphatase activity co-fractionates with galactosyltransferase activity, and that the high level found in the Golgi-enriched fraction is not due to cross-contamination with heavy microsomes. When beta-labelled C95 Dol-P-P and the C95 allylic polyisoprenyl pyrophosphate (Poly-P-P) were compared as substrates for the Golgi-enriched light microsomal and heavy microsomal fractions, similar Km values were calculated for the two pyrophosphorylated substrates for each membrane fraction. Based on these kinetic analyses, the enzyme(s) catalysing this reaction do not distinguish between substrates containing saturated or allylic alpha-isoprene units. When Dol-P-P phosphatase activity was assessed in submicrosomal fractions obtained from rat liver by two separate procedures, the highest specific activity was also detected in the Golgi-enriched fraction. While the specific activities for Dol-P-P phosphatase and sialyltransferase were in the relative order of Golgi greater than smooth endoplasmic reticulum (ER) greater than rough ER, the relative order of dolichol kinase was rough ER greater than smooth ER greater than Golgi.(ABSTRACT TRUNCATED AT 250 WORDS)

Prosomatostatin is processed in the Golgi apparatus of rat neural cells

Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.

Pretranslational and posttranslational regulation of the EGF receptor during the prereplicative phase of liver regeneration

We studied the regulation of the epidermal growth factor receptor mRNA and the number of epidermal growth factor binding sites in subcellular compartments involved in the biosynthesis and endocytosis of the epidermal growth factor receptor during the prereplicative phase of liver regeneration. The epidermal growth factor receptor mRNA, quantified by solution hybridization, decreased after partial hepatectomy, with a nadir of about 35% 18 hr after hepatectomy. An even stronger decrease in the number of epidermal growth factor binding sites after partial hepatectomy was observed in a Golgi-enriched low-density membrane fraction, reflecting available newly synthesized epidermal growth factor receptors. It is suggested that this decrease in newly synthesized available epidermal growth factor receptors is caused primarily, but not entirely, by decreased epidermal growth factor receptor mRNA levels and the additional down-regulation of epidermal growth factor binding sites may involve posttranslational mechanisms such as intracellular occupation by transforming growth factor-alpha. The observation that the number of specific epidermal growth factor binding sites after partial hepatectomy was only moderately reduced in prelysosomal endosomes and in lysosomes, compared with the newly synthesized receptors, may indicate that a pool of receptors targeted for lysosomes exists and these receptors are regulated in a different manner than the receptor pool targeted for the cell surface. Furthermore, at least two separable endocytic subcompartments are involved in the transport of the epidermal growth factor/epidermal growth factor receptor complex in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)

Sphingomyelin is synthesized in the cis Golgi

We have employed in vitro a truncated ceramide analogue with 8 carbon atoms in the sphingosine and the fatty acyl residue, each, to investigate the activity of various membrane fractions to synthesize truncated sphingomyelin. This shortened ceramide readily diffuses through membranes and therefore can easily find access to the lumina of intact organelles. Sphingomyelin synthase activity resides in the Golgi apparatus, and after sucrose density gradient centrifugation of Golgi-enriched fractions sphingomyelin synthesis follows a cis Golgi marker enzyme.

Subcellular distribution of small GTP binding proteins in pancreas: identification of small GTP binding proteins in the rough endoplasmic reticulum.

Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[gamma-35S]) was assayed in each fraction. Enrichment of GTP[gamma-35S] binding was greatest in the interfacial "smooth" microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a [alpha-32P]GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP[gamma-35S] binding, as well as the small GTP binding proteins detected by the [alpha-32P]GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMs were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP[gamma-35S] binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.

Synthetic fragments of beta-casein as model substrates for liver and mammary gland casein kinases.

The octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu, corresponding to the 14-21 sequence of bovine beta-casein A2 and 11 shorter and/or modified derivatives were synthesized and used as model substrates for three casein kinases: rat liver casein kinases 2 and 1 and a casein kinase isolated from the golgi-enriched fraction of lactating mammary gland (GEF-casein kinase). Casein kinase-2 readily phosphorylates the octapeptide at its Ser-4 residue with a Vmax value comparable to those obtained with protein substrates and Km values of 85 microM and 11 microM in the absence and presence of polylysine, respectively. These are the most favourable kinetic parameters reported so far with peptide substrates of casein kinase-2. Stepwise shortening of the octapeptide from its N terminus promotes both a gradual decrease of Vmax and an increase of Km, this being especially dramatic in passing from the hexapeptide Leu-Ser-Ser-Ser-Glu-Glu (Km 210 microM) to the pentapeptide Ser-Ser-Ser-Glu-Glu (Km 2630 microM). The tetrapeptide Ser-Ser-Glu-Glu is the shortest derivative still phosphorylated by casein kinase-2, albeit very slowly, and the tripeptides Ser-Glu-Glu and Glu-Leu-Ser were not substrates at all. Furthermore, the pentapeptide Ser-Ser-Ser-Glu-Glu was found to be a better substrate than Ser-Ser-Ala-Glu-Glu, Ser-Ala-Ser-Glu-Glu and Ser-Ala-Ala-Glu-Glu by virtue of its lower Km value. These data, while confirming that the motif Ser-Xaa-Xaa-Glu is specifically recognized by casein kinase-2, strongly suggest that additional local structural features can improve the phosphorylation efficiency of serine-containing peptides which are devoid of the large acidic clusters recurrent in many phosphorylation sites of casein kinase 2. In particular, predictive structural analysis as well as NMR and C18 reverse-phase HPLC elution profile data support the hypothesis that a beta-turn conformation is responsible for the remarkable suitability of the octapeptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu and some of its shorter derivatives to phosphorylation mediated by casein kinase-2. While neither the peptide Glu-Ser-Leu-Ser-Ser-Ser-Glu-Glu nor any of its derivatives were affected by casein kinase-1, a rapid phosphorylation of the octapeptide by GEF-casein kinase at Ser-5 (not Ser-4) was obtained.(ABSTRACT TRUNCATED AT 400 WORDS)

Selective effect of nerve growth factor on some Golgi and lysosomal enzyme activities of rat pheochromocytoma (PC12) cells

Treatment with neuronal growth factor (NGF) results in the growth of neuronal processes by PC12 cells and a concomitant 70% increase in the area of the Golgi apparatus. To define the observed morphologic changes in biochemical terms, we investigated the effect of NGF treatment on some Golgi and lysosomal enzyme activities of PC12 cells. Enzyme activities characteristic of the Golgi apparatus, lysosomes, plasma membranes, mitochondria, and endoplasmic reticulum were measured in cell homogenates, in post-mitochondrial supernatants, and in Golgi-enriched fractions from control and from NGF-stimulated PC12 cells. Treatment of PC12 cells with NGF did not change the level of the Golgi activity of UDPGal:GlcNAc galactosyltransferase while that of CMP-sialic acid:lactosylceramide sialyltransferase was increased three- to fivefold in all fractions studied. For lysosomal enzymes, NGF treatment resulted in a two- to threefold higher level of arylsulfatase activity compared to either acid phosphatase or acid α-mannosidase activities. These results indicate that there is a selective increase of at least one Golgi and one lysosomal activity as a result of NGF stimulation of PC12 cells. Both of these enzymes are involved in glycolipid metabolism. It is possible that the dramatic morphologic changes observed during NGF-induced differentiation of PC12 cells are associated not only with increased synthesis in the Golgi apparatus of plasma membrane components such as gangliosides, but also with increased degradation in lysosomes of other plasma membrane components such as sulfatide.

Chapter 14 Lectin—Colloidal Gold-Induced Density Perturbation of Membranes: Application to Affinity Elimination of the Plasma Membrane

Publisher Summary In an effort to document the composition of the membranes of the Golgi complex and compare and contrast this organelle with the rough endoplasmic reticulum, this chapter describes a subcellular-fractionation procedure for rat myeloma cells that effectively eliminates rough microsomes from Golgi-enriched fractions. The differential and isopycnic-sedimentation protocol yields Golgi-enriched fractions of considerable purity. The chapter develops a novel density perturbation procedure to eliminate these plasma membrane contaminants. This protocol makes use of the specific interaction of the plant lectin, wheat germ agglutinin (WGA), with carbohydrates on the surface of intact cells. This lectin is known to bind oligosaccharides with nonreducing terminal D-GlcNAc and clustered sialic acid residues. The binding of the lectin conjugate to the cell surface makes it possible to achieve, during subcellular fractionation, extensive sedimentation of alkaline phosphatase activity, the lectin conjugate itself, and iodinated cell surface proteins to the mitochondrial pellet.