Role of Sec61alpha in the regulated transfer of the ribosome-nascent chain complex from the signal recognition particle to the translocation channel.

Targeting of ribosome-nascent chain complexes to the translocon in the endoplasmic reticulum is mediated by the concerted action of the signal recognition particle (SRP) and the SRP receptor (SR). Ribosome-stripped microsomes were digested with proteases to sever cytoplasmic domains of SRalpha, SRbeta, TRAM, and the Sec61 complex. We characterized protein translocation intermediates that accumulate when Sec61alpha or SRbeta is inactivated by proteolysis. In the absence of a functional Sec61 complex, dissociation of SRP54 from the signal sequence is blocked. Experiments using SR proteoliposomes confirmed the assembly of a membrane-bound posttargeting intermediate. These results strongly suggest that the Sec61 complex regulates the GTP hydrolysis cycle of the SRP-SR complex at the stage of signal sequence dissociation from SRP54.

Identification, purification, and characterization of the rat liver golgi membrane ATP transporter.

Phosphorylation of secretory and integral membrane proteins and of proteoglycans also occurs in the lumen of the Golgi apparatus. ATP, the phosphate donor in these reactions, must first cross the Golgi membrane before it can serve as substrate. The existence of a specific ATP transporter in the Golgi membrane has been previously demonstrated in vitro using intact Golgi membrane vesicles from rat liver and mammary gland. We have now identified and purified the rat liver Golgi membrane ATP transporter. The transporter was purified to apparent homogeneity by a combination of conventional ion exchange, dye color, and affinity chromatography. An approximately 70,000-fold purification (2% yield) was achieved starting from crude rat liver Golgi membranes. A protein with an apparent molecular mass of 60 kDa was identified as the putative transporter by a combination of column chromatography, photoaffinity labeling with an analog of ATP, and native functional size determination on a glycerol gradient. The purified transporter appears to exist as a homodimer within the Golgi membrane, and when reconstituted into phosphatidylcholine liposomes, was active in ATP but not nucleotide sugar or adenosine 3'-phosphate 5'-phosphosulfate transport. The transport activity was saturable with an apparent Km very similar to that of intact Golgi vesicles.

Identification and purification of the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter.

Glycosylation of glycoproteins, proteoglycans, and glycosphingolipids occurs mainly in the lumen of the endoplasmic reticulum and the Golgi apparatus. Nucleotide sugars, donors of all the sugars involved in Golgi glycosylation reactions, are synthesized in the cytoplasm and require specialized transporters to be translocated into the lumen of the Golgi apparatus. By controlling the supply of sugar nucleotides in the lumen of the Golgi apparatus, these transporters directly regulate the glycosylation of macromolecules transiting the Golgi. We have identified and purified the rat liver Golgi membrane UDP-N-acetylgalactosamine transporter. The transporter was purified to apparent homogeneity by a combination of conventional and dye color chromatography. An approximately 63,000-fold purification (6% yield) was achieved starting from crude rat liver Golgi membranes and resulting in a protein with an apparent molecular mass of 43 kDa. The transporter was active when reconstituted into phosphatidylcholine vesicles and could be specifically photolabeled with P3-(4-azidoanilido)-uridine-5'-[P1-32P]triphosphate, an analog of UDP-N-acetylgalactosamine. Native functional size determination on a glycerol gradient suggested that the transporter exists as a homodimer within the Golgi membrane.

Transporters of nucleotide sugars, nucleotide sulfate and ATP in the Golgi apparatus.

Proteins and glycolipids are glycosylated, sulfated and phosphorylated in the lumen of the Golgi apparatus. The nucleotide substrates of these reactions must first be translocated from the cytosol into the Golgi lumen by specific transporters (antiporters). These are hydrophobic, transmembrane spanning proteins that appear to regulate post-translational modifications in the Golgi lumen.

A mutant yeast deficient in Golgi transport of uridine diphosphate N-acetylglucosamine.

Mannan chains of Kluyveromyces lactis mannoproteins are similar to those of Saccharomyces cerevisiae except that they have terminal alpha1-->2-linked N-acetylglucosamine and lack mannose phosphate. In a previous study, Douglas and Ballou (Douglas, R. K., and Ballou, C. E. (1982) Biochemistry 21, 1561-1570) characterized a mutant, mnn2-2, which lacked terminal N-acetylglucosamine in its mannoproteins. The mutant had normal levels of N-acetylglucosaminyltransferase activity, and the partially purified enzyme from wild-type and mutant cells had the same apparent size, heat stability, affinity for substrates, metal requirement, and subcellular location. No qualitative or quantitative differences were found between mutant and wild-type cells in endogenous mannan acceptors and pools of UDP-GlcNAc. Chitin was synthesized at similar rates in wild-type and mutant cells, and the latter did not have a soluble inhibitor of the N-acetylglucosaminyltransferase or a hexosaminidase that could remove N-acetylglucosamine from mannoproteins. Together, the above observations led Douglas and Ballou ((1982) Biochemistry 21, 1561-1570) to postulate that the mutant might have a defect in compartmentation of substrates involved in the biosynthesis of mannoproteins. We determined whether the above mutant phenotype is the result of defective transport of UDP-GlcNAc into Golgi vesicles from K. lactis. Golgi vesicles which were sealed and of the same membrane topographical orientation as in vivo were isolated from wild-type and mnn2-2 mutant cells and incubated with UDP-GlcNAc in an assay in vitro. The initial rate of transport of UDP-GlcNAc into Golgi vesicles from wild-type cells was temperature dependent, saturable with an apparent Km of 5.5 microM and a Vmax of 8.2 pmol/mg of protein/3 min. No transport of UDP-GlcNAc was detected into Golgi vesicles from mutant cells. However, Golgi vesicles from both cells translocated GDP-mannose at comparable velocities, indicating that the above transport defect is specific. In addition to the above defect in mannoproteins, mutant cells were also deficient in the biosynthesis of glucosamine containing lipids.

Indomethacin, esculetin and nordihydroguaiaretic acid modify arachidonate biosynthesis in rat adrenocortical cells.

We have studied the duration of the ACTH inhibition effect on the incorporation and transformation of [1-14 C] eicosatrienoic acid, in isolated adrenocortical cells of normal rats. The effect of esculetin, indomethacin and nordihydroguaiaretic acid alone, or in the presence of ACTH or diBucAMP on arachidonate biosynthesis was also investigated. ACTH and diBucAMP produced a significant inhibition in arachidonic acid biosynthesis. The inhibition produced by ACTH on delta 5 desaturating activity was considered to be a short-term effect. Nordihydroguaiaretic acid and esculetin provoked a depression in the uptake of 20:3 (n-6) acid and an inhibition in the formation of 20:4 (n-6) acid by adrenocortical cells. This effect was potentiated when the cells were currently treated with either ACTH or diBucAMP. Indomethacin produced no changes in the uptake of 20:3 (n-6) acid, while induced an increment on delta 5 desaturation activity. This effect would indicate that, normally, the metabolites produced by the cyclooxygenase pathway would operate by depressing arachidonic acid biosynthesis. Considering the negative regulation of the delta 5 desaturase activity system produced by ACTH through cAMP, and the positive modulation inferred by the results obtained in this work, it is possible to assume that there are, at least, two mechanisms that take place on 20:4 (n-6) acid formation. These mechanisms seem to work independently from one another and they probably interact when effecting a bi-directional control.

Indomethacin, esculetin and nordihydroguaiaretic acid modify arachidonate biosynthesis in rat adrenocortical cells.

We have studied the duration of the ACTH inhibition effect on the incorporation and transformation of [1-14 C] eicosatrienoic acid, in isolated adrenocortical cells of normal rats. The effect of esculetin, indomethacin and nordihydroguaiaretic acid alone, or in the presence of ACTH or diBucAMP on arachidonate biosynthesis was also investigated. ACTH and diBucAMP produced a significant inhibition in arachidonic acid biosynthesis. The inhibition produced by ACTH on delta 5 desaturating activity was considered to be a short-term effect. Nordihydroguaiaretic acid and esculetin provoked a depression in the uptake of 20:3 (n-6) acid and an inhibition in the formation of 20:4 (n-6) acid by adrenocortical cells. This effect was potentiated when the cells were currently treated with either ACTH or diBucAMP. Indomethacin produced no changes in the uptake of 20:3 (n-6) acid, while induced an increment on delta 5 desaturation activity. This effect would indicate that, normally, the metabolites produced by the cyclooxygenase pathway would operate by depressing arachidonic acid biosynthesis. Considering the negative regulation of the delta 5 desaturase activity system produced by ACTH through cAMP, and the positive modulation inferred by the results obtained in this work, it is possible to assume that there are, at least, two mechanisms that take place on 20:4 (n-6) acid formation. These mechanisms seem to work independently from one another and they probably interact when effecting a bi-directional control.

Purification of the Golgi adenosine 3'-phosphate 5'-phosphosulfate transporter, a homodimer within the membrane.

Sulfation of proteoglycans, secretory and membrane proteins, and glycolipids occurs in the lumen of the Golgi apparatus. Adenosine 3'-phosphate 5'-phosphosulfate (PAPS), the sulfate donor in these reactions, must be transported from the cytosol, its site of synthesis, into the lumen of the Golgi apparatus. We have identified and purified to apparent homogeneity the rat liver Golgi membrane PAPS transporter by a combination of conventional and affinity chromatography as well as photoaffinity radiolabeling with adenosine 3',5'-bisphosphate, a competitive inhibitor of PAPS transport. The transporter, a 75-kDa protein, was purified 70,000-fold over homogenate (6% yield) and transported PAPS into phosphatidylcholine liposomes selectively and in a saturable manner (apparent Km of 1.7 microM). Radiation target-inactivation analyses of the transport activity in rat liver Golgi vesicles, together with the above described biochemical approaches, demonstrate that the PAPS transporter within the Golgi membrane is a homodimer.

A monomeric protein in the Golgi membrane catalyzes both N-deacetylation and N-sulfation of heparan sulfate.

Recent studies have shown that the rat liver heparan sulfate N-deacetylase/N-sulfotransferase is a glycoprotein encoded by a single polypeptide chain of 882 amino acids. Using radiation inactivation analyses, we have now determined that in rat liver Golgi vesicles the target size for the N-deacetylase is 88 +/- 14 kDa, whereas that of the N-sulfotransferase is 92 +/- 8 kDa. These results, together with previous biochemical and molecular cloning approaches, demonstrate that 1) in rat liver Golgi membranes there exists only on population of molecules expressing both activities, 2) the active protein in the Golgi membrane functions as a monomer, and 3) there is no evidence that a large independent protein acts as a regulator of either activity.

Long-chain acyl-CoA synthetase of rat testis microsomes. Substrate specificity and hormonal regulation.

We investigated long-chain fatty-acyl-CoA synthetase activity in rat testicular microsomes. The apparent Michaelis constants (Km's) for the substrate fatty acids increased while their corresponding maximal velocities decreased in the order 18:3(n-3), 20:3(n-6), and 18:0. The reaction with 20:3 as substrate was diminished in the presence of a constant amount of either 18:0, 18:2(n-6), or 18:3(n-3) in a manner consistent with their action as simple competitive inhibitors, with the Ki values for 18:0 and 18:3(n-3) being of the same order of magnitude as their respective Km's. Adrenocorticotrophin and/or dexamethasone administration to intact rats caused a significant decrease in the thioesterification of all three substrates without producing any alteration in the fatty-acid composition of the microsomal membranes. These results indicate the presence of a broad-specificity activating enzyme in testis whose function is subject to hormonal regulation.

Guanosine diphosphatase is required for protein and sphingolipid glycosylation in the Golgi lumen of Saccharomyces cerevisiae.

Current models for nucleotide sugar use in the Golgi apparatus predict a critical role for the lumenal nucleoside diphosphatase. After transfer of sugars to endogenous macromolecular acceptors, the enzyme converts nucleoside diphosphates to nucleoside monophosphates which in turn exit the Golgi lumen in a coupled antiporter reaction, allowing entry of additional nucleotide sugar from the cytosol. To test this model, we cloned the gene for the S. cerevisiae guanosine diphosphatase and constructed a null mutation. This mutation should reduce the concentrations of GDP-mannose and GMP and increase the concentration of GDP in the Golgi lumen. The alterations should in turn decrease mannosylation of proteins and lipids in this compartment. In fact, we found a partial block in O- and N-glycosylation of proteins such as chitinase and carboxypeptidase Y and underglycosylation of invertase. In addition, mannosylinositolphosphorylceramide levels were drastically reduced.

Arachidonic acid biosynthesis in non-stimulated and adrenocorticotropin-stimulated Sertoli and Leydig cells.

The biosynthesis of arachidonic acid in Sertoli and Leydig cells isolated from the testes of mature rats has been investigated. Both types of cells incorporated [2-14C]eicosatrienoic acid from the incubation medium and transformed it into arachidonic acid. The administration of adrenocorticotropin (ACTH) to the rats decreased the delta 5 desaturating activity in the isolated testicular cells, while ACTH produced no changes in the uptake of the substrate. Similar results were obtained when ACTH was added to the incubation medium of cells isolated from non-hormone treated rats. The total fatty acid composition of the Sertoli cells isolated from ACTH-treated rats showed a significant increase in the relative percentage of 18:2n-6 and a decrease in the C20 and C22 polyenes. This may indicate that ACTH exerts an inhibitory effect on delta 6, delta 5 and delta 4 desaturase activities. Addition of corticosterone to the incubation medium also produced a significant decrease in arachidonic acid biosynthesis. Because ACTH is known to stimulate the release of corticosterone in vivo, both hormones may act cumulatively in the regulation of arachidonic acid metabolism in Sertoli and Leydig cells.

Subcellular localization and membrane topology of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase in mouse liver.

Serine palmitoyltransferase, 3-dehydrosphinganine reductase and sphinganine N-acyltransferase are responsible for the first steps in sphingolipid biosynthesis forming 3-oxosphinganine, sphinganine, and dihydroceramide, respectively. We confirmed the localization of these enzymes in the endoplasmic reticulum (ER) using highly purified mouse liver ER and Golgi preparations. Mild digestion of sealed "right-side out" mouse liver ER derived vesicles with different proteolytic enzymes under conditions where latency of mannose-6-phosphatase was 90% produced approximately 60-80% inactivation of serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase activities. These sphingolipid biosynthetic activities (serine palmitoyltransferase, 3-dehydrosphinganine reductase, and sphinganine N-acyltransferase) are not latent, indicating that they face the cytosolic side of the ER, so that substrates have free access to their active sites. Moreover, the membrane-impermeable compound, 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid, which binds to a large number of ER proteins, inhibits serine palmitoyltransferase and sphinganine N-acyltransferase activities by 30-70%.

Sphingolipid biosynthesis in cultured neurons. Down-regulation of serine palmitoyltransferase by sphingoid bases.

Addition of exogenous sphingosine homologues (D-erythro configuration) with different alkyl chain lengths (12 and 18 carbon atoms) to the medium of primary cultured cerebellar cells resulted in a decrease of serine palmitoyltransferase activity in a time- and concentration-dependent manner. This enzyme catalyzes the first committed step in sphingolipid biosynthesis. Half-maximal reduction of enzyme activity occurred after a 4-h treatment with 25 microM sphingoid bases. Maximal decrease (approx. 80%) was obtained after treating the cells for 4-8 h with 50 microM long-chain bases. When a biosynthetically inert sphingoid, azidosphingosine (10-50 microM), was fed to the cells, decrease of 3-ketosphinganine formation was much slower, reaching its maximum (approx. 80%) after 24 h. In contrast to D-erythro-sphingosine, L-threo-C18-sphingosine did not yield any decrease of serine palmitoyltransferase activity when added to the cells under identical experimental conditions. Decrease of serine palmitoyltransferase activity was fully reversible after removal of the long-chain bases from the culture medium. Activities of other enzymes of lipid metabolism, ceramide synthase, long-chain acyl-CoA synthase and choline phosphotransferase, were not affected by the addition of sphingoid bases, indicating that the down regulation of serine palmitoyltransferase is quite specific.

Abnormal metabolism of polyunsaturated fatty acids in adrenal glands of diabetic rats.

Studies carried out on the adrenal glands of experimental diabetic rats have shown an important inhibition in polyenoic fatty acid biosynthesis. This effect was demonstrated by testing the activities of long-chain fatty acyl-CoA synthetase, the delta 5- and delta 6-desaturases of the (n-6) essential fatty-acid series and the delta 6-desaturase of the (n-3) series in liver and adrenal microsomes. The depression in desaturating activity in the insulin-deprived animals was independent of that produced on acyl-CoA-thioester biosynthesis. Experiments measuring the incorporation and transformation of [1-14C]eicosa-8,11,14-trienoic acid in adrenocortical cells isolated from streptozotocin-diabetic animals demonstrated a significant inhibition of arachidonic acid biosynthesis compared to controls. Insulin injections in diabetic rats partially restored the delta 5- and delta 6-desaturase activities. This effect could result from direct action by the hormone since the restoration was reproduced when arachidonic acid biosynthesis was measured after insulin was added to the incubation medium of adrenocortical cells isolated from diabetic animals. The results of the present study provide new information about the implication of this abnormal metabolism in the adrenal gland of diabetic rats.

Biosynthesis of polyunsaturated fatty acids of (n-6) and (n-3) series in isolated adrenocortical cells of rats. Effect of ACTH.

Both the capacity of isolated adrenocortical cells to incorporate and transform [1-14C]linoleic and [1-14C]alpha-linolenic acids and the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from [1-14C]alpha-linolenic acid were investigated. The cells were able to incorporate both labeled precursor acids and convert them into higher homologs. This transformation increases along the incubation time tested. When linoleic acid was the precursor, the biosynthesis of higher homologs was carried out following the desaturating-elongating route. Both pathways, the desaturating-elongating and the elongating, were detected when the substrate was alpha-linolenic acid. The results proved the existence of delta 6, delta 5 and delta 4-desaturases in this type of cells. Isolated adrenocortical cells obtained from rats treated with ACTH showed an increase in the amount of [1-14C]alpha-18:3 that remained in the cells without metabolization and, consequently, a decrease in the last product formed (20:5 n-3) was evident compared to the controls. Simultaneously, the desaturation-elongation products decreased significantly. Similar results were obtained when cells isolated from untreated rats were incubated for 3 h in the presence of ACTH. In this case, the values obtained returned to normal levels 6 h after incubation. These results were mimicked by dibutyryl-cyclic AMP. It can be concluded that the effect of ACTH on the biosynthesis of polyunsaturated fatty acids from alpha-linolenic acid was mediated through an enhancement of the intracellular levels of cAMP.

Long-chain fatty acyl-CoA synthetase of rat adrenal microsomes. Effect of ACTH and epinephrine.

Acyl-CoA synthetase activity with various long-chain fatty acid substrates and its kinetic properties were measured in rat adrenal microsomes. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order eicosa-8,11,14-trienoic acid less than alpha-linolenic acid less than linoleic acid less than palmitic acid. The maximum velocities with these fatty acids decreased in the order linolenic greater than eicosa-8,11,14-trienoic acid greater than palmitic acid. The synthesis of radioactivity palmitoyl-CoA, linoleyl-CoA, alpha-linolenyl-CoA and eicosa-8,11,14-trienoyl-CoA from the respective radioactive substrates decreased in the presence of all the other fatty acids mentioned above. These effects were inversely correlated with their apparent Km values. These results support the idea of a single long-chain fatty acyl-CoA synthetase in the adrenal microsomal fraction for the acid tested. After testing the influence of different hormones, it was shown that the administration of epinephrine, ACTH and dexamethasone caused a significant decrease in the activity of the long-chain fatty acid-CoA synthetase. This inhibition is independent of the one produced by the same hormones on the desaturation of linoleic to gamma-linolenic acid.

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, "fluidity" and delta 6 desaturation activity in kidney and lung.

High carbohydrate and high protein diets administered to rats for only three days decreased and increased, respectively, the arachidonate/linoleate ratio (20:4 n6/18:2 n6) in total lipids of lung, kidney, and liver microsomes. In liver and kidney this correlated with a significant decrease in the first dietary condition, and with an increase in the second, of delta 6 desaturase activity, measured by the rate of conversion of 1-14C linoleate n6 to 1-14C linolenate n6. Such an enzymatic activity was not detectable in lung microsomes, probably because of the low capacity of this tissue to produce the coenzyme A ester of the substrate used, since in lung the effects of dietary manipulation on the 20:4/18:2 ratios were as large as in liver. Significant differences were observed in microsomes of the three tissues examined in the steady state fluorescence anisotropy (rs) of diphenylhexatriene, which correlated with their cholesterol/phospholipid ratios. Both were lower in microsomes from liver than from the other two tissues, and both remained unchanged in each tissue under the dietary regimens studied. The results indicate that the observed effects of high carbohydrate and high protein diets on fatty acid delta 6 desaturation activity do not lead to apparent alterations in the physical properties of microsomal membranes.

Effect of high carbohydrate and high protein diets on microsomal fatty acid composition, [quot ]fluidity[quot ] and delta 6 desaturation activity in kidney and lung.

High carbohydrate and high protein diets administered to rats for only three days decreased and increased, respectively, the arachidonate/linoleate ratio (20:4 n6/18:2 n6) in total lipids of lung, kidney, and liver microsomes. In liver and kidney this correlated with a significant decrease in the first dietary condition, and with an increase in the second, of delta 6 desaturase activity, measured by the rate of conversion of 1-14C linoleate n6 to 1-14C linolenate n6. Such an enzymatic activity was not detectable in lung microsomes, probably because of the low capacity of this tissue to produce the coenzyme A ester of the substrate used, since in lung the effects of dietary manipulation on the 20:4/18:2 ratios were as large as in liver. Significant differences were observed in microsomes of the three tissues examined in the steady state fluorescence anisotropy (rs) of diphenylhexatriene, which correlated with their cholesterol/phospholipid ratios. Both were lower in microsomes from liver than from the other two tissues, and both remained unchanged in each tissue under the dietary regimens studied. The results indicate that the observed effects of high carbohydrate and high protein diets on fatty acid delta 6 desaturation activity do not lead to apparent alterations in the physical properties of microsomal membranes.

ACTH depresses delta 6 and delta 5 desaturation activity in rat adrenal gland and liver.

The in vivo and in vitro effect of ACTH on the biosynthesis of polyunsaturated fatty acid of rat adrenal gland and liver was studied. The administration of ACTH to intact rats produced a significant decrease in the conversion of [1-14C]linoleic acid to gamma-linolenic acid, [1-14C]alpha-linolenic acid to octadeca-6,9,12,15-tetraenoic acid, and [1-14C]eicosa-8,11,14-trienoic acid to arachidonic acid in liver and adrenal microsomes. Isolated adrenocortical cells and hepatocytes obtained from animals treated with ACTH showed a decrease in the incorporation and desaturation of exogenous [1-14C]eicosa-8,11,14-trienoic acid. The addition of ACTH to the incubation medium of adrenocortical cells and hepatocytes isolated from untreated rats also caused a decrease in delta 5 desaturation activity. The effect of ACTH on adrenal and liver desaturases could be produced as a consequence of the release of glucocorticoids, already measured in the experiments. However, the in vitro experiments carried out with hepatocytes isolated from untreated rats, where corticosterone was absent, indicated that ACTH can depress delta 5 desaturation per se.