Subcellular sites of enzymes catalyzing the phosphorylation-dephosphorylation of dolichol in the central nervous system

The subcellular locations of several enzymes involved in dolichyl monophosphate (Dol-P) metabolism in brain have been investigated. Dolichol kinase is highly enriched in a heavy microsomal fraction from calf brain, while 71% of the Dol-P phosphatase activity was recovered with the light microsomes. Lower amounts of the phosphatase activity were also found in the heavy microsomal, mitochondrial-lysosomal, and synaptic plasma membrane fractions. Since the light microsomal fraction also contained substantial acetylcholinesterase activity, an axon plasma membrane marker, an axolemma-enriched fraction, was prepared from rat brain by a second procedure. A comparison with microsomal and mitochondrial-lysosomal fractions revealed that the axolemma-enriched fraction contained the highest specific activity of Dol-P phosphatase, indicating that the enzyme was present in the axon plasma membrane. The tunicamycin-sensitive UDP-N-acetylglucosamine:Dol-P N- acetylglucosaminylphosphotransferase , glucosyl- phosphoryldolichol (Glc-P-Dol) synthase, Glc-P-Dol:oligosaccharide glucosyltransferase, and the oligosaccharyltransferase were all found predominantly in the heavy microsomes. These results indicate that the enzymes responsible for the initiation and termination of biosynthesis, as well as the transfer of dolichol-linked oligosaccharides, reside in the rough endoplasmic reticulum (ER) of central nervous tissue. Evidence that at least some Dol-P molecules formed by dolichol kinase are accessible to multiple glycosyltransferases in the rough ER of brain is also presented.     

Recycling of dolichyl monophosphate to the cytoplasmic leaflet of the endoplasmic reticulum after the cleavage of dolichyl pyrophosphate on the lumenal monolayer

During protein N-glycosylation, dolichyl pyrophosphate (Dol-P-P) is discharged in the lumenal monolayer of the endoplasmic reticulum (ER). Dol-P-P is then cleaved to Dol-P by Dol-P-P phosphatase (DPPase). Studies with the yeast mutant cwh8Delta, lacking DPPase activity, indicate that recycling of Dol-P produced by DPPase contributes significantly to the pool of Dol-P utilized for lipid intermediate biosynthesis on the cytoplasmic leaflet. Whether Dol-P formed in the lumen diffuses directly back to the cytoplasmic leaflet or is first dephosphorylated to dolichol has not been determined. Incubation of sealed ER vesicles from calf brain with acetyl-Asn-Tyr-Thr-NH(2), an N-glycosylatable peptide, to generate Dol-P-P in the lumenal monolayer produced corresponding increases in the rates of Man-P-Dol, Glc-P-Dol, and GlcNAc-P-P-Dol synthesis in the absence of CTP. No changes in dolichol kinase activity were observed. When streptolysin-O permeabilized CHO cells were incubated with an acceptor peptide, N-glycopeptide synthesis, requiring multiple cycles of the dolichol pathway, occurred in the absence of CTP. The results obtained with sealed microsomes and CHO cells indicate that Dol-P, formed from Dol-P-P, returns to the cytoplasmic leaflet where it can be reutilized for lipid intermediate biosynthesis, and dolichol kinase is not required for recycling. It is possible that the flip-flopping of the carrier lipid is mediated by a flippase, which would provide a mechanism for the recycling of Dol-P derived from Man-P-Dol-mediated reactions in N-, O-, and C-mannosylation of proteins, GPI anchor assembly, and the three Glc-P-Dol-mediated reactions in Glc(3)Man(9)GlcNAc(2)-P-P-Dol (DLO) biosynthesis.     

The CWH8 gene encodes a dolichyl pyrophosphate phosphatase with a luminally oriented active site in the endoplasmic reticulum of Saccharomyces cerevisiae

Mutations in the CWH8 gene, which encodes an ER transmembrane protein with a phosphate binding pocket in Saccharomyces cerevisiae, result in a deficiency in dolichyl pyrophosphate (Dol-P-P)-linked oligosaccharide intermediate synthesis and protein N-glycosylation (van Berkel, M. A., Rieger, M., te Heesen, S., Ram, A. F., van den Ende, H., Aebi, M., and Klis, F. M. (1999) Glycobiology 9, 243-253). Genetic, enzymological, and topological approaches were taken to investigate the potential role of Cwh8p in Dol-P-P/Dol-P metabolism. Overexpression of Cwh8p in the yeast double mutant strain, lacking LPP1/DPP1, resulted in an impressive increase in Dol-P-P phosphatase activity, a relatively small increase in Dol-P phosphatase activity, but no change in phosphatidate (PA) phosphatase activity in microsomal fractions. The Dol-P-P phosphatase encoded by CWH8 is optimally active in the presence of 0.5% octyl glucoside and relatively unstable in Triton X-100, distinguishing this activity from the lipid phosphatases encoded by LPP1 and DPP1. Stoichiometric amounts of P(i) and Dol-P are formed during the enzymatic reaction indicating that Cwh8p cleaves the anhydride linkage in Dol-P-P. Membrane fractions from Sf-9 cells expressing Cwh8p contained a 30-fold higher level of Dol-P-P phosphatase activity, a slight increase in Dol-P phosphatase activity, but no increase in PA phosphatase relative to controls. This is the first report of a lipid phosphatase that hydrolyzes Dol-P-P/Dol-P but not PA. In accord with this enzymatic function, Dol-P-P accumulated in cells lacking the Dol-P-P phosphatase. Topological studies using different approaches indicate that Cwh8p is a transmembrane protein with a luminally oriented active site. The specificity, subcellular location, and topological orientation of this novel enzyme are consistent with a role in the re-utilization of the glycosyl carrier lipid for additional rounds of lipid intermediate biosynthesis after its release during protein N-glycosylation reactions.     

Characterization and Localization of a Long-Chain Isoprenyltransferase Activity in Porcine Brain: Proposed Role in the Biosynthesis of Dolichyl Phosphate

Pig brain microsomes catalyzed the enzymatic transfer of radiolabeled isoprenyl groups from [1-14C]isopentenyl pyrophosphate [( 1-14C]I-P-P) into long-chain polyisoprenyl pyrophosphates (Poly-P-P) and unidentified neutral lipids. The brain isoprenyltransferase activity synthesizing the Poly-P-P (1) required 5 mM Mg2+ and 10 mM vanadate ions for maximal activity; (2) exhibited an apparent Km of 8 microM for I-P-P; (3) utilized exogenous farnesyl pyrophosphate and two stereoisomers of geranylgeranyl pyrophosphate as substrates; (4) was optimal at pH 8.5; and (5) was stimulated by dithiothreitol. The major products were identified as C90 and C95 allylic Poly-P-P on the basis of the following chemical and chromatographic properties: (1) the intact product co-chromatographed with authentic Poly-P-P on silica-gel-impregnated paper; (2) the major product was converted to a compound chromatographically identical to polyisoprenyl monophosphate (Poly-P) by alkaline hydrolysis; (3) treatment of the labeled Poly-P with wheat germ acid phosphatase or mild acid yielded neutral labeled products; (4) the KOH hydrolyzed product coeluted with authentic Poly-P from lipophilic Sephadex LH-20; and (5) the labeled lipids produced by enzymatic dephosphorylation had mobilities identical to fully unsaturated polyisoprenols containing 18 (C90) and 19 (C95) isoprene units when analyzed by reverse-phase chromatography. When subcellular fractions from rat brain gray matter were compared, the highest specific activity was found in the heavy microsomes. These results demonstrate that brain contains an isoprenyltransferase activity, associated with the rough endoplasmic reticulum, capable of synthesizing long-chain Poly-P-P. The enzymatic reactions by which the Poly-P-P intermediate is converted to dolichyl phosphate remain to be elucidated.     

Identification and characterization of a cDNA encoding a dolichyl pyrophosphate phosphatase located in the endoplasmic reticulum of mammalian cells

The CWH8 gene in Saccharomyces cerevisiae has been shown recently (Fernandez, F., Rush, J. S., Toke, D. A., Han, G., Quinn, J. E., Carman, G. M., Choi, J.-Y., Voelker, D. R., Aebi, M., and Waechter, C. J. (2001) J. Biol. Chem. 276, 41455-41464) to encode a dolichyl pyrophosphate (Dol-P-P) phosphatase associated with crude microsomal fractions. Mutations in CWH8 result in the accumulation of Dol-P-P, deficiency in lipid intermediate synthesis, defective protein N-glycosylation, and a reduced growth rate. A cDNA (DOLPP1, GenBank accession number AB030189) from mouse brain encoding a homologue of the yeast CWH8 gene is now shown to complement the defects in growth and protein N-glycosylation, and to correct the accumulation of Dol-P-P in the cwh8Delta yeast mutant. Northern blot analyses demonstrate a wide distribution of the DOLPP1 mRNA in mouse tissues. Overexpression of Dolpp1p in yeast, COS, and Sf9 cells produces substantial increases in Dol-P-P phosphatase activity but not in dolichyl monophosphate or phosphatidic acid phosphatase activities in microsomal fractions. Subcellular fractionation and immunofluorescence studies localize the enzyme encoded by DOLPP1 to the endoplasmic reticulum of COS cells. The results of protease sensitivity studies with microsomal vesicles from the lpp1Delta/dpp1Delta yeast mutant expressing DOLPP1 are consistent with Dolpp1p having a luminally oriented active site. The sequence of the DOLPP1 cDNA predicts a polypeptide with 238 amino acids, and a new polypeptide corresponding to 27 kDa is observed when DOLPP1 is expressed in yeast, COS, and Sf9 cells. This study is the first identification and characterization of a cDNA clone encoding an essential component of a mammalian lipid pyrophosphate phosphatase that is highly specific for Dol-P-P. The specificity, subcellular location, and topological orientation of the active site described in the current study strongly support a role for Dolpp1p in the recycling of Dol-P-P discharged during protein N-glycosylation reactions on the luminal leaflet of the endoplasmic reticulum in mammalian cells.     

Biochemical activity and morphology of the Golgi apparatus in alloxan diabetic rats.

In Golgi-enriched membrane fraction isolated from alloxan diabetic rat liver, the protein content is lower but the protein composition is similar to normal. The specific activity of galactosyl transferase in this membrane fraction is higher than normal, but the total activity of the enzyme in the whole liver is normal. Great dispersion is found among the individual values. Differences in the specific activities of some other marker enzymes were also found in the Golgi-enriched membrane fraction. The morphology of the Golgi complex examined in either thin sections of Golgi-enriched fraction or ultra-thin sections of livers appears normal. A slight reduction of the rough endoplasmic reticulum is observed. The results obtained are discussed in comparison with those obtained for streptozotocin diabetic rat liver.     

Characterization of polyisoprenyl phosphate phosphatase activity in rat liver

The polyisoprenyl phosphate dephosphorylating activity of rat liver has been investigated with regard to substrate specificity, subcellular distribution, and transmembrane orientation. Total liver microsomes were employed as a source of enzymatic activity against a variety of 32P-labeled substrates. Susceptibility to dephosphorylation followed the order solanesyl phosphate greater than alpha-cis-polyprenyl 19-phosphate = alpha-trans-polyprenyl 19-phosphate = dihydrosolanesyl phosphate greater than (S)-dolichyl 19-phosphate = (R)-dolichyl 19-phosphate = (R,S)-dolichyl 11-phosphate. There appeared to be no major effect of chain length from 11 to 20 isoprenes. Data obtained from inhibition studies using solanesyl [32P]phosphate as substrate were consistent with the substrate specificity studies and suggested that a single activity is responsible. With dolichyl [32P]phosphate as substrate, the phosphatase specific activity of the subcellular fractions prepared from rat liver was found to follow the sequence Golgi = smooth endoplasmic reticulum greater than plasma membrane greater than lysosomes = rough endoplasmic reticulum greater than nuclei greater than mitochondria. Transmembrane topography studies, using enzyme latency as a criterion, were consistent with an orientation of the active site facing the cytoplasm.     

Brain dolichyl pyrophosphate phosphatase. Solubilization, characterization, and differentiation from dolichyl monophosphate phosphatase activity

Dolichyl [beta-32P]pyrophosphate ([beta-32P]Dol-P-P) has been prepared chemically to study Dol-P-P phosphatase in calf brain. Calf brain microsomes catalyze the enzymatic release of 32Pi from exogenous [beta-32P]Dol-P-P by a bacitracin-sensitive reaction. [32P]Pyrophosphate was not detected with the water-soluble product even when 1 mM sodium pyrophosphate was added to impede pyrophosphatase activity. A substantial fraction of the Dol-P-P phosphatase activity can be solubilized by treating brain microsomes with 3% Triton X-100. The detergent extracts catalyze the enzymatic release of 32Pi from [beta-32P]Dol-P-P and the conversion of [14C]undecaprenyl pyrophosphate to [14C]undecaprenyl monophosphate. The solubilized Dol-P-P phosphatase activity: 1) is optimal at neutral pH; 2) is inhibited by Mn2+ and stimulated by EDTA; 3) exhibits an apparent Km = 20 microM for Dol-P-P; 4) is competitively inhibited by undecaprenyl pyrophosphate, and 5) is blocked by bacitracin. Solubilized Dol-P-P phosphatase activity differs from Dol-P phosphatase activity present in the same detergent extracts with respect to: 1) thermolability at 50 degrees C, 2) effect of 20 mM EDTA, and 3) sensitivity to phosphate and fluoride ions. These studies describe the chemical synthesis of [beta-32P]Dol-P-P for use in a convenient assay of Dol-P-P phosphatase activity. A procedure for the solubilization of Dol-P-P phosphatase activity from microsomes is presented, and an enzymological comparison indicates that Dol-P-P and Dol-P phosphatase are separate enzymes in calf brain.     

Changes in electronegativity of lysosomal hydrolases during intracellular transport. An isoelectric-focusing study in subcellular fractions of rat kidney.

Isoelectric focusing was used to investigate the multiple forms of acid phosphatase, arylsulfatase, beta-glucuronidase, beta-galactosidase and beta-N-acetylhexosaminidase in the following, previously characterized subcellular fractions from rat kidney: a special rough microsomal fraction, enriched up to 9-fold over the homogenate in acid hydrolases; a smooth microsomal fraction; a Golgi membrane fraction enriched about 2.5-fold in acid hydrolases and 10- to 20-fold in several glycosyl transferases; and a lysosomal fraction enriched up to 25-fold in acid hydrolases. The electro-focusing behavior of the hydrolases in these fractions was markedly sensitive to the autolytic changes that occur under acidic conditions, even at 4 degrees C. Autolysis was minimized by extracting fractions in an alkaline medium (0.2% Triton X-100, 0.1 M sodium glycinate buffer, pH 10, 0.1 % p-nitrophenyloxamic acid) and adding p-nitrophenyloxamic acid (0.1 %), AN INHIBITOR OF LYSOSOMAL NEURAMINIDASE AND cathepsin D, to the pH gradient. The enzymes in the lysosomal fraction displayed a characteristic bimodal or trimodal distribution. Arylsulfatase, beta-glucuronidase and beta-N-acetylhexosaminidase occurred in an acidic form with an isoelectric point of 4.4, and a basic form with an isoelectric point of 6.2, 6.7 and 8.0, respectively. Acid phosphatase and beta-galactosidase occurred in an acidic, intermediate and basic form with isoelectric points of about 4. 1, 5.6 and 7.4, respectively. In the special rough microsomal fraction these enzymes were mostly in a basic form with isoelectric points between 7.5 and 9; these were 1-2 units higher than the corresponding basic forms in the lysosomal fraction. Treatment of extracts of the rough microsomal fraction with bacterial neuraminidase raised the isoelectric points of all five hydrolases by 1-2.5 units, indicating the presence of some N-acetylneuraminic acid residues in these basic glycoenzymes. The hydrolases in the Golgi fraction were largely in an acidic form with isoelectric points similar to or lower than those of the corresponding acidic components in the lysosomal fraction. The hydrolases in the smooth microsomal fraction showed isoelectric-focusing patterns intermediate between those in the rough microsomal and the Golgi fractions. These findings support the following scheme for the synthesis, transport and packaging of the lysosomal enzymes. Each hydrolase is synthesized in a restricted portion of the r     

Physicochemical modifications of lysosomal hydrolases during intracellular transport

1. The following fractions were prepared from rat kidney and characterized ultrastructurally, biochemically and enzymically: (a) an ordinary rough microsomal (RM(1)) fraction; (b) a special rough microsomal (RM(2)) fraction enriched seven- to nine-fold in acid hydrolases over the homogenate; (c) a smooth microsomal (SM) fraction; (d) a Golgi (GM) fraction enriched 2.5-fold in acid hydrolases and 10-, 15- and 20-fold in sialyltransferase, N-acetyl-lactosamine synthetase and galactosyltransferase respectively; (e) a lysosomal (L) fraction enriched 15- to 23-fold in acid hydrolases. The frequency of Golgi sacs and tubules seen in the electron microscope and the specific activity of the three glycosyltransferases in these fractions increased in the order: RM(2)相似文献   

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.     

Ultrastructure and polysome content of the microsomal subfractions of mouse plasmacytoma cells

Summary The endoplasmic reticulum (ER) of MPC-11 cells released as vesicles upon cell disruption by nitrogen cavitation was separated from the bulk of mitochondria, lysosomes and plasma membranes by a low speed centrifugation. The ER membranes were fractionated on discontinuous sucrose gradients into heavy rough (HR), light rough (LR) and smooth (S) membranes. The morphology of subcellular fractions was studied by electron microscopy and the ER membranes were shown to be virtually free of contaminating organelles. The S fraction was easily distinguishable because of the lack of ribosomes but there were no apparent morphological differences between the HR and LR fractions. Of total activity in the microsomal subfractions, 70% of the UDPase and 67% of the 5′-nucleotidase activity was associated with the S fraction. Polysomes were present in the HR, LR and nuclear-associated ER fractions but not in the S fraction. The HR and LR fractions did not appear to be contaminated to any great extent with free polysomes. RNA/protein and RNA/phospholipid ratios of the HR fraction were higher than those of the LR fraction, indicating a greater density of ribosomes in the former fraction. These ratios were much lower in the S fraction reflecting the low ribosome content.     

Multiple forms of synthase D phosphatase and phosphorylase a phosphatase in liver and regulatory effects of metabolites on their activities

The smooth endoplasmic reticulum (ER) and cytosol fractions of liver homogenates exhibit phosphoprotein phosphatase activity towards glycogen synthase D and phosphorylase a. The following observations suggest that liver contains multiple forms of these phosphatases. Synthase phosphatase activity in either fraction was more readily inactivated by heating than phosphorylase phosphatase activity. Both synthase phosphatase and phosphorylase phosphatase activities in smooth ER were non-competitively inhibited by Mg2+, but were activated by this ion in the cytosol. Synthase phosphatase activities in cytosol and smooth ER were stimulated by a number of sugar phosphates, particularly glucose-1-phosphate, galactose-6-phosphate and fructose-6-phosphate. Erythrose-4-phosphate stimulated synthase phosphatase activity in the cytosol, but inhibited the microsomal enzyme. Phosphorylase phosphatase activities in either fraction were inhibited by most sugar phosphates. Adenosine mono-, di- and tri-phosphates inhibited phosphatase activities in both fractions. Low concentrations of AMP and ADP inhibited phosphorylase phosphatase activities to a greater extent than synthase phosphatase activities. Chromatography of the smooth ER fraction on DEAE-cellulose resulted in the separation of synthase phosphatase from phosphorylase phosphatase, as soluble proteins. The elution profile for the microsomal phosphatase was different from that for the cytosol enzymes. It is concluded that: both synthase phosphatase and phosphorylase phosphatase in liver have at least two isoenzyme forms; synthase phosphatase and phosphorylase phosphatase are separate enzymes; the different behaviour of microsomal and cytosol phosphatases towards divalent cations and sugar phosphates provides a potential mechanism for the differential regulation of these activities in liver.     

The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver

Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Flow kinetics of a nucleoside phosphatase common to endoplasmic reticulum, Golgi apparatus, and plasma membrane of rat liver

Nucleoside mono-, di- and triphosphatase activities of highly purified endoplasmic reticulum (ER), Golgi apparatus, and plasma membrane fractions of rat liver were compared. The highest rates of hydrolysis were always in ER or plasma membrane. Golgi apparatus activity was intermediate between those of ER and plasma membrane. This relationship was true for both freshly isolated fractions and salt-extracted membranes. Detergent solubilization of the membranes, polyacrylamide gel electrophoresis of the solubilized proteins, and localization of the enzyme activities on the gel revealed bands of enzyme activity which had identical mobilities in all three membrane fractions as well as other bands of activity that occurred only in ER and to a lesser degree in the Golgi apparatus. Antibodies raised against one of the phosphatase bands of plasma membrane which was common to all three membrane fractions cross-reacted with the corresponding phosphatase band in ER and Golgi apparatus. The anti-nucleoside phosphatase was utilized in combination with pulse-chase techniques to investigate the flow kinetics of transfer of newly synthesized enzyme among different cell compartments. Label first appeared in nucleoside phosphatase within the ER. Maximum specific activity was observed at about 5 min after injection of label and was followed by rapid loss of label. This was followed by appearance of label in Golgi apparatus 15 to 25 min after injection of label and by subsequent rapid loss of label. Plasma membranes were labeled last with no evidence of either rapid accumulation of label or of rapid turnover. Flow of nucleoside phosphatase from its site of synthesis and insertion into the membrane at the endoplasmic reticulum to the plasma membrane via the Golgi apparatus is indicated but in a manner whereby a significant fraction of the protein may be processed (removed?) from the membrane concomitant with the flow process.     

Involvement of dolichol phosphates as intermediates in the mannosyl and galactosyl transferases of rat testicular germ cell Golgi apparatus membranes

Isolated Golgi apparatus membranes from the germinal elements (spermatocytes and early spermatids) of rat testis were examined for their ability to incorporate [14C]mannose and [14C]galactose into glycolipid and glycoprotein fractions. Transfer of mannose from GDP-[14C]mannose into a Lipid I fractions (GPD:MPP mannosyl transferase activity), identified as mannosyl phosphoryl dolichol, showed optimal activity at 1.5 mM manganese and at pH 7.5. Low concentrations of Triton X-100 (0.1%) stimulated transferase activity in the presence of exogenous dolichol phosphate (Dol-P); however, inhibition occurred at Triton X-100 concentrations greater than 0.1%. Maximal activity of this GDP:MPP mannosyl transferase occurred at 25 microM Dol-P. Activity using endogenous acceptor was 2.34 pmole/min/mg, whereas in the presence of 25 microM Dol-P the specific activity was 284 pmole/min/mg, a stimulation of 125-fold. Incorporation of mannose into a Lipid II (oligosaccharide pyrophosphoryl dolichol) and a glycoprotein fraction was also examined. In the absence of exogenous Dol-P, rapid incorporation into Lipid I occurred with a subsequent rise in Lipid II and glycoprotein fractions suggesting precursor-product relationships. Addition of exogenous Dol-P to galactosyl transferase assays showed only a minor stimulation, less than twofold, in all fractions. Over the concentration range of 9.4 to 62.5 micrograms/ml Dol-P, only 1% of radioactive product accumulated in the combined lipid fractions. These observations suggest that the mannose transfer involves Dol-P intermediates and also that spermatocyte Golgi membranes may be involved in formation of the oligosaccharide core as well as in terminal glycosylations.     

Submicrosomal distribution of dolichol kinase and dolichyl phosphate phosphatase in the microsomes of germinating soybeans

The availability of dolichyl phosphate is a major factor in the rate of formation of N-linked glycoproteins in mammalian cells. Recent studies in our laboratory suggested that glycoproteins required for seed germination and early plant development are formed via the dolichyl phosphate pathway. Soybean microsomes contain dolichol kinase and dolichyl phosphate phosphatase, enzymes that regulate dolichyl phosphate levels by interconversion of dolichyl phosphate and dolichol. In the present study, soybean microsomes were fractionated into rough and smooth endoplasmic reticulum and Golgi, and the activities of dolichol kinase and dolichyl phosphate phosphatase were measured in each. Submicrosomal fractions were obtained using a procedure developed for rat liver, and were characterized by marker enzymes, RNA content and electron microscopy. The site of N-glycosylation, the rough endoplasmic reticulum, contained high levels of both dolichol kinase and dolichyl phosphate phosphatase. This makes possible a mechanism whereby glycoprotein formation during seed germination is regulated by availability of dolichyl phosphate.     

Presence of adenylate cyclase activity in Golgi and other fractions from rat liver. II. Cytochemical localization within Golgi and ER membranes

The presence of adenylate cyclase (AC) in liver Golgi and microsomal fractions from ethanol-treated rats was tested cytochemically using 5'- adenylyl imidodiphosphate (AMP-PNP) lead phosphate method. Parallel biochemical assays showed that rat liver Golgi AC was only partially inhibited by lead: in the presence of 1 mM Pb++ 80% of the enzyme was preserved, while when 2 mM Pb++ was used 25% remained. No cAMP was formed when the AMP-PNP medium was incubated in the presence of 1 or 2 mM Pb++ but in the absence of cell fractions, indicating that at these concentrations Pb++ does not cause the nonenzymatic hydrolysis of AMP- PNP. Therefore, the reaction product observed by cytochemical localization is not due to the nonenzymatic hydrolysis of AMP-PNP by Pb++. In Golgi subfractions, lead phosphate reaction product was widely distributed among Golgi elements: it was seen in association with the majority of the very low density lipoprotein-filled secretory droplets which predominated in the two lightest Golgi fractions (GF1 and GF2) as well as within the majority of the cisternae found in the heaviest Golgi fraction (GF3). In the latter, reaction product was heaviest along the dilated peripheral rims of the cisternae. In all cases, the reaction product was localized to the outside or cytoplasmic face of the Golgi membranes. When microsomes were incubated cytochemically for AC, deposits were found on the cytoplasmic surface of smooth endoplasmic reticulum (ER) membranes, but none were observed on rough ER membranes. The results confirm the biochemical data reported previously indicating the presence of AC in Golgi and smooth microsomal fractions from rat liver and further demonstrate that the activity is indeed indigenous to Golgi elements and not due to plasma membrane contaminants. They also indicate that AC is widely distributed among Golgi and smooth ER elements. Thus, AC is not restricted in its distribution to plasma membranes as usually assumed.