Investigation of a possible correlation between rates of secretion and microsomal membrane association of plasma proteins synthesized by rat liver.

The rates of secretion of complement C3, haptoglobin and plasminogen have been determined after pulse labelling with [3H]leucine, and compared to the secretion of prothrombin, albumin and transferrin investigated previously (Kvalvaag, A.H., Tollersrud, O.K. and Helgeland, L. (1988) Biochim. Biophys. Acta 937, 319-327). To study membrane association, rough microsomes were treated with increasing concentrations of saponin, sodium deoxycholate or Triton X-100. All six proteins were quantitated in the soluble and membrane fraction by enzyme immunoassays. At concentrations of saponin from 0.08% to 0.32%, each secretory protein showed a characteristic distribution, almost identical to that obtained with 0.05% sodium deoxycholate or 0.08% Triton X-100. Albumin and transferrin with half-times for secretion (t1/2) 30 and 75 min, respectively, are both almost exclusively found in the luminal fraction (greater than 95%). Prothrombin and plasminogen, which both show an intermediate t1/2 (approx. 55 min), are partially associated with the membranes, as only about 60% was released. Haptoglobin and complement C3 also show some association with the membranes (80-85% released). C3 is secreted at the same rate as prothrombin and plasminogen (t1/2 = 55 min), whereas haptoglobin is secreted more rapidly (t1/2 = 40 min). Accordingly, no correlation between kinetics of secretion and membrane association was demonstrated.     

Hormonal regulation during secretion of alpha-fetoprotein in hepatoma cells grown in synthetic medium

The variant cell line of H4-II-E-C3 cells derived from the Reuber H-35 hepatoma cells has been established using protein- and lipid-free synthetic medium. This H4-II-E-C3-V line can synthesize and secrete considerable amounts of alpha-fetoprotein (AFP) and albumin. The addition of 5 X 10(-7) M dexamethasone to the medium stimulated the excretion of AFP without increasing total AFP synthesis, whereas 8.7 X 10(-8) M insulin inhibited the excretion of AFP without a significant inhibition of intracellular AFP synthesis. However, neither dexamethasone nor insulin altered either the cellular or secreted levels of albumin. Cells were pulse labeled with [35S]methionine and then chased after addition of excess unlabeled methionine. AFP appeared in the medium after 10 min, and 50% of the protein was secreted after 110 min. The rate of secretion of AFP was much slower than that of albumin, 50% of which was secreted after 25 min. Dexamethasone, 5 X 10(-7) M, caused a marked enhancement in the rate of AFP secretion, with 50% released after 75 min. Insulin, 8.7 X 10(-8) M, by contrast, caused a marked delay in AFP secretion with only 20% released after 180 min and then a plateau was approached. Since the intracellular AFP was excreted 55% after 180 min the remaining 25% of newly made AFP was suggested to be degraded during secretion. The kinetics of movement of AFP during secretion and endoglycosidase H treatment of intracellular and secreted AFP suggested that insulin impeded the transport of AFP from the rough endoplasmic reticulum to the Golgi apparatus.     

The biosynthesis of rat transferrin. Evidence for rapid glycosylation, disulfide bond formation, and tertiary folding

The transit time of newly synthesized transferrin in the liver is markedly longer than that of albumin. We sought to learn the basis of this difference by the use of labeled leucine and mannose in vivo and by isolation of newly formed transferrin from rough microsomes of rat liver. Albumin and alpha 1-antitrypsin, a second glycoprotein, were also studied for comparison. Minimal hepatic transit times were 17, 23, and 31 min for albumin, alpha 1-antitrypsin, and transferrin, respectively. The delay in the case of transferrin was found to occur chiefly in the rough endoplasmic reticulum and to be paralleled by an increase in the amount of transferrin relative to albumin in that organelle. Initial glycosylation of transferrin was as rapid as that of alpha 1-antitrypsin, and essentially all of the transferrin in the rough endoplasmic reticulum contained glycans which bound to concanavalin A and were removed by endoglycosidase H. Only 3% of the transferrin isolated from the rough microsomes came from the plasma by endocytosis or adsorption. Rapidity of disulfide bond formation in rough microsomes was evident from the presence of only 1.3 cysteine thiols/molecule of rough microsomal transferrin (total of 19 cystines) and the absence of mixed disulfides. Peptide patterns upon mild proteolysis were consistent with a native configuration of disulfide bond pairing. The ability of rough microsomal transferrin to bind and deliver iron through interaction with transferrin receptors on reticulocytes suggests that considerable tertiary structure is present. Thus, initial glycosylation, disulfide bridging, and tertiary folding are all rapid processes. The cause for the slow release of transferrin from the rough endoplasmic reticulum may lie with a rate-limiting transfer mechanism.     

Changes in the random distribution of sialic acid at the surface of the myeloid sinusoidal endothelium resulting from the presence of diaphragmed fenestrae

Frog exocrine pancreatic tissue was studied in vitro under conditions which maintain the differences between tissues from fasted and fed animals. Sodium dodecyl sulfate (SDS) gel electrophoresis after labeling with [14C]amino acids showed that feeding stimulated the synthesis of secretory proteins to the same relative degree as the overall protein synthesis. The intracellular transport of secretory proteins was studied by electronmicroscopy autoradiography after pulse-labeling with [3H]leucine. It was found that the transport route is similar under both feeding conditions. After their synthesis in the rough endoplasmic reticulum (RER), the proteins move through the peripheral elements and cisternae of the Golgi system into the condensing vacuoles. The velocity of the transport increases considerably after feeding. When frogs are fasted, the release of labeled proteins from the RER takes greater than 90 min, whereas after feeding, this happens within 30 min. Comparable differences were observed for transport through the Golgi system. The apparent differences between the frog and mammalian pancreas in the regulation of synthesis, intracellular transport, and secretion of proteins are discussed.     

Effect of corynetoxin isolated from parasitized annual ryegrass on albumin and transferrin synthesis and secretion by cultured fetal rat hepatocytes

A group of glycolipid toxins, corynetoxin (CT), isolated from parasitized annual ryegrass, was shown to suppress the synthesis of both albumin and transferrin by cultured fetal rat hepatocytes. Based on [³H]leucine incorporation, inhibition of transferrin synthesis was greater than that of both albumin and total protein synthesis. As a result, the secretion of albumin and transferrin was decreased. The incorporation of [³H]N-AcGlc into cellular glycoproteins was only marginally affected by CT, although a dramatic reduction was observed with respect to the secreted proteins. Transferrin secreted into the culture medium was substantially non-glycosylated, judging by the absence of [³H]N-AcGlc. These studies suggested that the toxin preferentially affects the synthesis, and hence the secretion of glycoproteins, although it did not block the secretion of the proteins albumin and transferrin, as these did not accumulate intercellularly. Since transferrin labelled with [³H]leucine but not [³H]N-AcGlc is detected in the culture medium of hepatocytes exposed to CT, it was concluded that glycosylation of the protein is not required for secretion. This study shows that the effects of CT on protein synthesis and secretion in cultured hepatocytes are similar to those reported for tunicamycin (TM).     

Albumin secreted by rat liver bypasses Golgi apparatus cisternae

Albumin was isolated immunologically from various subcellular fractions from livers of adult male rats receiving an intraperitoneal injection of [³H]leucine to investigate the kinetics and pathway of subcellular transfer of newly synthesized albumin during secretion. At appropriate time intervals, livers were excised and fractionated into endoplasmic reticulum and Golgi apparatus. Golgi apparatus were further subfractionated into cisternae and secretory vesicles. In endoplasmic reticulum fractions, labeled albumin appeared within 7.5 min of injection of isotope, followed by a rapid decline in specific activity. Albumin in Golgi apparatus was labeled and concentrated in secretory vesicles over 25 min. The radioactivity in albumin per mg total protein was highest in secretory vesicles and insignificant in the cisternal fraction. Labeled albumin was present in serum by 30 min and radioactivity in serum albumin reached a plateau within 60–90 min after injection of isotope. Results provide evidence for the migration of albumin from its site of synthesis on endoplasmic reticulum membrane-bound polyribosomes to its site of secretion into the circulation via the Golgi apparatus. The pathway of albumin transport to secretory vesicles is suggested to involve peripheral elemenst of the Golgi apparatus. Secretory vesicle formation and maturation required 20 to 30 min for completion, via a mechanism whereby the inner spaces of the central saccules may be bypassed.     

Variability in transport rates of secretory glycoproteins through the endoplasmic reticulum and Golgi in human hepatoma cells

We have previously shown that newly synthesized liver secretory proteins are exported at three distinct characteristic rates, with intracellular retention half-times of 110-120 min (e.g. transferrin), 75-80 min (e.g. ceruloplasmin), and 30-40 min (e.g. alpha 1-protease inhibitor) (J. B. Parent, H. Bauer, and K. Olden (1985) Biochim. Biophys. Acta, in press). In the present study we have determined the average time required for specific glycoproteins to move through the various compartments of the intracellular transport pathway, consisting of endoplasmic reticulum and Golgi complex. Localization in particular compartments was monitored by the use of the following complementary approaches: (i) Percoll density gradient fractionation of the subcellular organelles, (ii) sensitivity of the glycan moiety of N-linked glycosylation to endo-beta-N-acetylglucosaminidase H, and (iii) by the lectin-binding characteristics. The cell fractionation studies revealed that alpha 1-protease inhibitor, ceruloplasmin, and transferrin were transported from the rough endoplasmic reticulum with a retention half-time of 10, 30, or 45 min, respectively. Measurements of the rate at which newly synthesized glycoprotein became endo H-resistant (an event localized near the medial region of Golgi) demonstrated that it took 60-70, 30, and 18 min for 50% of transferrin, ceruloplasmin, and alpha 1-protease inhibitor, respectively, to reach the medial Golgi. Consistent with this finding, maximal binding of transferrin to wheat germ agglutinin (also a medial Golgi event) and Ricinus communis agglutinin I (a trans Golgi event) required 75 and 90 min, respectively, and maximal binding of ceruloplasmin to both lectins occurred in approximately 30 min. Maximal binding of alpha 1-protease inhibitor to wheat germ agglutinin and Ricinus communis agglutinin I required 15 and 30 min, respectively. The results presented here clearly indicate that (i) the time required for protein secretion cannot be entirely accounted for by lag in transport from the rough endoplasmic reticulum to the Golgi since the glycoproteins examined are retained in the former organelle for no more than two-fifths of the total intracellular retention half-time, and (ii) the variability in rates of protein secretion is not due solely to differences in rates of transport from the rough endoplasmic reticulum to the Golgi as variability in retention within the Golgi is also demonstrated. The results are discussed in terms of their compatibility with receptor-mediated transport of glycoproteins in both the endoplasmic reticulum and Golgi.     

Movement of zymosterol, a precursor of cholesterol, among three membranes in human fibroblasts

Where examined, cholesterol is synthesized in the endoplasmic reticulum; however, its precursor, zymosterol, is found mostly in the plasma membrane. The novel implication of these disparate findings is that zymosterol circulates within the cell. In tracing its movements, we have now established the following: (a) in human fibroblasts, zymosterol is converted to cholesterol solely in the rough ER. (b) Little or no zymosterol or cholesterol accumulates in the rough ER in vivo. (c) Newly synthesized zymosterol moves to the plasma membrane without a detectable lag and with a half-time of 9 min, about twice as fast as cholesterol. (d) The pool of radiolabeled zymosterol in the plasma membrane turns over rapidly, faster than does intracellular cholesterol. Thus, plasma membrane zymosterol is not stagnant. (e) [3H]Zymosterol pulsed into intact cells is initially found in the plasma membrane. It is rapidly internalized and is then converted to [3H] cholesterol. Half of the [3H]cholesterol produced returns to the plasma membrane within 30 min of the initial [3H]zymosterol pulse. (f) Nascent zymosterol accumulates in a buoyant sterol-rich intracellular membrane before it reaches the plasma membrane. This membrane also acquires nascent cholesterol, exogenous [3H]zymosterol pulsed into intact cells, and [3H]cholesterol synthesized from the exogenous [3H] zymosterol. These results suggest that at least one sterol moves rapidly and in both directions among the rough endoplasmic reticulum, a sterol-rich intracellular membrane bearing nascent cholesterol, and the plasma membrane.     

Glucose removal from N-linked oligosaccharides is required for efficient maturation of certain secretory glycoproteins from the rough endoplasmic reticulum to the Golgi complex

1- Deoxynojirimycin is a specific inhibitor of glucosidases I and II, the first enzymes that process N-linked oligosaccharides after their transfer to polypeptides in the rough endoplasmic reticulum. In a pulse- chase experiment, 1- deoxynojirimycin greatly reduced the rate of secretion of alpha 1-antitrypsin and alpha 1-antichymotrypsin by human hepatoma HepG2 cells, but had marginal effects on secretion of the glycoproteins C3 and transferrin, or of albumin. As judged by equilibrium gradient centrifugation, 1- deoxynojirimycin caused alpha 1- antitrypsin and alpha 1-antichymotrypsin to accumulate in the rough endoplasmic reticulum. The oligosaccharides on cell-associated alpha 1- antitrypsin and alpha 1-antichymotrypsin synthesized in the presence of 1- deoxynojirimycin , remained sensitive to Endoglycosidase H and most likely had the structure Glu1- 3Man9GlcNAc2 . Tunicamycin, an antibiotic that inhibits addition of N-linked oligosaccharide units to glycoproteins, had a similar differential effect on secretion of these proteins. Swainsonine , an inhibitor of the Golgi enzyme alpha- mannosidase II, had no effect on the rates of protein secretion, although the proteins were in this case secreted with an abnormal N- linked, partially complex, oligosaccharide. We conclude that the movement of alpha 1-antitrypsin and alpha 1-antichymotrypsin from the rough endoplasmic reticulum to the Golgi requires that the N-linked oligosaccharides be processed to at least the Man9GlcNAc2 form; possibly this oligosaccharide forms part of the recognition site of a transport receptor for certain secretory proteins.     

Synthesis and secretion of albumin by a synchronized rat hepatoma cell line.

The rat hepatoma cell H4-12 which synthesizes and secretes albumin was synchronized by growth in isoleucine-deficient medium followed by a second block with excess thymidine. Albumin synthesis and secretion was measured in the synchronized cells at different time intervals representative of early S, late S, G2, mitosis, early G1 and late G1 phases of the cell cycle. Maximal albumin synthesis occurred during G1 although significant synthesis also occurred during the other cell cyle phases. Most (75--80%) of the radioactive albumin produced during a 15 min pulse incubation with L-[4,5-3H] leucine was found in the microsomal cell fraction and this nascent albumin was secreted into the incubation medium during a 160 min chase period. Fifty percent of the nascent albumin was secreted by 50--55 min and this pattern of secretion did not change during the cell cycle. These data indicate that albumin synthesis occurs throughout the cell cycle but that it is preferred during G1. The rate of intracellular transport and secretion of albumin does not vary during the different phase of the cell cycle.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [3H]leucine or [3H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [3H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [3H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [3H]mannose and [14C]leucine confirmed this. The decreased incorporation of [3H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Intrahepatic assembly of very low density lipoproteins. Rate of transport out of the endoplasmic reticulum determines rate of secretion

To identify the rate-limiting step(s) in the hepatic production of very low density lipoproteins (VLDL), we investigated the intracellular distribution and rate of intracellular transport of de novo synthesized apolipoprotein B (apoB). For all secretory proteins examined (i.e. albumin, large molecular weight apoB, and small molecular weight apoB) the rough and smooth microsomes contained the majority of intracellular de novo synthesized protein, while the Golgi subfraction contained 10% or less. Pulse-chase analysis of the intracellular movement of apoB and albumin showed that the first order rate constant (in terms of half-life) describing the rate of movement out of the smooth and rough microsomes determined the overall rate of movement out of the cell. These data suggest that movement out of the endoplasmic reticulum, the site where VLDL is assembled, determines the overall rate of secretion. Furthermore, compared to albumin, the rate of intracellular transport of apoB was approximately two times slower suggesting that processing unique to VLDL apoB occurring in the endoplasmic reticulum was responsible. Additional studies show that essentially all of the de novo synthesized 35S-labeled albumin (produced from a pulse of [35S]methionine) lost from the cell during the chase period could be recovered in the culture medium. In contrast, much less of large molecular weight apoB (36%) and small molecular weight apoB (60%) was recovered in the culture medium. Since these cultured rat hepatocytes do not take up or degrade newly secreted apoB, these data suggest that a significant amount of apoB is degraded intracellularly.     

Accelerated secretion of human lysozyme with a disulfide bond mutation.

The mutant human lysozyme, [Ala77, Ala95]lysozyme, in which the disulfide bond Cys77-Cys95 is eliminated, is known to exhibit increased secretion in yeast, compared to wild-type human lysozyme [Taniyama, Y., Yamamoto, Y., Nakao, M., Kikuchi, M. & Ikehara, M. (1988) Biochem. Biophys. Res. Commun. 152, 962-967]. To investigate this phenomenon, mammalian cells were used to analyze the secretion kinetics of [Ala77, Ala95]lysozyme and wild-type human lysozyme. The secretion rate of [Ala77, Ala95]lysozyme during the 150-min chase period was significantly accelerated [half-life (t1/2) = 29 min] compared to that of wild-type human lysozyme (t1/2 = 83 min), when expressed at the same levels within the cells. In contrast, after the 150-min chase, the rates of disappearance of both wild-type and mutant human lysozymes within the cells were similar, and considerably slower (t1/2 = 220 min), respectively. The remaining intracellular wild-type human lysozyme was localized mainly in the endoplasmic reticulum, whereas accelerated transport of the [Ala77, Ala95]lysozyme mutant protein from the endoplasmic reticulum to the Golgi apparatus was observed. Also in yeast cells, similar secretion kinetics and the differences in t1/2 for wild-type and mutant human lysozymes during the early chase period were observed. The two-phase kinetics of disappearance of intracellular human lysozymes suggest that only a proportion of the proteins becomes secretion competent soon after synthesis and is completely secreted during the early chase period, whereas others enter the distinct, slow pathways of intracellular transport and/or degradation. Increased secretion of [Ala77, Ala95]lysozyme is possibly due to enhanced competence for secretion acquired in the endoplasmic reticulum at the early stage of transport events, which is closely connected with the removal of a disulfide bond.     

Cytological events involved in glycoprotein synthesis in cellular and syncytial trophoblast of human placenta. An electron microscope autoradiographic study of [3H]galactose incorporation

Electron microscope autoradiography was used to study glycoprotein synthesis in cellular trophoblast (cytotrophoblast) and syncytial trophoblast of term human placental villi incubated in vitro with D-[1-3H]galactose ([3H]gal). Autoradiographs were analyzed using the hypothetical grain analysis of Blackett and Parry (1973. J. Cell Biol. 57:9-15). The results of this study indicated that [3H]gal incorporation into term placental villi was predominantly localized to cytotrophoblast. Utilization of [3H]gal by term syncytial trophoblast was extremely low and yielded too few grains for a quantitative grain analysis. This result is in striking contrast to that found in the preceding study of [3H]leucine incorporation (Nelson, D. M., A. C. Enders, and B. F. King. 1978). Within cytotrophoblast, the rough endoplasmic reticulum incorporated the most [3H]gal into glycoprotein. The Golgi apparatus was another site of [3H]gal incorporation. The vast majority of the [3H]gal incorporated into cytotrophoblast during the pulse incubation remained intracellular through the duration of the experiment. There was little autoradiographic evidence for secretion of tritiated macromolecules. Cytotrophoblast incubated for the longest time period studied (4 h+) showed a substantial concentration of tritiated macromolecules in the Golgi complex and in the ground plasm but not in the rough endoplasmic reticulum.     

The hormonal control of protein N-glycosylation in the developing rabbit mammary gland and its effect upon transferrin synthesis and secretion

Pregnant rabbit mammary gland explants cultured with insulin, prolactin and cortisol, synthesise and secrete transferrin radiolabelled with [³H]leucine or [³H]mannose. Omission of prolactin from the culture medium inhibited the incorporation of [³H]leucine into casein but not transferrin. Total transferrin secreted under these conditions was approx. 75% of the control (+ prolactin) value measured by rocket immunoelectrophoresis. Little incorporation of [³H]mannose into transferrin was seen in the absence of prolactin suggesting a lack of glycosylation of the protein. Dual label experiments with [³H]mannose and [¹⁴C]leucine confirmed this. The decreased incorporation of [³H]mannose into dolichol linked intermediates suggests a general effect on protein N-glycosylation in the absence of prolactin. Thus, while the synthesis of the polypeptide backbone of transferrin does not require prolactin its glycosylation does.     

Biosynthesis of high density lipoprotein by chicken liver: conjugation of nascent lipids with apoprotein A1

To study the assembly of newly synthesized lipids with apoprotein A1, we administered [2-3H]glycerol to young chickens and determined the hepatic intracellular sites of lipid synthesis and association of nascent lipids with apoprotein A1. [2-3H]glycerol was rapidly incorporated into hepatic lipids, reaching maximal levels at 5 min, and this preceded the appearance of lipid radioactivity in the plasma. The liver was fractionated into rough and smooth endoplasmic reticulum and Golgi cell fractions. The isolated cell fractions were further subfractionated into membrane and soluble (content) fractions by treatment with 0.1 M Na2CO3, pH 11.3. At various times, the lipid radioactivity was measured in each of the intracellular organelles, in immunoprecipitable apoprotein A1, and in materials that floated at buoyant densities similar to those of plasma lipoproteins. Maximal incorporation occurred at 1 min in the rough endoplasmic reticulum, at 3-5 min in the smooth endoplasmic reticulum, and at 5 min in the Golgi cell fractions. The majority (66-93%) of radioactive glycerol was incorporated into triglycerides with smaller (4-27%) amounts into phospholipids. About 80% of the lipid radioactivity in the endoplasmic reticulum and 70% of that in the Golgi cell fractions was in the membranes. The radioactive lipids in the content subfraction were distributed in various density classes with most nascent lipids floating at a density less than or equal to 1.063 g/ml. Apoprotein A1 from the Golgi apparatus, obtained by immunoprecipitation, contained sixfold more nascent lipids than did that from the endoplasmic reticulum. These data indicate that [2-3H]glycerol is quickly incorporated into lipids of the endoplasmic reticulum and the Golgi cell fractions, that most of the nascent lipids are conjugated with apoproteins A1 in the Golgi apparatus, and that very little association of nascent lipid to apoprotein A1 occurs in the endoplasmic reticulum.     

The secretion of tropoelastin by chick-embryo artery cells.

Chymotryptic fingerprint analyses of tropoelastin a and tropoelastin b demonstrated a very close relationship between these two polypeptides synthesized in a cell-free system under the direction of chick-embryo polyribosomal mRNA. A similar study on tropoelastin polypeptides extracted in their hydroxylated and under-hydroxylated forms from artery cells incubated with [3H]valine in the absence and presence of alpha alpha'-bipyridine or 3,4-dehydroproline confirmed this close relationship and suggested that tropoelastins a and b are likely to be the products of a single gene. Pulse-chase experiments in which the synthesis and secretion of tropoelastin by artery cells were monitored demonstrated that, after a pulse with [3H]proline, the polypeptides rapidly appeared in the medium and the half-time of tropoelastin secretion was approx. 30 min. Further pulse-chase studies, in which [3H]tropoelastin contents of subcellular fractions were determined, showed that rough and smooth microsomal fractions contained maximal amounts of tropoelastin at different times. The quantity of tropoelastin in the smooth-microsomal fraction was always only a small proportion of that in the rough-microsomal fraction, suggesting rapid translocation of the polypeptides to the plasma membrane. Incubation of the cells with 0.1 mM-colchicine did not markedly alter the rate of secretion or the distribution of tropoelastin between the subcellular fractions, whereas when 1 microM-monensin was included in the incubations the polypeptides were retained in the rough microsomal fraction. The results are consistent with the proposal that tropoelastin may follow a pathway of secretion from rough endoplasmic reticulum to the plasma membrane via secretory vesicles.     

Secretion of proalbumin by canavanine-treated Hep-G2 cells

The two processing sites in the conversion of preproalbumin to albumin are marked by arginine residues. Therefore, to study the mechanisms of albumin processing and secretion, the arginine residues of nascent albumin were replaced with canavanine by the incubation of Hep-G2 cells with this arginine analog. During a 4-h interval, canavanine inhibited (67%) the secretion of nascent albumin and increased the intracellular transit time of albumin secretion from 24 to 39 min. At 1 h, canavanine inhibited total protein synthesis by 19% and albumin synthesis by about 40%. Both the intracellular and secreted albumin produced by canavanine-treated cells were analyzed by DEAE-cellulose chromatography and were found to be more acidic than normal proalbumin and albumin. Further analysis on sodium dodecyl sulfate polyacrylamide gel electrophoresis indicated that the albumin produced and secreted by canavanine-treated cells appeared to have a larger molecular weight (by 4000) than serum albumin. The canavanine-treated cells were incubated with L-[3H]leucine and L-[3H]phenylalanine and the location of radioactive L-leucine and L-phenylalanine in the 30 NH2-terminal amino acid residues of secreted albumin was determined. The results indicated that canavanine-treated cells secreted proalbumin (79%) and also some fully processed albumin (21%). Preproalbumin was not secreted. Untreated Hep-G2 cells mostly secreted fully processed serum albumin (93%) with only traces of proalbumin (7%).     

Colchicine inhibition of plasma protein release from rat hepatocytes

Colchicine, both in vitro and in vivo, inhibits secretion of albumin and other plasma proteins. In vitro, secretion by rat liver slices is inhibited at 10-minus 6 M with maximal effect at 10-minus 5 M. Inhibition of secretion is accompanied by a concomitant retention of nonsecreted proteins within the slices. Colchicine does not inhibit protein synthesis at these concentrations. Vinblastine also inhibits plasma protein secretion but lumicolchicine, griseofulvin, and cytochalasin B do not. Colchicine also acts in vivo at 10-25 mumol/100 g body weight. Inhibition of secretion is not due to changes in the intracellular nucleotide phosphate levels. Colchicine, administered intravenously, acts within 2 min and its inhibitory effect lasts for at least 3 h. Colchicine has no effect on transport of secretory proteins in the rough or smooth endoplasmic reticulum but it causes these proteins to accumulate in Golgi-derived secretory vesicles.     

Biosynthesis of high density lipoprotein by chicken liver: intracellular transport and proteolytic processing of nascent apolipoprotein A-1

To study the in vivo processing and secretion of Apolipoprotein A-I (Apo A-I), young chickens were administered individual L-[3H]amino acids intravenously and the time of intracellular transport of nascent Apo A-I from rough endoplasmic reticulum (RER) to the Golgi apparatus was measured. Within 3 to 9 min there was maximal incorporation of radioactivity into Apo A-I in both the RER and the Golgi cell fractions. By contrast, the majority of radioactive albumin was also present in the RER by 3 to 9 min, but did not reach peak amounts in the Golgi fraction until 9 to 25 min. Both radioactive Apo A-I and albumin appeared in the blood at about the same time (between 20 and 30 min). NH2-terminal amino acid sequence analysis of nascent intracellular Apo A-I showed that it contains a pro-hexapeptide extension identical to that of human Apo A-I. After 30 min of administration of radioactive amino acids radioactive Apo A-I was isolated by immunoprecipitation from the liver and serum. NH2-terminal sequence analysis of 20 amino acids indicated that chicken liver contained an equal mixture of nascent pro-Apo A-I and fully processed Apo A-I, whereas the serum only contained processed Apo A-I. Further studies showed that the RER only contained pro-Apo A-I, whereas a mixture of pro-Apo A-I and processed Apo A-I was found in the Golgi complex. These results indicate that, in chicken hepatocytes, there is a more rapid transport of Apo A-I than of albumin from the RER to the Golgi cell fractions, and that Apo A-I remains in the Golgi apparatus for a longer period of time before it is secreted into the blood. In addition these studies show that the in vivo proteolytic processing of chicken pro-Apo A-I to Apo A-I occurs in the Golgi cell fractions.