Biogenesis of the polymeric IgA receptor in rat hepatocytes. I. Kinetic studies of its intracellular forms

The polymeric IgA receptor (or secretory component [SC]) is a major biliary secretory protein in the rat. It was identified as an 80,000-mol-wt (80 K) glycoprotein by coprecipitation (with IgA) by anti-IgA antibodies (Sztul, E. S., K. E. Howell, and G. E. Palade, 1983, J. Cell Biol., 97:1582-1591) and was used as antigen to raise anti-SC antibodies in rabbits. Pulse labeling with [35S]cysteine in vivo, followed by the immunoprecipitation of solubilized total microsomal fractions with anti-SC sera, made possible the identification of three intracellular forms of SC (all apparently membrane proteins) and the definition of their kinetic and structural interrelations. At 5 min postinjection of [35S]cysteine, a major band of Mr 105,000 was maximally labeled. This peptide lost radioactivity concomitantly with the appearance of a radioactive doublet of Mr 116,000 and 120,000 at 15-30 min postinjection. Loss of radioactivity from 116K paralleled increased labeling of the 120K peptide which appears to be the mature form of the receptor. The 105K form was sensitive to endoglycosidase H which converted it to a 96K peptide. The 116K and 120K forms were resistant to endoglycosidase H but sensitive to endoglycosidase F which converts them to 96K and 100K forms, respectively. Taken together, these findings support the following conclusions: (a) All rat hepatic SC forms are the products of a single gene; (b) all SC forms are N-glycosylated; (c) the 116K form is the result of the terminal glycosylation of the 105K form; and (d) the 120K peptide is probably produced by modifications at other sites than its complex oligosaccharide chains.     

Binding and uptake of 125I-insulin into rat liver hepatocytes and endothelium. An in vivo radioautographic study

Electron microscope radioautography has been used to study hormone-receptor interaction. At intervals of 3, 10, and 20 min after the injection of 125I-insulin, free hormone was separated from bound hormone by whole body perfusion with modified Ringer's solution. The localization of bound hormone, fixed in situ by perfusion with glutaraldehyde, was determined. At 3 min, 125I-insulin has been shown to be exclusively localized to the hepatocyte plasmalemma (Bergeron et al., 1977, Proc. Natl. Acad. Sci. U. S. A., 74:5051--5055). In the present study, quantitation indicated that 10(5) receptors were present per cell and distributed equally along the sinusoidal and lateral segments of the hepatocyte plasmalemma. At later times, label was found in the Golgi region. At 10 min, both secretory elements of the Golgi apparatus and lysosome-like vacuoles were labeled, and at 20 min the label was especially concentrated over the latter vacuoles. Acid phosphatase cytochemistry showed that the vacuoles did not react and therefore were presumed not to be lysosomal. These Golgi vacuoles may constitute a compartment involved in the initial degradation and/or site of action of the hormone. Control experiments were carried out at all time intervals and consisted of parallel injections of radiolabeled insulin with excess unlabeled hormone. At all times in controls, label was diminished over hepatocytes and was found primarily over endothelial cells and within the macropinocytotic vesicles and dense bodies of these cells. Kupffer cells and lipocytes were unlabeled after the injection of 125I-insulin with or without excess unlabeled insulin.     

Intracellular and transcellular transport of secretory component and albumin in rat hepatocytes

The intra- and transcellular transports of hepatic secretory and membrane proteins were studied in rats in vivo using [3H]fucose and [35S]cysteine as metabolic precursors. Incorporated radioactivity in plasma, bile, and liver subcellular fractions was measured and the labeled proteins of the Golgi complex, bile, and plasma were separated by SDS PAGE and identified by fluorography. 3H-radioactivity in Golgi fractions peaked at 10 min postinjection (p.i.) and then declined concomitantly with the appearance of labeled glycoproteins in plasma. Maximal secretion of secretory fucoproteins from Golgi occurred between 10 and 20 min p.i. In contrast, the clearance of labeled proteins from Golgi membrane subfractions occurred past 30 min p.i., indicating that membrane proteins leave the Golgi complex at least 30 min later than the bulk of content proteins. A major 80,000-dalton form of secretory component (SC) was identified in the bile by co-precipitation with (IgA)2 by an anti-IgA antibody. An antibody (raised in rabbit) against the biliary 80,000-dalton peptide recognized two larger forms (116,000 and 94,000 dalton), presumably precursors, in Golgi membranes. A comparative study of kinetics of transport of 35S-SC and 35S-albumin showed that albumin peaked in bile at approximately 45 min p.i., whereas the SC peak occurred at 80 min p.i., suggesting that the transit time differs for plasma and membrane proteins that are delivered to the bile canaliculus.     

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.     

Sec23p and a novel 105-kDa protein function as a multimeric complex to promote vesicle budding and protein transport from the endoplasmic reticulum.

A cell-free protein transport reaction has been used to monitor the purification of a functional form of the Sec23 protein, a SEC gene product required for the formation or stability of protein transport vesicles that bud from the endoplasmic reticulum (ER). Previously, we reported that Sec23p is an 84-kDa peripheral membrane protein that is released from a sedimentable fraction by vigorous mechanical agitation of yeast cells and is required for ER to Golgi transport assayed in vitro. We have purified soluble Sec23p by complementation of an in vitro ER to Golgi transport reaction reconstituted with components from sec23 mutant cells. Sec23p overproduced in yeast exists in two forms: a monomeric species and a species that behaves as a 250- to 300-kDa complex that contains Sec23p and a distinct 105-kDa polypeptide (p105). Sec23p purified from cells containing one SEC23 gene exists solely in the large multimeric form. A stable association between Sec23p and p105 is confirmed by cofractionation of the two proteins throughout the purification. p105 is a novel yeast protein involved in ER to Golgi transport. Like Sec23p, it is required for vesicle budding from the ER because p105 antiserum completely inhibits transport vesicle formation in vitro.     

Calnexin acts as a molecular chaperone during the folding of glycoprotein B of human cytomegalovirus.

Human cytomegalovirus glycoprotein B (gB) is synthesized as a 105-kDa nonglycosylated polypeptide and cotranslationally modified by addition of N-linked oligosaccharides to a 160-kDa precursor in the endoplasmic reticulum (ER). It is then transported to the Golgi complex, where it is endoproteolytically cleaved to form the disulfide-linked mature gp55-116 complex. Pulse-chase experiments demonstrate that the 160-kDa gB precursor was transiently associated with calnexin, a membrane-bound chaperone, in the ER. The association was maximal immediately after synthesis, and they dissociated with a half-time of 15 min. Complete inhibition of binding by tunicamycin or castanospermine indicates the importance of N-linked oligosaccharides for it. Nonreducing sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated that during an initial stage in the biogenesis, the 160-kDa gB precursor was first synthesized as a fully reduced form and rapidly converted to an oxidized form, with a half-time of 18 min. Both forms of the gB precursor could bind to calnexin. The kinetics of the conversion from the fully reduced to the oxidized form coincided with that of dissociation of the 160-kDa gB precursor from calnexin, suggesting that the two steps are closely related.     

Non-conventional trafficking of the cystic fibrosis transmembrane conductance regulator through the early secretory pathway.

The mechanism(s) of cystic fibrosis transmembrane conductance regulator (CFTR) trafficking from the endoplasmic reticulum (ER) through the Golgi apparatus, the step impaired in individuals afflicted with the prevalent CFTR-DeltaF508 mutation leading to cystic fibrosis, is largely unknown. Recent morphological observations suggested that CFTR is largely absent from the Golgi in situ (Bannykh, S. I., Bannykh, G. I., Fish, K. N., Moyer, B. D., Riordan, J. R., and Balch, W. E. (2000) Traffic 1, 852-870), raising the possibility of a novel trafficking pathway through the early secretory pathway. We now report that export of CFTR from the ER is regulated by the conventional coat protein complex II (COPII) in all cell types tested. Remarkably, in a cell type-specific manner, processing of CFTR from the core-glycosylated (band B) ER form to the complex-glycosylated (band C) isoform followed a non-conventional pathway that was insensitive to dominant negative Arf1, Rab1a/Rab2 GTPases, or the SNAp REceptor (SNARE) component syntaxin 5, all of which block the conventional trafficking pathway from the ER to the Golgi. Moreover, CFTR transport through the non-conventional pathway was potently blocked by overexpression of the late endosomal target-SNARE syntaxin 13, suggesting that recycling through a late Golgi/endosomal system was a prerequisite for CFTR maturation. We conclude that CFTR transport in the early secretory pathway can involve a novel pathway between the ER and late Golgi/endosomal compartments that may influence developmental expression of CFTR on the cell surface in polarized epithelial cells.     

Lectin labeling of sprouting neurons. I. Regional distribution of surface glycoconjugates

Well-defined ferritin-conjugated lectins were used to map glycoconjugates on the surface of sprouting neurons from rat superior cervical ganglion (SCG) and spinal cord (SC). The cultured neurons were exposed to the markers and processed for electron microscopy, and the number of ferritin particles per unit area of plasmalemma was measured in three different regions: perikaryon, neuritic shaft, and growth cone. Three different binding patterns are observed for different lectin: equal receptor density throughout the plasmalemma of the growing neuron (e.g., Ricinus communis agglutinin I in SCG neurons), gradual decrease (e.g., wheat-germ agglutinin in SCG and SC neurons) and gradual increase (e.g., Ricinus communis agglutinin II in SC neurons) in the density of lectin receptors as one moves from the perikaryon to the growth cone. Furthermore, lectin receptor densities differ in the two types of neurons analyzed. We can conclude that the plasmalemma of the growth cone has biochemical properties different from those of the perikaryon, and that the neuron's structural polarity is expressed in its surface glycoconjugates. This phenomenon may be related to the growth cone's special functional properties and to the process of expansion of the plasma membrane.     

Estimation of Golgi membrane flow rates in ovary glands of aptenia cordifolia using cytochalasin B

Ovary gland cells of Aptenia cordifolia were exposed to 100 micrograms/ml cytochalasin B (cyt B) for 30 or 60 min during the phase of granulocrine polysaccharide secretion. The drug caused a congestion of Golgi vesicles around the dictyosomes, probably resulting from an inhibition of the vesicle migration towards the plasma membrane. The ultrastructural feature of the Golgi apparatus in control and cyt B treated cells was analyzed using stereological methods in order to estimate the mean area of vesicular membrane produced by a single dictyosome during a 30 min period of effective cyt B action. Assuming that the rate of vesicle congestion can be equated with the rate of vesicle production, the 236 dictyosomes found to be present in the non-growing ovary gland cells form 7517 vesicles in total, or approximately 32 vesicles each within a period of 30 min. This corresponds to a membrane turnover rate of 70.4 micrometers/min (this equals approximately 10% of the total plasma membrane area per min), since the mean vesicle surface area was calculated to be 0.281 microns2. The turnover time of a single Golgi cisterna was determinated to be 7.34 min, and the average vesicle life time to be 8.86 min. Discussion focuses upon the way by which the relatively high amount of vesicular membrane material incorporated into the plasmalemma is recycled into the endomembrane system. Since a bulk membrane retrieval in the form of vesicles, as well as a bulk vesicle migration from the ER to the dictyosomes could not be observed, we suggest that a transfer of membrane subunits is involved in the maintenance of membrane equilibrium in the Golgi apparatus.     

Effect of colchicine on the uptake of prolactin and insulin into Golgi fractions of rat liver

In previous studies we have shown that 125I-labeled prolactin is taken up by a receptor-dependent process and concentrated in an intact form in Golgi elements from female rat liver (J. Biol. Chem., 1979, 254:209- 214). In this study we have examined the effect of colchicine on this uptake process into Golgi elements. Colchicine [25 mumol (10 mg)/100 gm body wt] was injected intraperitoneally in adult female rats, and hepatic Golgi fractions were prepared at 1, 2, and 3 h postinjection. The enzyme recoveries and morphological appearance of fractions from colchicine-treated and control (alcohol alone) animals were similar. At times greater than 1 h after colchicine there was a marked (greater than 60%) inhibition of uptake of 125I-ovine prolactin (125I-oPRL) into Golgi light and intermediate fractions but no inhibition of uptake into Golgi heavy and plasmalemma elements. At times from 2 to 45 min postinjection, 125I-oPRL was extracted from Golgi elements and found to be largely intact as judged by rebinding to receptors. The inhibitory effect of colchicine was seen at doses ranging from 0.25 mumol to 25 mumol/100 g body wt. Vincristine also inhibited 125I-oPRL uptake into the Golgi light and intermediate fractions but lumicolchicine had no inhibitory effect. There was a smaller effect of colchicine both at early (1 h) and later (3 h) times on the extent and pattern of 125I- insulin uptake. Colchicine treatment did not produce a significant change in lactogen receptor levels in the Golgi fractions. These results demonstrate that colchicine treatment inhibited the transfer of prolactin into Golgi vesicular elements. The much smaller effect on insulin uptake suggests that there may be differences in the manner in which the two hormones are handled in the course of internalization.     

The rate of bulk flow from the endoplasmic reticulum to the cell surface

Tripeptides containing the acceptor sequence for Asn-linked glycosylation (Asn-X-Ser/Thr) were added to CHO and HepG2 cells. The tripeptides were glycosylated in the ER and then secreted into the medium, via the Golgi complex in which the oligosaccharide chains were processed. The half-time for secretion, approximately 10 min, was faster than that of known proteins transported through the same pathway. Since much evidence suggests that oligosaccharide chains are not signals for transport, it appears that no signal is necessary for rapid and efficient transport from the ER to the Golgi, or from the Golgi to the cell surface. Rather, it appears that proteins retained as permanent residents en route through the ER-Golgi transport pathway must contain specific retention signals.     

Endogenous glycosphingolipids move to the cell surface at a rate consistent with bulk flow estimates.

The bulk flow model of intracellular trafficking predicts that forward transport from the ER through the Golgi to the plasma membrane proceeds by default without a special signal being required (Wieland, F.T., Gleason, M. L., Serafini, T. A., and Rothman, J. E. (1987) Cell 50, 289-300). We tested a crucial prediction of this model, which is that the endogenous lipid components of the transport vesicles would reach the plasma membrane at the rapid rate of bulk flow. The rate at which endogenous glycosphingolipids moved from the ER through the Golgi to the plasma membrane was determined in Chinese hamster ovary cells using metabolic labeling with tritiated palmitate and oxidation of cell surface ganglioside NeuAc alpha 2----3Gal beta 1----4Glc beta 1----4Cer (GM3) with periodate. Whereas radioactive precursor became incorporated into ceramide and glucosyl ceramide without a detectable lag, synthesis of labeled lactosyl ceramide and ganglioside GM3 did not begin until 5-6 min and 11-12 min, respectively, after addition of labeled precursor. Labeled GM3 reached the plasma membrane 5-6 min following its synthesis. Overall, approximately 18 min transpired from the time that the ceramide precursor was synthesized in the ER until labeled GM3 reached the plasma membrane. These results indicate that lipid transport vesicles move rapidly to the plasma membrane at a rate consistent with bulk flow estimates.     

Membrane and secretory proteins are transported from the Golgi complex to the sinusoidal plasmalemma of hepatocytes by distinct vesicular carriers

From rat livers labeled in vivo for 30 min with [35S] cys-met, we have isolated two classes of vesicular carriers operating between the Golgi complex and the basolateral (sinusoidal) plasmalemma. The starting preparation is a Golgi light fraction (GLF) isolated by flotation in a discontinuous sucrose density gradient and processed through immunoisolation on magnetic beads coated with an antibody against the last 11 aa. of the pIgA-R tail. GLF and the ensuing subfractions (bound vs nonbound) were lysed, and the lysates processed through immunoprecipitation with anti-pIgA-R and anti-albumin antibodies followed by radioactivity counting, SDS-PAGE, and fluorography. The recovery of newly synthesized pIgA-R was > 90% and the distribution was 90% vs 10% in the bound vs nonbound subfractions, respectively. Albumin radioactivity was recovered to approximately 80%, with 20% and 80% in bound vs nonbound subfractions, respectively. Other proteins studied were: (a) secretory-apolipoprotein-B, prothrombin, C3 component of the complement, and caeruloplasmin; (b) membrane-transferrin receptor, EGR- receptor, asialoglycoprotein receptor, and the glucose transporter. In all the experiments we have performed, the secretory proteins distributed up to 85% in the nonbound subfraction (large secretory vacuoles), whereas the membrane proteins were segregated up to 95% in the bound subfraction (small vesicular carriers). These results suggest that in hepatocytes, membrane and secretory proteins are transported from the Golgi to the basolateral plasmalemma by separate vesicular carriers as in glandular cells capable of constitutive and regulated secretion.     

Intracellular transport and processing of lysosomal cathepsin B

Intracellular transport and processing of lysosomal cathepsin B was investigated in the subcellular fractions of rat liver by pulse-labeling experiments with [35S]methionine in vivo. A newly synthesized procathepsin B with a molecular weight of 39 kDa firstly appeared in the rough microsomal fraction at 10 min postinjection of label. This procathepsin B moved from the microsomal fractions to the Golgi subfractions at 30 min postinjection, and then a processed mature enzyme appeared in the lysosomal fraction at 60 min. These results suggest that the propeptide-processing of procathepsin B takes place in lysosomes in the course of intracellular transport from endoplasmic reticulum through Golgi complex to lysosomes.     

Epidermal growth factor receptor kinase translocation and activation in vivo

The rat liver epidermal growth factor (EGF) receptor was assessed for EGF-dependent autophosphorylation as well as phosphorylation of a defined exogenous substrate in purified plasmalemma and Golgiendosome fractions isolated from rat liver homogenates. While EGF-dependent kinase activity was readily detected in plasmalemma the corresponding activity in Golgi-endosome fractions required detergent. Consequent to the systemic injection of EGF in vivo, the majority (approximately 60%) of receptor as evaluated by 125I-EGF binding was rapidly lost (T 1/2 approximately 8 min) from the plasmalemma and correspondingly accumulated in the Golgi-endosome fraction in a dose-dependent manner. Electron microscope radioautography of 125I-EGF uptake into Golgi-endosome fractions identified internalization into lipoprotein-filled vesicles of heterogenous size and shape but not into stacked saccules of the Golgi apparatus. Evaluation of receptor kinase activity in plasmalemma fractions isolated at various times after EGF injection in vivo showed more rapid loss of EGF-dependent autophosphorylation activity (T 1/2 approximately 10 s) than of receptor content (T 1/2 approximately 8 min). In contrast to the EGF receptor kinase of the plasmalemma fraction, kinase activity accumulating in endosomes was activated, i.e. maximally stimulated, in the absence of EGF or Triton X-100 in vitro. Furthermore, following the peak time of accumulation of EGF receptor kinase in endosomes (5-15 min) EGF-dependent autophosphorylation activity and EGF receptor content were lost more slowly (T 1/2 approximately 27 and 87 min for the loss of autophosphorylation activity and receptor content, respectively). The rapidity of translocation of activated EGF receptor into endosomes (30 s) and the dose response to low levels (1 microgram) of EGF injected are consistent with a physiological role for internalized EGF receptor kinase activity.     

Immunoisolation and Characterization of a Subdomain of the Endoplasmic Reticulum That Concentrates Proteins Involved in COPII Vesicle Biogenesis

Rubella virus E1 glycoprotein normally complexes with E2 in the endoplasmic reticulum (ER) to form a heterodimer that is transported to and retained in the Golgi complex. In a previous study, we showed that in the absence of E2, unassembled E1 subunits accumulate in a tubular pre-Golgi compartment whose morphology and biochemical properties are distinct from both rough ER and Golgi. We hypothesized that this compartment corresponds to hypertrophied ER exit sites that have expanded in response to overexpression of E1. In the present study we constructed BHK cells stably expressing E1 protein containing a cytoplasmically disposed epitope and isolated the pre-Golgi compartment from these cells by cell fractionation and immunoisolation. Double label indirect immunofluorescence in cells and immunoblotting of immunoisolated tubular networks revealed that proteins involved in formation of ER-derived transport vesicles, namely p58/ERGIC 53, Sec23p, and Sec13p, were concentrated in the E1-containing pre-Golgi compartment. Furthermore, budding structures were evident in these membrane profiles, and a highly abundant but unknown 65-kDa protein was also present. By comparison, marker proteins of the rough ER, Golgi, and COPI vesicles were not enriched in these membranes. These results demonstrate that the composition of the tubular networks corresponds to that expected of ER exit sites. Accordingly, we propose the name SEREC (smooth ER exit compartment) for this structure.     

Synthesis of membrane glycoproteins in rat small-intestinal villus cells. Redistribution of l-[1,5,6-3H]-fucose-labelled membrane glycoproteins among Golgi, lateral basal and microvillus membranes in vivo

The biogenesis of plasmalemma glycoproteins of rat small-intestinal villus cells was studied by following the incorporation of l-[1,5,6-(3)H]fucose, given intraperitoneally with and without chase, into Golgi, lateral basal and microvillus membranes. Each membrane fraction showed distinct kinetics of incorporation of labelled fucose and was differently affected by the chase, which produced a much greater decrease in incorporation of label into Golgi and microvillus than into lateral basal membranes. The kinetic data suggest a redistribution of newly synthesized glycoproteins from the site of fucosylation, the Golgi complex, directly into both lateral basal and microvillus membranes. The observed biphasic pattern of label incorporation into the microvillus membrane fraction may be evidence for a second indirect route of incorporation. The selective effect of the chase suggests the presence of two different pools of radioactive fucose in the Golgi complex that differ in (1) their accessibility to dilution with non-radioactive fucose, and (2) their utilization for the biosynthesis of membrane glycoproteins subsequently destined for either the microvillus or the lateral basal parts of the plasmalemma. The radioactively labelled glycoproteins of the different membrane fractions were separated by sodium dodecyl sulphate/polyacrylamide-slab-gel electrophoresis and identified by fluorography. The patterns of labelled glycoproteins in Golgi and lateral basal membranes were identical at all times. At least 14 bands could be identified shortly after radioactive-fucose injection. Most seemed to disappear at later times, although one of them, which was never observed in microvillus membranes, increased in relative intensity. All but two of the labelled glycoproteins present in the microvillus membrane corresponded to those observed in Golgi and lateral basal membranes shortly after fucose injection. The patterns of labelled glycoproteins in all membrane fractions were little affected by the chase. These data support a flow concept for the insertion of most surface-membrane glycoproteins of the intestinal villus cells.     

Reconstitution of protein transport from the endoplasmic reticulum to the Golgi complex in yeast: the acceptor Golgi compartment is defective in the sec23 mutant

Using either permeabilized cells or microsomes we have reconstituted the early events of the yeast secretory pathway in vitro. In the first stage of the reaction approximately 50-70% of the prepro-alpha-factor, synthesized in a yeast translation lysate, is translocated into the endoplasmic reticulum (ER) of permeabilized yeast cells or directly into yeast microsomes. In the second stage of the reaction 48-66% of the ER form of alpha-factor (26,000 D) is then converted to the high molecular weight Golgi form in the presence of ATP, soluble factors and an acceptor membrane fraction; GTP gamma S inhibits this transport reaction. Donor, acceptor, and soluble fractions can be separated in this assay. This has enabled us to determine the defective fraction in sec23, a secretory mutant that blocks ER to Golgi transport in vivo. When fractions were prepared from mutant cells grown at the permissive or restrictive temperature and then assayed in vitro, the acceptor Golgi fraction was found to be defective.     

Topology of asialoglycoprotein receptor in rat liver subcellular fractions: a ferritin immunoelectron microscopic study

Direct ferritin immunoelectron microscopy was used to visualize the asialoglycoprotein receptor in various rat liver subcellular fractions. The cytoplasmic surfaces of cytoplasmic organelles such as the rough and smooth microsomes, Golgi cisternae and lysosomes showed hardly any ferritin label exception for the slight labeling of secretory granules found mainly in the light Golgi fraction (GF1). Occasionally, however, open membrane sheet structures, smooth vesicular or tubular structures heavily labeled with ferritin, were present in all these subcellular fractions. These structures probably correspond to fragmented sinusoidal or lateral hepatocyte plasma membranes recovered to these subcellular fractions. When the limiting membranes of the secretion granules were partially broken by mechanical force, a number of ferritin particles frequently were seen attached in large clusters to the luminal surface of the membrane, the cytoplasmic surface of the corresponding domain being slightly labeled. These observations are strong evidence that the receptor protein is never translocated vertically throughout the intracellular transport from ER to plasma membrane via Golgi apparatus and from plasma membrane back to trans-Golgi elements and also in lysosomes, always exposing the major antigenic sites to the luminal or extracellular surface and the minor counterparts to the cytoplasmic surface of the membranes. The receptor protein also is suggested to be concentrated in clusters on the luminal surface of secretion granules when they form on the trans-side of the Golgi apparatus.     

Pulse-chase studies of the synthesis and intracellular transport of apolipoprotein B-100 in Hep G2 cells

The synthesis and secretion of apolipoprotein B-100 (apoB-100) have been studied in a human hepatoma cell line, the Hep G2 cells. The time needed for the synthesis of apoB-100 was estimated to be 14 min, which corresponds to a translation rate of approximately 6 amino acids/s. ApoB-100 was compared with albumin and alpha 2-macroglobulin as to the distribution between the membrane and the luminal content in the endoplasmic reticulum (ER) and the Golgi apparatus. The results suggested that apoB-100 approximately followed the distribution of these secretory proteins in the Golgi, while the ratios between the percent membrane-bound apoB-100 and percent membrane-bound albumin or alpha 2-macroglobulin were 3-4:1 in the ER. This may suggest that apoB-100 occurs in a membrane-associated form in ER prior to the integration in the lipoproteins. Pulse-chase studies combined with subcellular fractionation was used to investigate the kinetics for the intracellular transfer of apoB-100. A 3-min pulse of [35S]methionine was followed by an increase in apoB-100 radioactivity in the ER during the first 10-15 min of chase. The following 10-15 min of chase were characterized by linear decrease in apoB-100 radioactivity with a decay rate of approximately 6%/min. The residence kinetics for apoB-100 in the ER differed from that of transferrin and probably also from that of albumin. By comparing the time for the pulse maximum in ER with that in the denser Golgi fractions the time needed for the transfer between ER and Golgi could be estimated to be 10 min. The time needed for the secretion of newly synthesized apoB-100 was estimated to be 30 min. This indicates that the transfer of the protein through the Golgi apparatus to the extracellular space requires 20 min.