Evidence for the Participation of the Golgi Apparatus in the Intracellular Transport of Nascent Albumin in the Liver Cell

Colchicine inhibits the secretion into plasma of lipoproteins (12,18), albumin (11), and other proteins (11) produced by the liver. Electron microscopy has shown that, after administration of colchicine, the electron-dense, nonsecreted lipoproteins accumulate in cytoplasmic vesicles (12) and also in the Golgi apparatus (18) of hepatocytes; however, the accumulation site of the electron-transparent, nonsecreted proteins is unknown. In the present report, we demonstrate that fibrinogen, an electron-transparent protein synthetized by the liver cells (16), accumulates not only in the Golgi apparatus, but also within the endoplasmic reticulum when its secretion into plasma is inhibited by colchicine.Fibrinogen was demonstrated in the hepatocytes by means of antirat fibrinogen antibodies labeled with peroxidase, a method which can be used to detect antigens under light and electron microscopy (3). MATERIALS AND METHODSAdult male rats were injected with colchicine, 2.5 mg/kg, intraperitoneally, and were sacrificed 4, 6, 8, 16, 24, or 32 h after injection; rats, injected with 0.9% NaCI or with 2.5 mg/kg of lumicolchicine (a mixture of colchicine isomers prepared by irradiation of colchicine with ultraviolet light [20]) and sacrificed at the same time intervals, acted as controls; the experimental groups corresponding to each time interval consisted of five animals.Antirat fibrinogen antibodies were prepared as follows: a sheep was immunized by an intradermic injection of 7 mg of rat fibrinogen added with Freund's complete adjuvant, followed by an intramuscular injection of fibrinogen with Freund's adjuvant every month for 4 mo; rat fibrinogen was prepared according to the method of Poison et al. (14). Antibodies against antigens other than fibrinogen were eliminated by an immunoadsorbent technique (4) using an immunoadsorbent prepared from normal rat serum; purified antirat fibrinogen antibodies were finally obtained by the same technique, using an immunoadsorbent prepared from normal rat plasma. From those purified antibodies, Fab fragments were prepared by digestion with I% papain (15) and purified by filtration on a diethylaminoethyl-cellulose column (DE 52, Whatman Biochemicals Ltd., Maidstone, England) with sodium phosphate buffer 0.01 M pH 8.0; the Fab fragments were concentrated and labeled with horseradish peroxidase (RZ 3.0, Sigma Chemical Co., St. Louis, Mo.) according to Avrameas (2). For control reactions, Fab fragments were also prepared from normal sheep gamma globulins obtained by ammonium sulfate precipitation followed by filtration on a diethylaminoethyl-cellulose column (DE 52) with sodium phosphate buffer 0.017 M pH 6.9; those Fab fragments were also concentrated and labeled with horseradish peroxidase.Liver specimens were processed according to a technique previously described (6). Briefly, the specimens were at once fixed in 10% paraformaldehyde (prepared as described by Karnovsky [10]) buffered with phosphate buffer 0.1 M pH 7.4 for 6 h at 4~ After fixation, the liver pieces were washed for 24-48 h...

Section: Discussionmentioning

confidence: 99%

Inhibition by colchicine of fibrinogen translocation in hepatocytes.

Feldmann¹,

Maurice²,

Sapin³

et al. 1975

“…Mannose, the other sugar which is closest to the protein, has also been shown to be incorporated mostly in the rough ER ; and the sugars situated at the terminal part of the chain are added in the smooth ER and the Golgi apparatus (9)(10)(11)(12)(13)(14)(15)(16) . Although albumin is not a glycoprotein, it still travels the same secretory route as glycoproteins in that it can be located in the rough and smooth ER and in the Golgi apparatus (29)(30)(31)(32)(33) . The experiments reported in this paper concentrate on the in vitro incorporation into endogenous protein of the sugars closest to the protein core, and these experiments attempt to distinguish between the immediate submicrosomal fate of newly formed hepatic glycoprotein and of albumin .…”

Section: Discussionmentioning

confidence: 99%

The Secretory Pathways of Rat Serum Glycoproteins and Albumin

Redman

Cherian

1972

147

These studies compare the secretory pathways of newly formed rat serum glycoproteins and albumin by studying their submicrosomal localization at early times after the beginning of their synthesis and also by determining the submicrosomal site of incorporation of N-acetylglucosamine, mannose, galactose, and leucine into protein . N-acetylglucosamine, mannose, and galactose were only incorporated in vitro into proteins from membraneattached polysomes and not into proteins from free polysomes . Mannose incorporation occurred in the rough endoplasmic reticulum, was stimulated by puromycin but not by cycloheximide, and 90% of the mannose-labeled protein was bound to the membranes .Galactose incorporation, by contrast, occurred in the smooth microsome fraction and 89% of the radioactive protein was in the cisternae . Albumin was mostly recovered (98%0 ) in the cisternae, with negligible amounts in the membranes . To determine whether the radioactive sugars were being incorporated into serum proteins or into membrane protein, the solubilized in vivo-labeled proteins were treated with specific antisera to rat serum proteins or to albumin. Immunoelectrophoresis of the "C-labeled leucine membrane and cisternal proteins showed that the membranes contained radioactive serum glycoprotein but no albumin, while the cisternal fraction contained all of the radioactive albumin and some glycoproteins . The results indicate that newly formed serum glycoproteins remain attached to the membranes of the rough endoplasmic reticulum after they are released from the membrane-attached polysomes, while albumin passes directly into the cisternae .

“…It is known (Glaumann and Ericsson, 1970 ;Peters et al ., 1971 ; see also Table II in , that secretion of labeled proteins into the blood begins approximately 15 min after [ 3 H]leucine administration . Shorter times of labeling are therefore needed to examine possible differences in the quality of newly synthesized aligned so that radioactivity peaks can be related with the corresponding stained bands .…”

Section: B Kinetics Of Labeling Of Microsornal Proteinsmentioning

confidence: 99%

“…In liver cells, secretory proteins destined for the bloodstream are thought to be manufactured in membrane-bound polysomes and to be directly discharged into the ER cisternae (Palade and Siekevitz, 1956 ;Campbell et al ., 1960 ;Peters, 1962 a,b ; Redman and Sabatini, 1966 ;Redman, 1969 ;Ganoza and Williams, 1969), where they may be modified by enzymes bound to the limiting membranes (Molnar et al ., 1965 ;DeLorenzo et al ., 1966 ;Wagner and Cynkin, 1971 ;Redman and Cherian, 1972), before being transferred to the Golgi apparatus (Glaumann, 1970 ;Glaumann and Ericsson, 1970;Schachter et al ., 1970 ;Peters et al ., 1972) . The intracisternal route provided by the ER may also be utilized by proteins retained intracellularly which are diverted from the secretory pathway into other membrane-bound compartments, such as lysosomes (de Duve and Wattiaux, 1966 ;Goldstone and Koenig, 1972), or peroxisomes (Higashi and Peters, 1963 a,b ; Kashiwagi et al ., 1971 ; but see Lazarow andde Duve, 1973) .…”

Section: Introductionmentioning

confidence: 99%

Selective Release of Content From Microsomal Vesicles Without Membrane Disassembly

Kreibich

Sabatini

1974

Rough and smooth microsomes were shown to have similar sets of polypeptide chains except for the proteins of ribosomes bound to the rough endoplasmic reticulum (ER) . More than 50 species of polypeptides were detected by acrylamide gel electrophoresis, ranging in molecular weight from 10,000 to approximately 200,000 daltons . The content of rough and smooth microsomes was separated from the membrane vesicles using sublytic concentrations of detergents and differential centrifugation . A specific subset of proteins which consisted of approximately 25 polypeptides was characteristic of the microsomal content . Some of these proteins showed high rates of in vivo incorporation of radioactive leucine or glucosamine, but several others incorporated only low levels of radioactivity within short labeling intervals and appeared to be long-term residents of the lumen of the ER . Seven polypeptides in the content subfractions, including serum albumin, contained almost 50% of the leucine radioactivity incorporated during 5 min and crossreacted with antiserum against rat serum . Almost all microsomal glycoproteins were at least partly released with the microsomal content .Smooth microsomes contained higher levels of albumin than rough microsomes, but after short times of labeling with [ 3 H]leucine the specific activity of albumin in the latter was higher, supporting the notion that newly synthesized serum proteins are transferred from rough to smooth portions of the ER . On the other hand, after labeling for 30 min with [ 3 H]glucosamine, smooth microsomes contained higher levels of radioactivity than rough microsomes . This would be expected if glycosidation of newly synthesized polypeptides proceeds during their transit through ER cisternae .The labeling pattern of membrane proteins in microsomes obtained from animals which received three daily injections of [ 3 H]leucine, the last administered 1 day before sacrifice, followed the intensity of bands stained with Coomassie blue,