J. Cell Sci. 39, 273-290 (1979) 273 Printed in Great Britain © Company of Biologists Limited IMMUNOELECTRON-MICROSCOPIC STUDIES OF ENDOPLASMIC RETICULUM-GOLGI RELATIONSHIPS IN THE INTRACELLULAR TRANSPORT PROCESS OF LIPOPROTEIN PARTICLES IN RAT HEPATOCYTES SHIRO MATSUURA AND YUTAKA TASHIRO* Department of Pliysiology, Kansai Medical University, 1 Fumizonocho, Moriguchi-shi, Osaka 570, Japan SUMMARY Endoplasmic reticulum (ER)-Golgi relationships in the intracellular transport process of secretory proteins in rat hepatocytes have been studied using lipoprotein particles as a marker for the secretory protein and cytochrome P-450 as a marker enzyme for the ER membranes. Ferritin immunoelectron-microscopic observation revealed that, while almot-t all the micro- somal vesicles derived from ER membranes are heavily labelled with ferritin anti-cytochrome P-450 antibody conjugates, labelling of the small peripheral vesicles containing lipoprotein particles, the stacks of Golgi saccules, especially the outermost saccule which is sometimes fenestrated, condensing vacuoles in the trans-Golgi region and the secretion droplets of lipo- protein were scanty and at the control level. Such a characteristic pattern of labelling was especially evident when these structures were prepared from phenobarbital-treated rats. These findings indicate that the membranes of the small peripheral vesicles do not contain cytochrome P-450 and that the cytochrome is probably not transferred to Golgi saccules in the transport process of lipoprotein from ER to Golgi. It is suggested, therefore, that the small peripheral vesicles are formed by budding of the special regions of ER membrane where micro- somal marker proteins such as cytochrome P-450 are excluded and the membrane proteins destined to the Golgi complexes are clustered. It is also shown that lysosomal membranes are not labelled with the anti P-450 antibody conjugates. INTRODUCTION It has been generally accepted that the pathway followed by secretory proteins leads from rough endoplasmic reticulum (ERj to the transitional elements of this system, then to the small peripheral vesicles (Golgi transporting vesicles) on the cis side of Golgi complexes and finally either to condensing vacuoles or to Golgi stacks (Palade, 1975; Jamieson & Palade, 1977). In order to make such a transport process possible, membrane should flow from ER to Golgi complexes (Palade, 1975; Jamieson & Palade, 1977). Recent biochemical evidence indicates, however, that there is no mixing among either the lipid (Keenan & Morre, 1970; Meldolesi, Jamieson & Palade, 1971) or the • Mailing address: Dr Yutaka Tashiro, Department of Physiology, Kansai Medical University, 1 Fumizonocho, Moriguchi-shi, Osaka 570, Japan. 274 S. Matsuura and Y. Tasliiro protein (Bergeron, Ehrenreich, Siekevitz & Palade, 1973; Van Golde, Fleischer, Fleischer, Azzi & Chance, 1971) components of the membranes of the 2 compartments in the pancreas and in the liver. This imposes stringent limitations on membrane interactions since it suggests that lateral diffusion of components is prevented at the time when the membrane of the 2 compartments establishes continuity. In order to clarify the molecular mechanisms involved in the ER-Golgi relation- ships, we have investigated the transport process from ER to Golgi of lipoprotein particles in rat hepatocytes, since the particles are easily identified by electron micros- copy, while cytochrome P-450, a marker enzyme for the ER membranes, can be localized by ferritin immunoelectron-microscopy (Matsuura, Fujii-Kuriyama & Tashiro, 1978). In addition, lipoprotein particles in hepatocytes have been exten- sively studied biochemically as well as electron microscopically by a number of investigators, such as Stein & Stein (1967), Jones, Ruderman & Herrera (1967), Claude (1970), Ehrenreich, Bergeron, Siekevitz & Palade (1973) and Glaumann, Bergstrand & Ericsson (1975) and Bergeron, Borts & Cruz (1978). In the previous paper (Matsuura et al. 1978) we have shown that, although almost all of the microsomal vesicles derived from ER membranes are markedly labelled, Golgi saccules and condensing vacuoles in trans-Golgi regions are hardly labelled with the ferritin antibody conjugates. An important question is whether or not the membranes of the small peripheral vesicles carrying lipoprotein particles have the cytochrome. The most direct approach to this problem would be to localize the antigens in ultrathin sections of the cells. Experimental approaches using frozen thin sectioning as reported by Painter, Toku- yasu & Singer (1973), Tokuyasu (1973), Sasaki & Tashiro (1976) as well as using embedding in albumin as reported by McLean & Singer (1970) and Kraehenbuhl, Racine & Jamieson (1977) have been tried without success. In the latter procedure the antigenicity of the cytochrome appears to be lost during embedding in concentrated albumin by polymerization with glutaraldehyde. We have, therefore, used cell organelles prepared by cell fractionation techniques for localization of antigens. The present immunoelectron-microscopic observations revealed that the small peripheral vesicles containing lipoprotein particles are not labelled with the ferritin antibody conjugates. This means that, when the transfer vesicles are formed on ER membrane, cytochrome P-450 molecules are excluded in advance from those regions of the ER membrane where the vesicles are to be formed by budding mechanisms. The physiological significance of such a finding with regard to the membrane flow accompanying the intracellular transport process of secretory proteins is discussed. MATERIALS AND METHODS Immunochemical procedures Preparation of the monospecific antibody to phenobarbital-induced cytochrome P-450 has been described previously (Matsuura, Fujii-Kuriyama & Tashiro, 1979). Ferritin and the anti- body were coupled according to the procedure of Kishida, Olsen, Berg & Prockop (1975) using glutaraldehyde as a coupling agent, and the ferritin anti P-450 antibody conjugates with the Intracellular transport of lipoprotein 275 molar ratio of immunoglobulin G (IgG) to ferritin of approximately unity were isolated by gel filtration on Bio-gel ArjM as described previously (Matsuura et al. 1978). Preparation of Golgi and microsome fractions Male non-starved rats (Sprague-Dawley) weighing ~2oo g were used, since the recovery of the Golgi apparatus was higher in these than in starved animals (Glaumann et al. 1975). Golgi fraction was prepared either from untreated or from phenobarbital-treated rats. In the latter case, rats were given a single daily intraperitoneal injection of phenobarbital (10 mg per 100 g body weight) for 4 days and were sacrificed without fasting. The procedures reported by Hino, Asano, Sato & Shimidzu (1978) were used for the preparation of Golgi fraction. Total, smooth and rough microsome fractions were prepared as described previously (Matsuura et al. 1978). Labelling of t/ie Golgi and microsome fractions The direct ferritin antibody method was used exclusively. The cell fractions were incubated for 30 min at 4 °C either with the antibody conjugates or with the control conjugates as de- scribed previously (Matsuura et al. 1978). The concentration of the specific antibodies in the antibody conjugates was adjusted to at least ~3- to 5-fold molar excess over the biochemically calculated number of cytochrome P-450 molecules in each fraction. Electron microscopy The incubated materials were washed by sucrose density gradient centrifugation (Matsuura et al. 1978). The membrane fractions were resuspended and centrifuged at 10000 g for 10 min, and the thin pellets were fixed with glutaraldehyde—osmium tetroxide mixture, dehydrated and embedded in Epon and sectioned as described previously (Matsuura et al. 1979). Samples of liver tissue from normal rats were fixed in 1 % OsO4 in distilled water, pH 6 (Claude, 1970; Ehrenreich et al. 1973), dehydrated, embedded and sectioned as above. The thin sections were observed with a Hitachi HU-12 electron microscope. RESULTS In situ localization of lipoprotein particles in rat hepatocytes In rat hepatocytes, the Golgi complexes are less orderly constructed and less clearly polarized than in other protein-secreting cells, as described by Ehrenreich et al. (1973). The stacks of cisternae (saccules) and the vacuoles located on the trans side have the usual Golgi features and are accordingly easily recognized, as shown in Fig. 1. Transitional elements of the type regularly found on the opposite or cis side of the stacks in other secretory cells, such as pancreatic exocrine cells, are, however, rarely encountered: their place is usually taken by irregularly distributed smooth ER elements. Fig. 1 also shows that in the Golgi apparatus, the peripheral dilated portion of the cisternae (star) and the trans Golgi vacuoles (condensing vacuoles) are loaded with a number of electron-opaque particles, 30—80 nm in diameter. The occurrence of such particles in the Golgi elements of mammalian hepatocytes has been described repeatedly and they have been identified as very low density lipoprotein (VLDL) particles or their precursor particles (Jones et al. 1967; Hamilton, Regen, Gray & LeQuire, 1967; Chapman, Mills & Taylaur, 1972). In addition to those found in Golgi complexes, similar particles are present singly or in pairs in the smooth ER in tubular configuration, as well as in the extended S. Matsuura and Y. Tashiro \ t i ' r 'S Fig. i. Lipoprotein particles in hepatocytes of non-starved rat. A Golgi complex appears in the vicinity