intact Golgi apparatus immobilized on nitrocellulose strips, transfer in vitro of radiolabeled membranes was demonstrated. Nucleoside triphosphates were ...
Proc. Natl. Acad. Sci. USA Vol. 84, pp. 6098-6102, September 1987 Biochemistry
Intracellular membrane flow: Reconstitution of transition vesicle formation and function in a cell-free system (Golgi apparatus/endoplasmic reticulum)
D. DAVID NOWACK*, DOROTHY M. MORRO*, MARK PAULIKt, THOMAS W. KEENANt, AND D. JAMES MORRu§¶ Departments of *Foods and Nutrition, tBiological Sciences, and WMedicinal Chemistry, Purdue University, West Lafayette, IN 47907; and *Department of Biochemistry and Nutrition, Virginia Polytechnic Institute and State University, Blacksburg, VA 24601
Communicated by Edwin Mertz, May 18, 1987 (received for review April 14, 1987)
ABSTRACT Transfer of membrane between endoplasmic reticulum and Golgi apparatus in situ is considered to occur via 60-nm transition vesicles derived from part-rough, partsmooth transition elements of the endoplasmic reticulum. A procedure is described for the isolation of a fraction enriched in these transition elements from rat liver. The isolated fraction generates small vesicles morphologically resembling transition vesicles when incubated with nucleoside triphosphate at 370C. In the cell-free system consisting of a donor fraction enriched in transition elements and an acceptor fraction consisting of intact Golgi apparatus immobilized on nitrocellulose strips, transfer in vitro of radiolabeled membranes was demonstrated. Nucleoside triphosphates were required for transfer, and transfer was facilitated by a cytosol fraction of Mr >10,000. In the presence of both nucleoside triphosphate and cytosol, radiolabeled proteins were transferred in a manner dependent upon both time and temperature. Transfer appeared to be both vectorial and specific in that, with Golgi apparatus (or endoplasmic reticulum) as both donor and acceptor, only negligible time and temperature-dependent transfer was observed. The test system described is expected to facilitate further investigation of the transfer process and to provide a convenient assay to guide transition vesicle isolation and characterization.
perature dependent and was enhanced by the presence of nucleoside triphosphate (plus a regenerating system) and a cytosol fraction of Mr >10,000.
MATERIALS AND METHODS Materials. Rats were 180-g males of the Holtzman strain from Harlan Sprague Dawley (Indianapolis). Sephadex G-25 was from Pharmacia. Nitrocellulose was from S & S Scientific (Keene, NH). All chemicals were from Sigma. Isolation and Labeling of the Donor Fraction. To label the endoplasmic reticulum from which the transition elements were obtained, ca. 1.5 x 0.3 x 0.3 mm slices were cut by hand with a razor blade from the freshly isolated livers. Approximately 5 g of slices were incubated in 5 ml of phosphatebuffered saline (0.9%, pH 7.2) with 0.5 or 1 mCi (1 Ci = 37 GBq) of [3H]leucine for 1 hr. The slices were collected on a Miracloth (Chicopee Mills, NY) filter and washed to remove unincorporated radioactivity. Endoplasmic reticulum fractions were isolated as described (9). Livers or liver slices were homogenized in 2 volumes of a medium containing 0.05 M Tris maleate (pH 6.5), 0.5 M sucrose, 5 mM MgCl2, and 1% dextran for 45 sec with a Polytron 20 ST homogenizer operated at 6000 rpm. The homogenates were centrifuged for 15 min at 6000 x g to remove nuclei, plasma membrane fragments, and Golgi apparatus, and the supernatant was diluted 1:5 with homogenization medium and centrifuged at 10,000 x g to remove mitochondria. The supernatant containing the endoplasmic reticulum was layered onto a discontinuous sucrose gradient consisting of 2.0, 1.5, and 1.3 M sucrose layers. The material from the 1.3 M sucrose/sample interface was withdrawn in a Pasteur pipette, pelleted by centrifugation, and used as the starting material for the donor fraction. Preparation of the Acceptor Fraction. To prepare the acceptor membranes, highly purified Golgi apparatus membranes isolated from the same livers as the donor membranes were adsorbed on nitrocellulose strips. Golgi apparatus, purified as described (10), were resuspended at a final concentration of 1-2 mg of protein per ml in 33 mM Hepes (pH 7.0) containing 2.5 mM magnesium acetate and 33 mM KCl [Hepes/Mg(OAc)2/KCl] and incubated with 1 cm X 1 cm nitrocellulose strips (10 strips per ml) at 37°C for 1 hr with continuous shaking. The squares then were transferred to Hepes/Mg(OAc)2/KCl containing 5% bovine serum albumin and incubated for 1 hr at 4°C with shaking. The strips, each loaded with about 100 jig of Golgi apparatus protein, were rinsed through four changes of Hepes/Mg(OAc)2/KCl, blotted, and added to the incubation medium. Incubations were in 8-ml glass shell vials, with 3 strips per vial. The strips were arranged vertically in the vial in the form of a triangle around the circumference. All solutions were maintained at 4°C until initiation of the reaction by transfer to 37°C. At the end of the
Transfer of membrane materials from endoplasmic reticulum to Golgi apparatus long has been considered from morphological evidence to be mediated by transition vesicles that bleb off specialized, part-rough, part-smooth regions of the endoplasmic reticulum (1, 2). These vesicles, covered by a nap-like coat material not containing clathrin (3-5), are thought to coalesce to form new Golgi apparatus cisternae or to fuse with existing Golgi apparatus membranes to effect delivery to the Golgi apparatus of materials derived from the endoplasmic reticulum (6). Operation of the segment of the exocytotic pathway has been assumed largely from static images provided from electron microscopy. To demonstrate the phenomenon in vitro, we used a cell-free incubation mixture patterned after that described by Rothman and colleagues (7, 8). With appropriately "primed" (complete incubation, 37°C) preparations of part-rough, part-smooth transitional regions of endoplasmic reticulum from rat liver, we observed the formation of small blebbing profiles similar to those associated with transition elements in situ (9). These vesicles were characterized by an electron-dense (but not clathrin-derived) coat material. In this paper, we report the reconstitution of membrane transfer in a cell-free environment using rat liver fractions. Radioactivity was transferred from transition elements metabolically labeled with [3H]leucine to Golgi apparatus immobilized on nitrocellulose strips. Transfer was time and temThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
$To whom reprint requests should be addressed. 6098
Biochemistry: Nowack et al. incubation, the strips were rinsed through four changes of
Hepes/Mg(OAc)2/KCl, blotted on Whatman No. 1 filter paper, and dried. The dried squares were placed individually in scintillation counter vials and, after addition of 10 ml of aqueous counting scintillant ACS (Amersham), radioactivity was determined. The Reconstituted Membrane Transfer System. The complete reconstituted cell system (total volume, 1 ml) consisted of (i) 250 1ul of resuspended radiolabeled membranes (3 mg of protein per ml), (ii) 250 1.l of gel-filtered soluble supernatant proteins (1 mg of protein per ml) prepared from a microsomefree supernatant of rat liver by centrifugation at 90,000 x g for 60 min (Beckman SW 50.1 rotor), and (iii) 250 ,l of Hepes/Mg(OAc)2/KCl, pH 7.0. The gel filtration consisted of passing 15 ml of the supernatant from high-speed centrifugation through a column (48 x 2.5 cm) containing Sephadex G-25 that had been equilibrated with 25 mM Tris'HCl (pH 8.0) containing 50 mM KCl. The original volume placed in the column was retrieved by concentrating the samples utilizing a Centricon YM 10 filter (Amicon). In addition, 250 1l of an ATP-regenerating system [33 mM Hepes/33 mM KCI/2.5 mM Mg(OAc)2/67 ,uM ATP/333 AM UTP/2 mM creatine phosphate/10 units of creatine phosphokinase (rabbit muscle) per ml (final pH = 7.0)] was added. Two identical membrane-containing systems were incubated in parallel. A complete mixture was maintained at 4°C as a control, and the other complete mixture was incubated at 37°C. Electron Microscopy. Immediately after animal sacrifice, liver tissue was fixed rapidly by immersion in 1% osmium tetroxide/0. 1 M sodium phosphate, pH 7.2, at 4°C. Fractions were fixed in 2.5% glutaraldehyde/0.1 M sodium phosphate, pH 7.2, followed by post-fixation in 1% osmium tetroxide in the same buffer. Dehydration was through an acetone series, with embedment in Epon (11). Thin sections were examined and photographed with a Philips EM 200 electron microscope.
RESULTS Transition Elements and Golgi Apparatus in situ and as Represented in the Cell-Free System. The part-rough (with ribosomes), part-smooth (lacking ribosomes) elements of the endoplasmic reticulum called transition elements that lie proximal to the cis face of the Golgi apparatus of rat liver (Fig. LA) are considered to be the source of the numerous 60-nm vesicles (transition vesicles) associated with the cis Golgi apparatus face (small arrows in Fig. 1A). These vesicles have been postulated to migrate to the Golgi apparatus and there to coalesce to form new Golgi apparatus cisternae (1, 2). The transition vesicles are distinguished from the more familiar clathrin-coated vesicles of the trans Golgi apparatus face by the nap-like rather than "spiny" appearance of their surfaces (Fig. 1A Inset). Structures morphologically resembling the in situ equivalents of transition vesicles were present in the fractions isolated from incubated liver slices and used as the donor membranes in the cell-free system (Fig. 1B). In the presence of nucleoside triphosphate, small 60-nm vesicles were observed to form from various membrane fragments within the preparations (see also ref. 9). Such vesicles and blebcontaining profiles were completely absent from preparations of rough-surfaced endoplasmic reticulum isolated and incubated under similar conditions. Purified Golgi apparatus were immobilized on cellulose nitrate strips (Fig. 1C) to serve as the acceptor fraction. The Golgi apparatus preparations formed a monolayer of Golgi apparatus stacks of approximately 100 ,ug of Golgi apparatus protein per strip. When incubated in the presence of either cytosol or serum albumin (1 mg of protein per ml), the amount of protein and the morphological appearance of the attached
Proc. Natl. Acad. Sci. USA 84 (1987)
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Golgi apparatus stacks remained constant for more than 1 hr of incubation at 370C. The Golgi apparatus preparations were typical of those prepared routinely in the laboratory and consisted of >90% Golgi apparatus-derived membrane material based on morphometry and analysis of marker enzymes. Transfer Comparing Primed and Unprimed Transition Element Preparations. With radiolabeled endoplasmic reticulum as the donor membranes (Fig. 1B) and unlabeled Golgi apparatus as the recipient membranes (Fig. 1C), transfer of radioactive membrane proteins from the primed donor preparations at 370C was time dependent (Fig. 2). Transfer at 370C was 4-8 times greater than with the unprimed preparations at 40C. The slow rate of transfer at 40C was linear for several hours, whereas that at 370C showed saturation kinetics with little additional transfer between 60 and 120 min. After 120 min, additional transfer could be initiated by adding new Golgi apparatus acceptor strips. When both donor and acceptor membranes were highly purified Golgi apparatus fractions, little or no temperaturedependent transfer of radioactivity was observed (Table 1). Similarly, with endoplasmic reticulum either as acceptor or as both donor and acceptor, no transfer was observed. Other conditions yielding no temperature-dependent transfer of radioactivity included (i) acceptor strips coated only with serum albumin (minus Golgi apparatus) and (ii) donor membranes labeled by radioiodination under oxidizing conditions. By morphometry, transition vesicles were estimated to occupy approximately 2% of the total membrane surface of the primed donor fractions utilized. Transfer from primed endoplasmic reticulum fractions to the nitrocellulose strips accounted for between 0.5% and 1.5% of the total radioactivity present in the starting primed donor fractions. Transfer from transition elements to Golgi apparatus was promoted by the presence of 2.5 ,.g of all-trans-retinol per ml, a compound known to influence transition vesicle abundance in situ (12). In contrast, retinol was without effect when Golgi apparatus were present as both donor and acceptor (Table 1). Dependence of Transfer and Response to Cytosol Fractions on Nucleoside Triphosphate. In the absence of nucleoside triphosphate and the regenerating system, very little temperature-dependent transfer between labeled donor fractions and unlabeled acceptor fractions occurred (Fig. 3). In the crude mixtures containing cytosol fractions and the regenerating system, other nucleoside triphosphates (e.g., GTP) also supported transfer. Increasing the nucleoside triphosphate concentration up to 10-fold that used in the complete reaction mixture increased transfer rates only slightly (ca. 30%). Transfer was accelerated by, but was not completely dependent upon, the presence of added cytosol fractions. Crude, unfractionated cytosol (microsome-free supernatant fractions) yielded variable results ranging from small stimulations to inhibition (Table 2). However, if the low molecular weight fractions were first removed by gel filtration, consistent stimulations were obtained (Fig. 3, Table 2). Using the crude and gel-filtered cytosol fractions, optimum transfer was obtained in the concentration range of 0.1-10 mg/ml (25-250 ,g of protein per reaction). DISCUSSION The search for defined cell-free systems that effect membrane transfers to Golgi apparatus received initial impetus from the work of Rothman and colleagues (7, 8, 13, 14) who demonstrated transfer of incompletely processed viral proteins from one compartment to a second compartment where processing was completed within Golgi apparatus preparations from cultured cells. Their system depended upon assay of the enzyme that adds N-acetylglucosamine to the G-protein of
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