View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by PubMed Central Biosynthesis and Processing of Ribophorins in the Endoplasmic Reticulum MELVIN G. ROSENFELD,* EUGENE E. MARCANTONIO,* JOHN HAKIMI,* VICTORIA M. ORT,* P. H. ATKINSON,* DAVID SABATINI7 and GERT KREIBICH ~ *Department of Cell Biology and The Kaplan Cancer Center, New York University, New York 10016; and *Department of Developmental Biology and Cancer, Albert Einstein College of Medicine, Bronx, New York 10461 ABSTRACT Ribophorins are two transmembrane glycoproteins characteristic of the rough endoplasmic reticulum, which are thought to be involved in the binding of ribosomes. Their biosynthesis was studied in vivo using lines of cultured rat hepatocytes (clone 9) and pituitary cells (GH 3.1) and in cell-free synthesis experiments. In vitro translation of mRNA extracted from free and bound polysomes of clone 9 cells demonstrated that ribophorins are made exclusively on bound polysomes. The primary translation products of ribophorin messengers obtained from cultured hepatocytes or from regenerating livers co-migrated with the respective mature proteins, but had slightly higher apparent molecular weights (2,000) than the ungly- cosylated forms immunoprecipitated from cells treated with tunicamycin. This indicates that ribophorins, in contrast to all other endoplasmic reticulum membrane proteins previously studied, contain transient amino-terminal insertion signals which are removed co-translation- ally. Kinetic and pulse-chase experiments with [35S]methionine and [3H]mannose demon- strated that ribophorins are not subjected to electrophoretically detectable posttranslational modifications, such as proteolytic cleavage or trimming and terminal glycosylation of oligosac- charide side chain(s). Direct analysis of the oligosaccharides of ribophorin I showed that they do not contain the terminal sugars characteristic of complex oligosaccharides and that they range in composition from MansGIcNAc to MansGIcNAc. These findings, as well as the observation that the mature proteins are sensitive to endoglycosidase H and insensitive to endoglycosidase D, are consistent with the notion that the biosynthetic pathway of the ribophorins does not require a stage of passage through the Golgi apparatus. It is now well established that many proteins destined for Golgi apparatus, since proteins of these different classes may different subcellular compartments share a common site of be processed by the same Golgi enzymes (12, 33), and some synthesis in ribosomes bound to membranes of the endo- have been found in the same Golgi cisternae (44, 53). On the plasmic reticulum (ER) I and are initially incorporated into other hand, it has not yet been determined if the biosynthesis this organelle (49). However, nothing is known about the of membrane polypeptides characteristic of the ER requires discriminating mechanisms that effect the sorting of these their transfer to the Golgi apparatus and if this organelle plays polypeptides and determine their different fates, or of the a role in ensuring the segregation of certain proteins within features of the polypeptides that serve as sorting signals in this the ER. Significant amounts of several ER proteins have been process. detected in fractions of Golgi membranes (5, 17-19, 20, 22, It is clear that the pathways followed by secretory, lysoso- 23), and it has been proposed (47) that an important function mal, and membrane proteins of different plasma membrane of the cis portion of the Golgi apparatus is the refinement of domains do not diverge before the polypeptides enter the a crude export from the ER, from which membrane proteins to be segregated in the ER must be continuously removed Abbreviations used in this paper. cl-9, clone 9; ER, endoplasmic and returned to this organelle. If this is the case, it may be reticulum. expected that some ER membrane polypeptides are subjected THE JOURNAL OF CELL BIOLOGY - VOLUME 99 SEPTEMBER1984 1076-1082 1076 © The Rockefeller University Press . 0021-952518410911076]07$1.00 to posttranslational modifications carried out by Golgi en- uing incubation in the presence of tunicamycin. zymes, such as those that affect oligosaccharide chains in For mannose labeling, hepatocytes (2 x 107 cells) were incubated for the indicated times in RPM1-10% horse serum supplemented with 2[aH]D-mannose glycoproteins. Of the many ER proteins characterized to date, at a concentration of l mCi/ml (24.3 Ci/mmol). however, only the ribophorins, which are restricted to the After labeling, monolayers were washed twice with Moscona's PBS (36) and rough ER and appear to play a role in ribosome binding (26- cells were scraped offthe dish into an SDS solution (20 mM Tris-HCl, 7.4, 2% 28), and HMG-CoA reductase, an enzyme of cholesterol SDS) and sonicated 10-30 s in a Heat Systems-Ultrasonics Instrument with a biosynthesis (31), have been found to bear covalently bound microtip (Heat Systems-Ultrasonics, Inc. Plainview, NY). The suspension was boiled for 2 min and centrifuged (2 min at 15,000 g) in an Eppendorf 5412 to oligosaccharides, and therefore may serve as natural probes remove unsolubilized material. The supernatant was diluted fourfold with 50 to study this question. mM Tris-HCl 7.4, 190 mM NaCI, 6 mM EDTA, 2.5% Triton X-100, 0.02% The biosynthesis of ribophorins is also of interest because NAN3, and 100 U/ml Trasylol (Solution A). of the role that these proteins appear to play in the binding Immuneprecipitation: Cell extracts were incubated with lgG frac- of ribosomes to the ER membranes and hence the co-trans- tions (300 ~g/ml) at room temperature for l h and at 4"C for 14 h before addition of protein-A-sepharose CL-4B beads. After incubation for 3 h at room lational incorporation of all proteins into the ER (49). Al- temperature, the beads were sedimented, washed three times with Solution A though ribophorins have been best characterized in rat liver containing 0.2% SDS, and boiled for 2 min in a solution of 10% SDS, l M microsomes, from where they can be recovered in association dithiothreitol, 2 mM EDTA. with the ribosomes after other membrane polypeptides are RNA Extraction: Clone 9 hepatocytes or GH 3.1 pituitary cells were dissolved by non-ionic detergents (28), they appear to be homogenized in a solution containing 5 M guanidine thiocyanate plus 100 mM universal components of rough ER membranes in all cell mercaptoethanol at pH 5.0 (58). RNA was separated by centrifugation through 0.2 vol of 5.7 M CsCl2 as described by Liu et al. (32). Similarly, total RNA was types, and basic structural features of both ribophorins seem prepared from control rats starved for 18 h or from rats sacrificed 15-24 h after to have been conserved during evolution (34). If ribophorins partial bepatectomy (l 6). are indeed necessary for the incorporation of proteins into Free and membrane-bound polysomes were prepared from cl-9 hepatocytes the ER, their synthesis may be part of the regulatory mecha- by a modification of the procedure of Ramsey and Steele (43). Polysome pellets were rinsed with sterile water and the RNA was extracted with 5 M guanidine nism that controls the development of the rough ER and the thiocyanate as described above. Poly A÷ mRNA was purified by chromatogra- extent to which synthesis of ER membrane proteins can take phy on oligo-dT cellulose (2). Preparations of messenger RNA from the place. pituitary cell line GH 3. l were fractionated on a methyl mercury gel (40) to In this paper we demonstrate that the ribophorins them- enrich for ribophorin encoding molecules. selves are products of bound polysomes which contain tran- Cell-free Protein Synthesis: Wheat germ extracts (35) containing sient amino-terminal insertion signals and acquire asparagine- 12.5 ~Ci of [3SS]methionine and 0.03 A26o U of mRNA in 0.025 ml were incubated at 25"C for 90 min. Dog pancreas microsomal membranes, prepared linked oligosaccharides during their co-translational insertion as described by Shields and Blobel (52), were added to a final concentration of into ER membranes. Because all posttranslational changes 2 A26o U/ml. Samples from translation mixtures (0.2 ml) were boiled in 2% that affect the ribophorins can be carried out by enzymatic SDS, centrifuged (2 min at 15,000 g), and diluted fourfold with solution A. activities found in the ER, the biosynthesis of these polypep- Immuneprecipitation was carried out as described above. tides does not require their transfer to the Golgi apparatus. Treatment with Endo H- and Endo D-N-acetyl-glucosamin- idases: Detergent extracts of rat liver rough microsomes were iodinated by the chloramine T procedure as previously described (34). After immuneprecip- MATERIALS AND METHODS itation of the ribophorins, the beads were boiled in 2% SDS and centrifuged. Guanidine thiocyanate was from Fluka (Hauppauge, NY), [3Sg]methionine Supernatants diluted with 1 M citrate-phosphate buffer (pH 5.0 or pH 6.4) to (specific activity 1,000 Ci/mmole), 2[3H]mannose (25 Ci/mmole), and [~25I]Na a final concentration of 50 mM were incubated with endogiycosidase H (8 x (100 Ci/mmole) from New England Nuclear (Boston, MA), Trasylol from l0 -4 U/ml) or with a crude preparation of Endoglycoside D (38) for l h at Mobay Chemical Corp. (New York, NY), oligo-dT cellulose from Collaborative 37"C at pH 5.0 or 6.4, respectively. Samples were then analyzed by PAGE (10%) followed by autoradiography. Research, Inc. (Lexington, MA), and tunicamycin from Calbiochem-Behring Corp. (La Jolla, CA). RPM! 1640, spinner minimal essential medium, horse Sizing Oligosaccharides: Oligosaccharides labeled with 2[3H]man- serum, and fetal calf serum were from Grand Island Biological Co. (Grand nose were released from giycopeptides derived from ribophorin I as previously Island, NY). Wheat germ was a gift of The General Mills Corp.