Biosynthesis of Plasma Retinol-binding Protein in Liver as a Larger Molecular Weight Precursor"

A study was performed to identify the first translated product of messenger RNA for retinol-binding protein (RBP), the specific plasma transport protein for vi- tamin A. Poly(A)+RNA was isolated from rat liver and translated in the rabbit reticulocyte in vitro protein- synthesizing system. RBP was identified and separated from other translated products by immunoprecipita-tion with specific rabbit anti-rat RBP antiserum. So- dium dodecyl sulfate-polyacrylamide gel electrophore-sis and fluorography of the immunoprecipitate consist- ently revealed one major product which migrated more slowly than purified rat serum RBP. The protein (preRBP) had an approximate molecular weight of 24,000. When dog pancreas microsomal membranes were cotranslationally present, the newly synthesized preRBP was processed to a protein which migrated coincidentally with purified rat serum RBP, approxi- mately 20,500 daltons. These results indicate that RBP is initially synthesized as a larger molecular weight precursor (preRBP) which is rapidly processed by the removal of a peptide of approximately 3,500 daltons to the size of the final RBP molecule that circulates in the plasma. Retinol-binding protein is a single polypeptide chain with a molecular weight of 20,000-21,000 (see Refs. 1 and 2 for recent reviews). This protein functions as the plasma transport pro- tein for retinol and serves to

A great deal is now known about the chemical structure, metabolism, and biological roles of RBP (1,2). RBP is syn-* This research was supported by Grants HL 21006 and AM 05968 from the National Institutes of Health. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
$ Postdoctoral trainee under Grant HL 07343 from the National Heart, Lung, and Blood Institute.
' The abbreviations used are: RBP, retinol-binding protein; SDS, sodium dodecyl sulfate; preRBP, precursor of RBP. thesized in the liver (5, 6 ) a n d is secreted into the plasma largely as the RBP-retinol complex; the rates of these processes are normally highly controlled. Only a limited amount of information is, however, available about the molecular mechanisms involved in RBP synthesis and secretion, and its regulation.
A number of secretory proteins of eukaryotic and prokaryotic origin have been demonstrated to be synthesized initially as larger molecular weight precursors (see Ref. 7 for review). These preproteins contain an NHZ-terminal extension usually ranging in size from 15 to 30 amino acid residues. For secreted proteins, the NH2-terminal signal or leader sequence is usually clipped cotranslationally by a microsomal protease, and the protein is translocated into the cisternae of the endoplasmic reticulum and eventually secreted (7-9). Plasma proteins secreted by the liver which have been shown to be synthesized initially as preproteins include albumin (lo), transferrin (111, prothrombin (12), and cockerel very low density apolipoprotein I1 (13).
We now report evidence that RBP is synthesized as the larger molecular weight preRBP, which is rapidly processed by the removal of a polypeptide of about 3500 daltons to the size of the final, secreted RBP molecule. These events thus emerge as possible sites of regulatory control of RBP synthesis and secretion.

EXPERIMENTAL PROCEDURES
RNA Isolation-Total RNA was isolated from the livers of 200-to 250-g male Holtzman rats by the guanidine-HC1 method of Chirgwin et al. (14). Poly(A)'RNA was obtained by subjecting the total liver RNA to oligo(dT)-cellulose affinity chromatography as described by Pickett et al. (15). RNA was quantitated utilizing the extinction coefficient of 1 A m unit/50 pg of RNA. All poly(A)+RNA samples had a 260/280 absorbance ratio of approximately 2.0.
Cell-free Protein Synthesis-The rat liver poly(A)+RNA was translated for 60 min at 28 "C in the micrococcal nuclease-treated rabbit reticulocyte lysate system (16), the components of which were purchased from Bethesda Research Laboratories. The reaction mixture, with a final volume of 120 p1, contained 40 81 of reticulocytes, 25 mM 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid, pH 7.4, 10 mM creatine phosphate, 48 mM K-acetate, 1.2 mM MgC12, 50 PM of each of 19 amino acids (without methionine), 80 pCi of (3sS)methionine (New England Nuclear; specific activity, >lo00 Ci/mmol) and 4 to 6 pg of poly(A)'RNA. Protein synthesis was determined by quantitation of the amount of ("S)methionine incorporated into trichloroacetic acidprecipitable protein in 2-81 samples of the reaction mixture as described by Mans and Novelli (17). The dog pancreas microsomal membranes used in some of the experiments were a generous gift of Dr. Philip Feigelson, Columbia University.
Immunoprecipitation-At the end of the 60-min incubation period, SDS was added to the translation mixture to a final concentration of 2%; the mixture was then boiled for 3 min, and diluted 1:lO with a solution containing 2.5% Triton X-100, 190 mM NaC1, 50 mM Tris-HCI, pH 7.4, and 6 mM EDTA. Samples were then first treated with 100 1.1 of a Protein-A Sepharose (Pharmacia) suspension plus 20 pl of nonimmune rabbit serum for 1.5 h, to reduce nonspecific binding (18). The Protein-A Sepharose was removed by centrifugation and 100 pl of fresh Protein-A Sepharose suspension and 20 pl of specific rabbit anti-rat RBP antiserum (4) were then added. The samples were allowed to incubate overnight at 4 "C with constant mixing. After incubation and formation of antigen-antibody (RBP-antiRBP) complexes bound to Protein-A Sepharose, the Protein-A Sepharose was collected on plastic filter discs (Isolab Inc., Akron, OH) followed by extensive washing with a solution containing 0.1 M Tris-HC1, pH 9.0, 0.5 M LiC1, and 1% P-mercaptoethanol. The antigen-antibody complexes were released from the Protein-A Sepharose by incubation for for 15 min in S D S sample buffer (5% SDS, 5.0 mM Tris-HCI, pH 6.8, 2% P-mercaptoethanol, 20% sucrose, and 0.05% bromphenol blue). After elution from the Protein-A Sepharose, the immunoprecipitated products were subjected to SDS-12.5% polyacrylamide slab gel electrophoresis (19). Gels were fixed, stained, soaked in EN'HANCE (New England Nuclear) and radioactive products were visualized by fluorography (20).
The purified rat RBP used in these experiments was isolated from rat serum in the manner described previously from this laboratory (21).
In Vivo Synthesized [35S]RBP-250-to 300-g male Holtzman rats were injected intraperitoneally with 500 pCi/100 g of body weight of ("S)methionine (New England Nuclear; specific activity, 1200 Ci/ mmol). Twelve min after injection (221, the livers were removed and homogenized in 3 volumes of 10 m~ Na-phosphate, pH 7.4, 125 mM NaCI, 1% Triton X-100. 1% Na-deoxycholate and lo-.' M phenylmethylsulfonyl fluoride. Samples were centrifuged for 2 h a t 100,OOO X g and the [:'"S]RBP in the supernatant immunoprecipitated in the same manner as described above.

RESULTS
Addition of the poly(A)'RNA to the mRNA-dependent rabbit reticulocyte lysate-translation system resulted in a 6to 10-fold stimulation in the incorporation of (~'"S)methionine into trichloroacetic acid-precipitable protein. The rate of protein synthesis was linear with time up to 60 min and was concentration-dependent for mRNA up to 50 pg of poly(A)'RNA added per ml of reaction mixture.
Immunoreactive RBP was separated from other mRNAdirected translation products by precipitation with a specific rabbit anti-rat RBP antiserum. Fig. 1 (Lune B ) shows that one major protein was immunoprecipitated by the anti-rat RBP serum. This protein consistently migrated more slowly than RBP isolated from rat serum (Fig. 1, position indicated by the arrow marked RBP). Thus, the immunoreactive product translated from liver mRNA was distinctly larger than serum RBP itself. In four separate experiments, the molecular weight estimate of the translated product was 24,000 f 700 (mean f SD), whereas that of pure RBP was 20,700 f 300. These findings suggest that RBP is first synthesized as a larger molecular weight precursor (preRBP). As can be seen in Fig. 1 (Lune A ) , this preRBP was not detected when the translation products were immunoprecipitated with nonimmune rabbit serum. A minor band at approximately 43,000 daltons (see Fig. 1) appeared in both the immune and nonimmune immunoprecipitates; accordingly, this band is considered to represent a nonspecific contaminant of the immunoprecipitation reaction. The intensity of this band varied considerably from reaction to reaction.
T o further establish that RBP is indeed first synthesized as a larger preprotein, the translation of the rat live. poly(A)'RNA was performed in the rabbit reticulocyte lysate system in the presence of dog pancreas microsomal membranes. When six A2,i0 units/ml of lysate of dog pancreas microsomal membranes were added cotranslationally to the reticulocytes and the samples analyzed as previously described, a considerable amount of the preRBP was processed to a smaller protein (Fig. 1, Lane C ) . This protein migrated exactly to the same position on the gel as did purified rat serum RBP (Fig. 1, position indicated by the arrow), and as did immunoprecipitated in vivo synthesized ['''SlRBP from rat liver homogenates (Fig. 1, Lune E ) . In several such experiments carried out, the extent of processing varied from about one-third to three-fourths of the preRBP. However, in the same experiment, when translation was carried out in the absence of microsomal membranes, the preRBP was the major immunoprecipitated band (Fig. 1, Lune B )  pancreas microsomal membranes did not process the preKBP to RBP (Fig. 1, Lane D). DISCUSSION Vitamin A mobilization from the liver is regulated by factors that control the rates of RBP synthesis and secretion. One factor which specifically regulates RBP secretion is the nutritional vitamin A status. Thus, studies in the rat have shown that retinol deficiency specifically blocks the hepatic secretion of RBP, which can then be stimulated rapidly by retinol repletion (5, 23, 24). This release of RBP is not blocked by inhibitors of protein synthesis. RBP in the liver is mainly found associated with microsomes, and is particularly enriched in the rough microsomal fraction (1). Recent studies suggest that the Golgi apparatus and secretory vesicles, and the microtubules are involved in the pathway of RBP secretion from the liver (24,25). The subcellular site where retinol normally interacts and forms a complex with RBP is not known.
The experiments reported here were undertaken as part of a research program aimed a t defining the molecular events involved in RBP synthesis and secretion, in order to subsequently identify and characterize sites and mechanisms of regulation. Our results show that immunoreactive RBP is initially synthesized from liver poly(A)'RNA as a product which migrates on gels more slowly than does pure rat serum RBP, and with an estimated molecular weight of 24,000. Similar results were also obtained (data not reported here in detail) with liver poly(A)'RNA isolated from vitamin A (retinol) deficient rats. Thus, RBP appears to resemble a number of other secretory proteins (7) in being synthesized initially as a larger molecular weight preprotein. In the case of RBP, however, it might be noted that the size of the prepiece in preRBP is rather large and at the upper limit of the size found for other secretory proteins (7, 26).
We next investigated the manner of processing of the larger molecular weight precursor to RBP. Two lines of evidence were obtained that suggest that preRBP is processed to a protein identical in size with serum RBP itself, presumably during translocation of the RBP across the endoplasmic reticulum bilayer (7-9). First, the cotranslational addition of dog pancreas microsomal membranes to the reticulocyte lysate resulted in the processing of a considerable proportion of the preRBP to a protein that migrated coincidentally with purified RBP. Second, posttranslational addition of the pancreas microsomal membranes did not result in the formation of a smaller protein (of the size of serum RBP).
Several secretory proteins, including albumin (27), parathyroid hormone (28), and insulin (29) have been demonstrated to be synthesized initially as preproproteins which are rapidly processed cotranslationally to more long-lived proproteins. Most of the immunoreactive albumin present in liver microsomes consists, for example, of the larger protein, proalbumin (30, 31). Studies conducted with immunoreactive RBP solubilized and isolated from rat liver microsomes have, however, suggested that RBP is not present in the microsomes as a larger, proRBP precursor (32). The results reported here further support this conclusion. First, as indicated above, cotranslational processing of preRBP with pancreatic microsomal membranes resulted in the production of a protein that appeared identical in size with purified RBP from serum. In addition, the 35S-labeled RBP immunoprecipitated from liver homogenates of rats injected with labeled methionine in vivo also appeared identical in size with purified RBP from serum. Thus, several lines of evidence all suggest that a proRBP intermediate does not exist during the biosynthesis of RBP in liver. Definitive evidence for this conclusion would, however, require the obtaining of NHn-terminal amino acid sequence information about the immediate product of the processing of preRBP, as compared to RBP isolated from serum.