Synthesis of Preprolactin and Conversion to Prolactin in Intact Cells and a Cell-free System*

Monolayer cultures of pituitary cells were pulse-labeled with [3H]leucine for several minutes and the incorporated radioactivity was analyzed by immunoprecipitation and electrophoresis on sodium dodecyl sulfate containing polyacrylamide gels. Following a 3-min labeling period, a peak of radioactivity with a mobility similar to that of preprolactin was observed, as well as radioactivity co-migrating with prolactin. Competition with unlabeled prolactin demonstrated the specificity of the immunoprecipitation reaction. After 5 min of pulse-labeling followed by 5-min chase in medium with unlabeled leucine, only a product with the mobility of prolactin remained. Addition of a membrane fraction from dog pancreas to a wheat germ cell-free translation system containing pituitary mRNA resulted in the conversion of preprolactin to prolactin. Partial sequence analysis demonstrated that the processed product contained the correct NH2 terminus of prolactin. Thus, both intact pituitary cells and a cell-free heterologous system are able to synthesize preprolactin and cleave it to prolactin offering strong evidence that preprolactin is the biosynthetic precursor to prolactin.


Monolayer
cultures of pituitary cells were pulse-labeled with r3H]leucine for several minutes and the incorporated radioactivity was analyzed by immunoprecipitation and electrophoresis on sodium dodecyl sulfate containing polyacrylamide gels. Following a 3-min labeling period, a peak of radioactivity with a mobility similar to that of preprolactin was observed,as well as radioactivity co-migrating with prolactin. Competition with unlabeled prolactin demonstrated the specificity of the immunoprecipitation reaction.
After 5 min of pulse-labeling followed by 5-min chase in medium with unlabeled leucine, only a product with the mobility of prolactin remained. Addition of a membrane fraction from dog pancreas to a wheat germ cell-free translation system containing pituitary mRNA resulted in the conversion of preprolactin to prolactin. Partial sequence analysis demonstrated that the processed product contained the correct NH2 terminus of prolactin. Thus, both intact pituitary cells and a cell-free heterologous system are able to synthesize preprolactin and cleave it to prolactin offering strong evidence that preprolactin is the biosynthetic precursor to prolactin.
The in vitro translation products of a number of secretory proteins have been found to be larger than the normally secreted form of the protein (for references see Ref. 1). The large cell-free translation products have been termed preproteins and it has been assumed that they represent precursors of the secretory proteins. However, it has been difficult to obtain evidence that the preproteins are actually synthesized in intact cells, presumably because they are very rapidly cleaved to a relatively stable intermediate storage size (proprotein)  in wheat germ extracts as described previously (9, 10). Dog pancreatic membranes were prepared and stored as described by Katz et al. (11). Following cell-free protein synthesis, reactions were diluted with an equal volume of NaCl/P,, 2% Triton X-100, 2% deoxycholate, 0.02 M leucine and then centrifuged at 10,000 x g for 10 min. Aliquots of the 10,000 x g supernatant were analyzed by immunoprecipitation. Zmmunoprecipitation and Analysis on Gels-For carrier immunoprecipitation, 5 pg of ['4C]prolactin were added to each sample and then an amount of specific rabbit anti-prolactin in excess of that required to precipitate the added carrier prolactin (9). After incubation overnight at 4"C, the immunoprecipitate was washed and pelleted as described previously (12). Previous studies have shown that the antisera used in this study are monospecific for prolactin (9, 12). For analysis on gels, the pellet was dissolved in 100 pl of 1% sodium dodecyl sulfate, 4 M urea, 0.05 M Tris-HCl, pH 7.4, 1% P-mercaptoethanol and heated to 90°C for 5 min. The sample was then electrophoresed on a 0.6 x 9 cm sodium dodecyl sulfate containing 12% polyacrylamide, 1.2% N,iV'-diallyltartardiamine gel using a discontinuous Tris/glycine buffer system (13). The gels were sliced and incubated overnight in 2% periodic acid, and the radioactivity was determined after addition of scintillation fluid. Alternatively, samples were combined with 0.02 ml of rabbit antiserum to rat prolactin and incubated overnight at 4°C. Then, 0.02 ml of a 10% suspension of Staphylococcus aureus, Cowan I strain, ATCC No. 12598, prepared as described by Kessler (14), was added to the samples and incubated for 15 min at 4'C. Antigen antibody complexes adsorbed to the Staphylococcus aureus were collected by centrifugation at 2,000 x g for 10 min. The pellet was washed four times by suspension in 0.75 ml of NaCl/P,, 1% Triton X-100, 1% deoxycholate, 0.01 M leucine followed by centrifugation at 2,000 X g for 10 min and transferred to a new tube for a final wash. The immunoprecipitated radioactivity was released from the pellet by incubation in 1% sodium dodecyl sulfate, 4 M urea, 0.05 M Tris, pH 7.4, 1% P-mercaptoethanol for 5 min at 90°C. After centrifugation at 10,000 x g for 10 min, the supernatant was analyzed on sodium dodecyl sulfate-containing polyacrylamide gels as described above. Sequence Analysis-Cell-free products synthesized in the presence ' The abbreviation used is: NaCl/P,, phosphate-buffered saline.
of dog pancreas membranes were labeled with [";'S]cystine (40 Ci/mmol) and ["Hlglycine (23 Ci/mmol). The cell-free product was immunoprecipitated in the presence of carrier prolactin and subjected to sequence analysis as described previously (10).

Synthesis of Preprolactin in Intact Cells-Monolayer cultures of dispersed pituitaries
were used in an effort to detect the synthesis of preprolactin in intact cells. Analysis by polyacrylamide gel electrophoresis of products labeled by a 3-min pulse with ["Hlleucine demonstrated the synthesis of several peaks of immunoreactive radioactivity (Fig. 1A). The major peak of radioactivity co-migrates with a prolactin standard. A peak of more slowly migrating radioactivity has an electrophoretic mobility similar to that of preprolactin synthesized in the cell-free wheat germ system (for comparison, see Fig.  3). The radioactivity which migrates more rapidly than prolactin probably represents incomplete peptide chains. The effects of competition with prolactin for binding to the antibody were next examined in order to be certain that all of the immunoreactive material synthesized during the short pulse actually contained the antigenic determinants of prolactin. Addition of unlabeled prolactin to the immunoprecipitation reaction almost completely abolished the binding of radioactivity to the antibody (Fig. 1B). These results strongly suggest the synthesis of preprolactin in intact pituitary cells. In order to examine the fate of preprolactin synthesized in intact cells, a brief labeling period (5 min) was followed by a chase (5 min) with unlabeled amino acids. Without the chase, the radioactivity is again found in preprolactin, prolactin, and some peptides ( Fig. 2A). Following the chase period, radioactivity was only found in prolactin (Fig. 2B) chase period appears to be sufficient to complete elongation of nascent chains and remove the precursor segments of all peptides labeled during the initial period. These findings support the view that preprolactin is a precursor of prolactin. Conversion of Preprolactin to Prolactin in a Cell-free System-As described previously (9,15,16), translation of pituitary RNA in a cell-free system from wheat germ results in the synthesis of preprolactin (Fig. 3A). Addition of a membrane preparation from dog pancreas to the wheat germ reaction mixture resulted in the synthesis of a substantial amount of cell-free product which co-migrates with prolactin (Fig. 3B). The membrane preparation apparently cleaved preprolactin to a size similar to that of prolactin. Addition of membranes to the wheat germ reaction mixture after completion of translation did not alter the mobility of preprolactin (data not shown). The fidelity of this apparent cleavage was examined by sequencing cell-free products synthesized in the presence of dog pancreas membranes and labeled with ["HIglycine and [""Slcystine.
The NH, terminus of the processed material contains cysteine at positions 4 and 9 and glycine at positions 6 and 7 (Fig. 4). This is identical to the sequence of these amino acids at the NH2 terminus of prolactin." The data also suggest the presence of a second sequence with a glycine at position 11. This aligns with the sequence of preprolactir? and is consistent with the observation that not all of the preprolactin is cleaved to prolactin by the dog pancreas membranes.

DISCUSSION
The present findings demonstrate that preprolactin is synthesized in pituitary cells and suggests that preprolactin is the biosynthetic precursor of prolactin. Furthermore, the demonstration of a cell-free system which synthesizes preprolactin and accurately cleaves it to prolactin is consistent with this view.
The function of the precursor segment of the preproteins is not known. The "signal hypothesis" suggests that the precursor segment is involved in recognition of endoplasmic reticulum and transport of secretory proteins (2). The recent finding that the primary translation product of ovalbumin mRNA does not contain a precursor segment demonstrates that not all secretory proteins contain such a peptide (17). At this time, it is not clear if ovalbumin is merely an exception which is secreted by an unusual mechanism or if the precursor segment is not involved in the secretory process.
Preprolactin synthesis was detected by pulse-labeling pituitary cells in monolayer cultures. Use of monolayer cultures allows the rapid disruption of cell membranes with detergents. As the enzyme which cleaves the precursor segment appears to be localized in a membrane fraction, rapid disruption of membranes may facilitate the ability to detect the synthesis Pulse-labeling of pituitary fragments rather than cell cultures failed to demonstrate the synthesis of preprolactin." It is possible that the time required to wash and homogenize tissue fragments allows the cleavage of preprolactin.
Although our studies demonstrate the synthesis of apparently complete preprolactin in intact cells, it has not been possible to determine if cleavage of preprolactin always occurs after completion of synthesis. Cleavage of the precursor segment could possibly occur at random times during the synthesis of a polypeptide.
Thus, some polypeptides might be synthesized as the complete preprotein and then cleaved, while the precursor segment might be removed before the completion of other polypeptides.
A heterologous cell-free system containing dog pancreas membranes and wheat germ extract is able to accurately cleave preprolactin to prolactin. Presently the enzyme recognition site which allows this accurate cleavage is not apparent. The amino acid sequence of preprolactin immediately preceding the prolactin NH, terminus differs from that of other precursors.' Analysis of the enzyme mechanisms which lead to the accurate removal of the precursor segment of secretory proteins will likely require development of a direct enzyme assay.
Addition of membranes to cell-free translation systems results in cleavage of preprolactin or other preproteins only when membranes are present during translation, not when they are present after translation (2,3,18,19). This has led to the suggestion that only nascent chains can be cleaved (19). However, the finding that intact cells synthesize the complete preprolactin molecule which is subsequently cleaved to prolactin raises the possibility that cleavage can occur following