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Cleavage of Honeybee Prepromelittin by an Endoprotease from Rat Liver Microsomes: Identification of Intact Signal Peptide

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Cleavage of Honeybee Prepromelittin by an Endoprotease from Rat Liver Microsomes: Identification of Intact Signal Peptide Proc. Natl. Acad. Sci. USA Vol. 79, pp. 2260-2263, April 1982 Biochemistry Cleavage of honeybee prepromelittin by an endoprotease from rat liver microsomes: Identification of intact signal peptide (precursor processing/signal peptidase/membrane protein/melittin) CHRISTA MOLLAY, ULRIKE VILAS, AND GUNTHER KREIL Institute for Molecular Biology, Austrian Academy of Sciences, Billrothstrasse 11, A5020 Salzburg, Austria Communicated by Paul D. Boyer, January 19, 1982 ABSTRACT It has previously been shown that rat liver mi- tivities, on a sucrose-deoxycholate gradient. This endoprotease crosomes contain a proteolytic enzyme that cleaves honeybee pre- cleaves prepromelittin at a single peptide bond, the pre- promelittin to yield promelittin. This enzyme has now been fur- pro junction. The-present communication describes the isola- ther purified by centrifugation on a sucrose-deoxycholate gradient tion ofthe second reaction product, intact signal peptide, from and then reconstituted into phospholipid vesicles. Incubation of such in vitro assays. prepromelittin with vesicles in the presence of melittin yields, in addition to promelittin, a hydrophobic peptide. The latter could be isolated by extraction with 1-butanol and paper electrophoresis MATERIALS AND METHODS in 30% formic acid and was shown to be intact signal peptide by analysis of peptic fragments and automated Edman degradation. Tritium-labeled amino acids ofthe highest specific activity cur- The microsomal enzyme is thus an endoprotease that hydrolyzes rently available were purchased from the Radiochemical Centre prepromelittin exclusively at the pre-pro junction. The precision (Amersham, England). Phospholipids were obtained from Lipid of this cleavage of an insect preprotein by a rat liver enzyme in- Products (Nutfield Nurseries, England), synthetic dipeptides dicates that we are dealing with the ubiquitous eukaryotic signal were from Bachem AG (Bubendorf, Switzerland), and other peptidase. chemicals were from standard suppliers. Phospholipase-free melittin was prepared from lyophilized bee venom (Firma The precursors of secreted polypeptides contain an amino-ter- Mack, Illertissen, Federal Republic of Germany) as described minal signal sequence that is not present in the final product (14). The fragment melittin1_19 was isolated by limited digestion (1, 2). In eukaryotic cells, cleavage of signal peptides is a co- of melittin with chymotrypsin (15). translational reaction that takes place early in the assembly of Preparation of Prepromelittin. Total RNA from queen b -e the polypeptide chain (3, 4). The protease that catalyzes this venom glands was translated in a cell-free system from wheat reaction, tentatively called signal peptidase, is neither tissue germ in the presence of one labeled and 19 unlabeled amino nor species specific, and correct processing of different pre- acids as described (16). Aliquots of these incubation mixtures proteins in vitro by, e.g., dog pancreas microsomes (5) or in vivo were used without further fractionation for the cleavage assays. in oocytes ofXenopus laevis (6) has been shown. Several groups Partial Purification of Signal Peptidasei. Rat liver rough of workers have, moreover, described the posttranslational microsomes were prepared according to Sandberg et aL (17), cleavage of signal peptides in the presence of microsomes and suspended in 1 M KCV/0.02% deoxycholate, and again collected detergents (7-10). These studies showed that signal peptidase by centrifugation. They were then dissolved in 0.4% deoxy- is an intracellular membrane-bound enzyme that, interestingly, cholate and layered onto a step gradient of 2-0.2 M sucrose/ is not inactivated by any ofthe typical protease inhibitors (11). 0.2% deoxycholate as described by Kaderbhai and Freedman In all these-experiments, only one of the reaction products, the (18). After centrifugation for 17 hr at 80,000 X g in a Beckman secreted polypeptide, could be detected, so that the question SW 41 rotor, the fraction present in the 1.4 M sucrose layer was of whether signal peptides are cleaved en bloc or in fragments collected. This fraction was added to dry lipid (phosphatidyl- remained unanswered. Evidence has, however, been pre- choline/phosphatidylethanolamine, 2.5:1; 14 mg ofphospho- sented that in vivo signal peptides, ifthey ever exist as separate lipid/1 mg of protein) and dialyzed for 4Whr against 0.1 M entities, must be extremely short-lived (12). Tris'HCVl1 mM o-phenanthroline, pH 8.0. The reconstituted We have investigated the cleavage ofhoneybee prepromelit- vesicles were collected by centrifugation. tin by rat liver microsomes and have shown that, in the presence Assay for Signal Peptidase. Labeled prepromelittin was in- ofdeoxycholate, promelittin with the correct NH2 terminus can cubated for 2 hr at 250C in dialysis buffer with reconstituted be generated in a slow reaction (8, 13). As reported briefly, an membranes containing 20-50 Ag ofprotein and melittin (1 moV 10-fold purification of a protease could be achieved after so- 50 mol ofphospholipid). In control samples, vesicles or melittin lubilization ofmicrosomal membraneswith detergents and sub- were omitted. The samples were then adjusted to pH 8.5 with sequent reassembly of membrane proteins with added phos- dilute NH3 and extracted three times with an equal volume of pholipids bydialysis (13). Even in these reconstituted membranes, 1-butanol. Under these conditions, promelittin remains in the this enzyme, which we consider to be the signal peptidase, is aqueous layer while uncleaved prepromelittin accumulates at acryptic enzyme requiringfor activity the presence ofdetergent the interface (16). or, as we have found, of melittin. Product Analysis. The purification of promelittin and its Signal peptidase from rough microsomes ofrat liver has now characterization by enzymatic hydrolysis and stepwise Edman .been partially purified, apparently free ofother proteolytic ac- degradation of the fragment derived from its amino end have been described (8). Essentially the same methods can be used The publication costs ofthis article were defrayed in part by page charge for analysis of prepromelittin (19). payment. This article must therefore be hereby marked "advertise- The butanol layer was applied to Whatman 3MM paper and ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. purified by high-voltage paper electrophoresis in 30% formic 2260 Downloaded by guest on October 2, 2021 Biochemistry: Mollay et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2261 E DNP Lys Glu Leu 20 A2 Al 2 10 I C. 0 2 4 6 8 Or CUL 0. 0 2 4 6 8 10 12 14 16 18 20 22 "0 cd 1 4 2 0 2 4 0 2 4 6 8 10 12 14 16 18 20 22 Distance from origin, cm FIG. 1. Analysis of the butanol extract from an experiment with prepromelittin labeled with [3H]alanine. (A) High-voltage paper electrophoresis in 30% formic acid. The butanol extracts from a sample ( ) and a control incubated without melittin (----) were applied to Whatman 3MM paper and fractionated at 30 V/cm for 2 hr. The dried paper was then cut into sections and the radioactivity present in each was determined in a liquid scintillation counter (toluene-based scintillator). (B) Electrophoretic separation of the peptic digest. Radioactive material present at the origin of A was hydrolyzed with pepsin overnight, and the digest (- ) was then fractionated by paper electrophoresis at pH 4.8. ----, Control. (C) Electro- phoretic separation of peptic fragments at pH 2.0. Neutral peptides (-) from the electrophoresis at pH 4.8 (B) were recovered by elution and rerun at lower pH. Two peptides, designated Al and A2, were present in about equal amounts. ----, Control. (D) Electrophoresis of peptides Al (-) and A2 (-) after Edman degradation. The two fragments were recovered by elution, shortened by one residue by Edman degradation, and again sep- arated by paper electrophoresis at pH 2.0 with synthetic dipeptides as references. MEL (1-19), melittin-119; E-DNP-Lys, E-dinitrophenyllysine. acid. The electropherogram was cut into sections, and the ra- Automated Edman Degradation. The cleaved signal peptide dioactivity present on each was determined in a liquid scintil- could also be partially purified by applying the butanol layer lation counter. The section corresponding to the origin was sub- to Whatman 3MM paper and then eluting with water to remove sequently soaked in dilute HCl (pH 1.8) containing 0.7-1 mg soluble radioactive contaminants. Signal peptide could subse- of pepsin/ml and incubated overnight at 30'C. The digest was quently be recovered by elution in sufficient yield with the sol- then dried and examined for the presence of peptic fragments vent mixture used for paper chromatography. The dried ma- ofthe signal peptide ofprepromelittin. This involved separation terial was dissolved in 0.4 ml of formic acid and subjected to by high-voltage paper electrophoresis at pH 2.0 (10% acetic acid automated Edman degradation in the presence of 3 mg of the adjusted with formic acid) or at pH 4.8 (1% pyridine/1% acetic nonprotein carrier Polybrene and 4 mg of apomyoglobin as de- acid) or by paper chromatography (butanol/acetic acid/water, scribed by Beyreuther et al. (20). 4:1:2) using suitable reference peptides as markers. Radioactive fragments were recovered by elution with 1% acetic acid/1% RESULTS 1-butanol, subjected to Edman degradation, and characterized Incubation of prepromelittin with rat liver microsomes in the by one of the above-mentioned separation methods. presence of detergent has previously been shown to yield pro- Downloaded by guest on October 2, 2021 2262 Biochemistry: Mollay et al. Proc. Natl. Acad. Sci. USA 79 (1982) melittin in a slow hydrolytic reaction (8, 13). After extraction cycle. Fragment Al thus has the structure X-Ala-Leu. Frag- with 1-butanol, this cleavage product can be purified from the ment A2 was also recovered by elution and shortened by one aqueous phase by paper chromatography and subsequently residue by Edman degradation. The resulting dipeptide is iden- characterized by enzymatic digestion and Edman degradation tical to Tyr-Ala on paper electrophoresis at pH 2.0 (Fig.
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