Membrane Biogenesis EVIDENCE THAT A SOLUBLE CHIMERIC POLYPEPTIDE CAN SERVE AS A PRECURSOR OF A MUTANT lac PERMEASE IN ESCHERZCHZA COLI*

(Received for publication, June 20, 1980)

Victor A. Fried From the Department of Microbiology, School of Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania 15261

A mutant in the Escherichia coli lac permease, called l?, appears to be defective in the biogenesis and proper assembly of this membrane protein. It was proposed that this defect led to the accumulation of a precursor of the mutant permease (Fried, V. A. (1977) J. Mol Bid. 114, 477-490). In this communication, evidence is pre- sented that the lacvf mutant accumulates a novel lac- specific soluble polypeptide with a molecular weight of 87,000. Detected by double-label analysis on sodium dodecyl sulfate gels, and identified as a lac-specific polypeptide on a two-dimensional gel system, this poly- peptide is immunoprecipitated by anti-transacetylase antibody. Pulse-chase experiments are consistent with the hypothesis that it is converted in vivo into a lac- specific membrane protein with an apparent molecular weight of 28,000, which appears to be the mutant lac permease. The results suggest that the 87,000-dalton soluble protein is a precursor of the mutant lac per- mease. It is proposed that this precursor is a polypro- tein chimera containing both the lacy and lacA gene products.

What are the properties of a protein that determine its cellular localization? What cellular processes are involved in determining this localization? Answers to these questions are fundamental to understanding membrane biogenesis. Progress has been made over the last few years toward answering these questions but the molecular details remain obscure. While an apolar protein could spontaneously self-assemble into the lipid milieu, there is abundant evidence that many proteins are assembled after or concomitant with posttranslational modi- fications of their primary gene products. Two general classes of such posttranslational mechanisms have been observed in a variety of systems. One type of mechanism involves proteins that are incorporated co-translationally from membrane- bound polysomes (1-5). Such proteins appear to be made with an NH2-terminal "signal sequence" that is proteolytically removed from the membrane protein as part of maturation. The other proteins are synthesized on free polysomes and then incorporated posttranslationally into the membrane. This latter mechanism may involve extensive proteolytic mod- ifications of a polyprotein precursor (6, 7). Other mechanisms may exist.

* This research was supported in part by Grants GM 23288 and RR-05416 from the National Institutes of Health, by Grant PCM- 8011740 from the National Science Foundation, and Grant U-22 from the Health Research and Services Foundation of Pittsburgh. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "aduertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

One approach to answering these questions is the study of mutants defective in the biogenesis of a specific membrane protein. In a previous study (a), I proposed that a mutant of the lac permease, the lacYf mutant, was defective in biogen- esis of the permease. This conclusion was based upon the unusual induction and deinduction kinetics of the Y' permease activity. When the lac operon was induced, P-galactosidase synthesis appeared normal but permease activity appeared slowly, reaching a maximum differential rate of appearance only after several generations. Upon deinduction, permease activity continued to appear. I proposed that the functional mutant permease arose from a slowly processed precursor form. As a consequence of this slow processing, the precursor form accumulated as a relatively stable species. After de nouo synthesis had been stopped by removing inducer, the precur- sor continued to be processed into the functional permease.

In this communication, I present evidence that the Yf permease is processed into its membrane form from a high molecular weight soluble precursor. This precursor appears to be a polyprotein or chimera which also contains the lacA gene product. Though this precursor does not represent a normal pathway for the biogenesis of the wild type permease (9), it does provide a model system for studying the properties of a protein that determine its cellular localization and the cellular mechanisms involved in posttranslational modifications.

EXPERIMENTAL PROCEDURES

Bacterial Strains and Culture Conditions-The Escherichia coli K-12 strain H3000 (wild type) lac ( lacZ+rA+) and the yf mutant (3000 X 19), now called Y'4 (lacTY'A-), have been described previ- ously (8). Strain H3000 is the parent of Y'4. Both strains are Su- as neither will plaque bacteriophage T4 carrying an amber or ochre mutation in gene 43, the DNA polymerase (T4 phage were a gift of Dr. J. D. Karam, Department of Biochemistry, Medical University of South Carolina). Cultures were grown at 30°C in M63 minimal salts medium (13.6 g of KH2P04, 2.0 g of (NH4)2S04, 0.5 mg of FeS04.7 H20, 2.9 g of NaC1, 0.25 g of MgS04. 7 H20 per liter, adjusted to pH 7.0 with KOH) containing 0.2% glycerol as carbon source and thymine HCI, 0.5 pg/ml. When required, the lac operon was induced with 1 mM IPTG.'

Cell Fractionation-The procedure used is a modification of that described by Showe and DeMoss (IO). Bacteria were washed once in 50 mM Tris-HCI, pH 7.9, 1 mM MgC12 (TM buffer) and pelleted in a 1.5-ml polypropylene Microfuge tube. Pellets, usually containing no more than 10"' bacteria, were resuspended in 0.25 ml of TM buffer and 50 pg/ml of egg white lysozyme, 2 pg/ml of DNAse, 1 mM PMSF. The suspension was freeze-thawed five times in an acetone/dry ice bath. The thawed sample was incubated in an ice bucket for 30 min and then separated into particulate and soluble fractions by centrif- ugation at 20,000 X g for 30 min at 4°C. The pellet was resuspended in TM buffer containing' lysozyme, DNAse, and PMSF, and the

The abbreviations used are: IPTG, isopropyl-P-D-thiogalactopy- ranoside; PMSF, phenylmethyl sulfonyl fluoride; SDS, sodium dode- cy1 sulfate; PAGE, polyacrylamide gel electrophoresis.

244

A Precursor of the 1acY'Permease 245

freeze-thaw procedure repeated. Supernatant fractions were pooled and the pellet was resuspended in 0.25 ml of TM buffer containing 1 mM PMSF. Both pellet and supernatant fractions were quick-frozen in liquid Na and kept a t -2O'C until used.

The effectiveness of this procedure in separating membrane and soluble components was assessed by measuring the distribution of some well characterized membrane and soluble protein markers. The wild type E. coli K-12 strain H3000 was used in the following exper- iments. Analyses of the pellet and soluble fractions are shown in Table I. Using this fractionation procedure, 60% of the total protein is present in the supernatant fraction. The two soluble enzymes of the lac operon, P-galactosidase and thiogalactoside transacetylase, were used as markers for soluble proteins. These enzymes represent an extreme of soluble protein sizes, since p-galactosidase is one of the largest E. coli proteins (subunit molecular weight = 1.16 X IO5; holoenzyme molecular weight = 4.64 X lo5) (11). while the lac thiogalactoside transacetylase is a relatively small protein (subunit molecular weight = 2.5 X 10"; holoenzyme molecular weight = 5.0 X 10') (12). About 95% of the activity of both enzymes is found in the supernatant fraction. Assay of NADH oxidase, a cytoplasmic mem- brane marker (13) and lipid, as labeled with [2-'H]glycerol, indicates that approximately 95% of the membrane has been recovered in the pellet fraction. These results demonstrate that this technique permits a nearly quantitative recovery of membrane and soluble fractions with little cross-contamination.

Labeling of Cultures-The experiments described in this commu- nication were performed with cultures either singly or doubly labeled with radioisotopes of leucine. I t was found that reproducible labeling patterns were obtained in the double-label experiments only when the concentration of the amino acid label was adjusted so that the rate of labeling was independent of the amino acid concentration and that the incorporation of the label was directly proportional to the increase in bacterial mass as indicated on differential plots. In such experiments, it was determined that approximately 4 nmol of leucine/ ml would be incorporated for an increase in the culture of 0.1 A 850 (1 A350 = 3 X lo8 bacteria/ml). The specific activity of the leucine label was adjusted to satisfy this condition, appropriate for the over which labeling was to be followed. Cultures were usually single-labeled with [jH]leucine a t a specific activity of 3 to 5 mCi/pmol. Labeling times usually ranged from 20 min to 1 h. When lac operon products were to be examined, the cultures were induced by addition of IPTG 20 min prior to addition of label.

For double-label experiments, an exponentially growing culture was divided into two portions when the culture reached 1.0 AZ5". In induction double-label experiments, IPTG was added to one of the

TABLE I Characterization of the pellet and supernatant fractions prepared

by the freeze-thaw procedure Recovery in:

Marker Total re- covery" Pellet ~~~~~~

Ph Ph Protein' 105 40 60 /%Galactosidase' 100 6 94 Thiogalactoside transacetylased 98 4 96 NADH oxidase' 90 96 4 Lipid as [2-'H]glycerol label' 98 95 5

a Total of activity recovered in pellet and supernatant fractions

' Protein was determined by the method of Lowry et al. (14) with

P-Galactosidase activity was measured as described previously

Thiogalactoside transacetylase activity was measured as de- scribed by Alpers et al. (16) but modified to include 2 m g / d of bovine serum albumin in all reagents.

'NADH oxidase was measured as described by Osborn et al. (13). 'A culture of H3000 grown in T-broth (14) was labeled with [2-

3H]glycerol (New England Nuclear) a t 10 pCi/ml (200 mCi/mmol) for one-half generation, chased with 1% unlabeled glycerol for one gen- eration, and washed several times by centrifugation. Fractionation was as described in the text. Aliquots of various fractions were solubilized directly in Aquasol-2 and radioactivity was determined in a scintillation counter. The radioactivity in the pellet fraction could be quantitatively extracted into chloroform/methanol.

relative to the sample before centrifugation.

bovine serum albumin as standard.

(15).

portions in order to induce the lac operon. Twenty minutes later, ["C]leucine was added to the uninduced culture and [3H]leucine was added to the induced culture. The specific activity of the [3H]leucine was adjusted so that it was 10-fold greater than the ['4C]leucine. The concentration of leucine in the cultures was the same, and was adjusted appropriately for the interval of labeling. At the end of the labeling period, unlabeled leucine was added to the cultures a t a concentration of 1 mg/ml, and the cultures were placed in ice. In double-label experiments, the 'H- and '"C-labeled cultures were mixed, washed twice by centrifugation at 4"C, and transferred to Microfuge tubes for fractionation into soluble and membrane com- ponents as described in the previous section.

Double-label pulse-chase experiments were performed as follows. Cultures were double-labeled as above; the pulse was usually for 20 min. For pulse-chase in the presence of inducer, unlabeled leucine was added to both cultures a t 1 mg/ml, and the cultures were mixed and allowed to grow together. Samples were removed a t appropriate times, washed, and fractionated as above. For double-label pulse- chase experiments with deinduction during the chase period, the cultures were combined and washed once by centrifugation at the end of the pulse. The mixed cultures were resuspended in medium without inducer and samples were taken as above.

Polyacrylamide Gel Electrophoresis-Polyacrylamide electropho- resis in the presence of SDS was performed using the discontinuous buffer system essentially as described by Laemmli (17) and modified for slab gels as described by Ames (18). Samples were prepared for electrophoresis by mixing the sample with an equal volume of 0.0625 M Tris/Cl pH 6.8,4% SDS, 10% P-mercaptoethanol, 20% glycerol, and a trace of bromphenol blue. This mixture was heated as indicated in the particular experiment before loading on the gels.

Two-dimensional gel electrophoresis was done as described by O'Farrell (19). The isoelectric focusing gel (pH range, 5 to 7) was run in 100-pI capillary pipettes. After isoelectric focusing, the gel was run in the second dimension on 0.75-mm-thick SDS slab gels as described above.

Gels were fixed in a solution of 45% methanol, 10% glacial acetic acid, 45% water (v/v); stained with a solution of 0.25% Coomassie blue (R-250) in 7.5% glacial acetic acid, 50% methanol; and destained in 7.5% acetic acid, 10% methanol. Apparent molecular weights were determined from the mobility of the following standards: P-galacto- sidase ( M , = 116,000); phosphorylase b ( M , = 94,000); bovine serum albumin ( M , = 68,000); catalase ( M , = 60,000); glutamic dehydrogen- ase (Mr = 53,000); ovalbumin ( M r = 43,000); aldolase ( M , = 40,000); lactate dehydrogenase ( M , = 35,000); carbonic anhydrase ( M , = 29,000); P-lactoglobulin ( M , = 18,400).

In order to measure radioactive proteins on tube gels, the gels were frozen directly after electrophoresis and sliced using a Mickle gel slicer. The slices were placed into scintillation vials and 10 ml of Econofluor (New England Nuclear) containing 5% Protosol (New England Nuclear) was added. The vials were incubated a t 45'C with gentle shaking overnight. After cooling, the samples were counted in a Mark 111 scintillation counter (Tracor Analytic). The scintillation counter was programmed with a series of quenched standards of 'H and I4C prepared in the scintillation fluid used above and containing blank gel slices. Data were corrected for quenching and, in double- label experiments, for spill-over between 'H and I4C counting chan- nels. All data were expressed in disintegrations per min for each isotope present in the slice. Slab gels were fixed, stained, destained, and dried under vacuum on a sheet of heavy filter paper. Sample lanes were gridded with a ruler in order to compare relative positions of slices across the gel, and the lanes were cut into slices with fine scissors. The slices were placed into scintillation vials, and 25 p1 of water was layered on each slice for 20 min. The Protosol/Econofluor scintillation fluid was added and treated as described above.

Fluorograms of slab gels were made using a modification of the procedure of Bonner and Laskey (20). Slab gels were fixed for 1 h. The fixed slab gel was placed in 100 ml of Enhance (New England Nuclear) for 1 h at room temperature with gentle shaking. After impregnation with Enhance, the gel was washed with several changes of water over 1 h, equilibrated for 30 min in a solution of 1% glycerol, 10% acetic acid, and dried under vacuum onto filter paper backing. The gels were exposed at -70°C to Kodak X-Omat XR-5 x-ray fdm which had been prefogged to AM(, = 0.15 according to the procedures of Laskey and Mills (21).

Analysis of Double-label Gels-In double-label experiments where the induced culture was labeled with "H-amino-acid and the unin- duced culture was labeled with the '"C-amino-acid, the lac-specific proteins were identified and quantitated by calculating the excess of

246 A Precursor of the 1acY’Permease

3H-labeled protein present in the gel slices relative to the normal distribution of 3H- and I4C-labeled proteins on the gel. The total 3H and I4C disintegrations per min on the gel were determined by summing the radioactive species in all the slices. The amount of 3H and “C in each slice was then expressed as the fraction (percentage) of the total isotope in the gel. The difference between the fraction of ’H and I4C for each slice was calculated. A plot of this difference across the gel would give positive peaks for the presence of lac- specific proteins and negative peaks due to the fact that induction of the lac operon introduces a new set of polypeptides not contributing to the normal protein distribution. The differential percentage plots were corrected for this by determining the percentage of excess represented by the lac protein species. The correction factor was determined by measuring the percentage of negative deflection rela- tive to the percentage of uninduced label in regions of the gel where lac-specific proteins were not detected. It was found that this correc- tion factor agreed closely with a correction factor based solely on the contribution of the major lac proteins (e.g. P-galactosidase). The differential percentage plots, A%, presented in this communication represent corrected values.

Nearly every double-label experiment reported in this communi- cation has also been performed with the labeling scheme reversed (i.e. I4C induced; 3H uninduced). The results of these experiments were indistinguishable from those shown, and thus the lac-specific peaks are unlikely to be artifacts of labeling and analysis procedures.

Preparation ofAntibodies to P-Galactosidase and the Thiogalac- toside Transacetylase-The wild type strain H3000, induced for the expression of the lac operon, was the source of the purified immu- nogens. P-Galactosidase was purified essentially as described by Cra- ven et al. (22). The purified P-galactosidase was greater than 98% homogeneous as determined by Coomassie blue staining of SDS gels. The lac thiogalactoside transacetylase was purified by a modification (12) of the procedures described by Zabin (23). The purified trans- acetylase was approximately 90% homogeneous as measured by Coo- massie blue staining of SDS gels.

The purified enzymes were mixed with an equal volume of Freund’s complete adjuvant (Difco). A total of 500 pg of protein was injected into the footpads of Dutch rabbits. On the 4th and 8th days, 200 pg of protein in Freund’s adjuvant was injected intramuscularly into the thighs. Four days later, purified antigen in normal buffered saline was injected subcutaneously (50-pg injections). The subcutaneous inoc- ulations were given every 4th day over a 3-week period. Two weeks after the final subcutaneous injection, serum was removed by heart puncture. After a 2-week rest, the subcutaneous injection regimen was repeated.

Serum was allowed to clot overnight at 4°C and the clot was removed by centrifugation. Complement was inactivated by heating at 56°C for 30 min and the precipitate was removed by centrifugation at 20,000 x g for 30 min. The y-globulin fraction was isolated by precipitation with ammonium sulfate (33%% saturation) and partially purified by gel filtration chromatography on Ultro-gel AcA-34 (LKB). This partially purified IgG fraction was stored at -70°C in aliquots at a concentration of 10 pg/pl of protein in 0.05 M Tris/Cl, pH 7.9, 0.1 M NaC1, 1 mM sodium azide.

In order to reduce the nonspecific background in the immunopre- cipitation experiments, the antibody preparations were preabsorbed before use. Unlabeled extracts of the uninduced H3000 strain were prepared by the freeze-thaw technique. Antibody, at a final concen- tration of 0.5 mg/ml, was mixed with the unlabeled total extract at a final concentration of 2 mg/ml of protein in IP buffer (1% Triton X- 100, 0.5% deoxycholate, 0.15 M NaCI, 50 mM Tris/CI, pH 7.5, 20 mM sodium azide, and 5 mM EDTA) containing 1 mM PMSF. The mixture was incubated at 4°C overnight and cleared of precipitated material by centrifugation at 20,000 X g for 45 min.

itation by mixing aliquots of the radioactively labeled protein fractions Immunoprecipitation-Samples were prepared for immunoprecip-

with aliquots of unlabeled extracts of the uninduced H3000 strain. The unlabeled protein was in approximately a 10’ mass excess. The mixture was mixed with an equal volume of 2% SDS in 0.0625 M Tris/ C1, pH 6.8, and heated at 100” for 2 min. A 100-pl volume of the SDS- treated sample in a 1.5-ml polypropylene Microfuge tube was diluted by addition of 1.0 ml of the IP buffer and cleared of particulate material by centrifugation at 20,000 X g for 30 min. Preadsorbed antibody was added to the sample and incubated at room temperature for 30 min. The amount of antibody used (usually 25 pg) was deter- mined to be in excess of antigen by pilot titration experiments. The antibody.antigen complexes formed in these incubations were re- covered using fixed Staphylococcus aureus (24). Two hundred micro-

liters of the S. aureus reagent (lo%, v/v) in IP buffer was added to each sample and the incubation was continued for an additional 30 min. The antib0dy.antigen.S. aureus complexes were recovered by centrifugation for 5 min in a Beckman Microfuge and the supernatants representing non-cross-reacting material were saved. The pellets were washed three times in IP buffer and then washed once in the same buffer without detergents. The precipitated material was dissociated by boiling the pellet for 2 min in 50 p1 of the electrophoresis sample buffer (0.0625 M Tris/Cl, pH 6.8, 2% SDS, 5% P-mercaptoethanol, in 10% glycerol) and the S. aureus was removed by centrifugation. Aliquots of the supernatant were counted in Aquasol-2 (New England Nuclear) and the remaining supernatant was stored at -2OOC until analyzed by SDS-PAGE.

In order to examine the material not immunoprecipitated, the first supernatants recovered above were precipitated by 10% trichloroace- tic acid, and the pellets were washed twice with ethanol/ethyl ether (1 : 1) and resuspended by boiling in the electrophoresis sample buffer.

RESULTS

Identification of lac-specific Proteins by Double-label Analysis-If the yf mutant were accumulating a stable pre- cursor of the permease, such a precursor should be identifiable as a new lac-specific protein species. Lac-specific proteins were identified by a double-label technique in which unin- duced cells and cells induced with IPTG were labeled with 14C- and 3H-amino-acid, respectively. The labeled proteins were resolved by SDS-PAGE and lac-specific polypeptides were detected as bands containing an excess of 3H relative to the normal distribution of I4C- and 3H-labeled polypeptides. This technique was used successfully in identifying the lac permease in haploid E. coli strains (25) and, more recently, in strains carrying the lacy gene in a plasmid vector in order to promote gene amplification (26).

The limits of the sensitivity of this technique are demon- strated by an analysis of a double-labeled, uninduced, wild type culture (Fig. 1). As expected, no major peaks of differ- ential labeling were seen, and the maximum difference in labeling for each slice across the gel was +0.1% of the total label recovered from the gel. Patterns similar to those shown in Fig. 1 also were obtained in induced double-label experi- ments with strains deleted for the entire lac operon.

FIG. 1. SDS-PAGE analysis of the double-label, uninduced, wild type strain. The wild type strain H3000 was double-labeled with leucine without induction and fractionated into soluble and membrane proteins as described under “Experimental Procedures.” Proteins were analyzed on 9% acrylamide tube gels. The gels were cut into 2-mm slices and analyzed by methods described in the text. The upper panels indicate actual radioactivity in each slice; the lower panels show differential percentage analysis. A, soluble proteins (l0OoC, 2 min). Total activity: 1.2 X IO6 dpm of 3H; 1.3 X IO5 dpm of

of 3H; 9.7 X lo4 dpm of I4C. 14C. B, membrane proteins (50”C, 30 min). Total activity: 1 X lo6 dpm

A Precursor of the 1acY‘Permease 247

The double-labeled analysis of soluble and membrane frac- tions of the induced wild type lac strain is shown in Fig. 2. The soluble fraction contains a major peak of lac-specific protein that represents 3% of the total leucine label in soluble protein (Fig. 2 A ) . This is the subunit of /?-galactosidase (Mr = 116,OOO), since it is quantitatively immunoprecipitated by anti-P-galactosidase antibody as described below. A peak rep- resenting about 0.25% of the label was found migrating at the dye front which in this gel contained all polypeptides with molecular weights 5 14,000.

A lac-specific peak representing the 25,000-dalton subunit of lac thiogalactoside transacetylase was not consistently ob- served in this analysis, although the enzymatic activity was found localized in the soluble portion. This was not surprising, since the transacetylase is translated with a 10-fold lower efficiency than is P-galactosidase, and has a relatively low leucine content (23). Based upon the specific activity of the transacetylase purified from this strain (12), the amount of transacetylase present in the wild type extracts was between 0.2 and 0.3% of the total soluble protein, and thus was at the limit of resolution of the double-label analysis.

The analysis of the membrane fraction of the wild type lac strain is shown in Fig. 2B (membrane solubilized in SDS at 100°C for 2 min) and Fig. 2C (membrane solubilized in SDS at 50°C for 30 min). Both analyses show a peak of lac-specific protein migrating with an apparent molecular weight of 116,000. This polypeptide was the subunit of ,&galactosidase since it was immunoprecipitated by anti-/?-galactosidase an- tibody.’ (The amount of P-galactosidase contaminating the membrane fraction varied in different experiments but a small amount was always recovered in the membrane fraction.)

In addition, a second lac-specific protein was observed, migrating with an apparent molecular weight of 32,000 in membrane samples prepared at 50°C but not in samples heated to 100°C. This lac-specific polypeptide, representing 0.8% of the leucine label in the membrane fraction, appears to be the lac permease, since 1) it is a lac-specific membrane protein; 2) it has the same apparent molecular weight as the lac permease observed by others (25-27); and 3) it is not present in strains deleted for the lac permease.’

The unusual solubilization of the lac permease in SDS was found to be a highly temperature-dependent process. The membrane fraction was solubilized in SDS at 23, 37, 50, and 70°C for 30 min, and at 100°C for 2 min, and analyzed as above. The 32,000-dalton, lac-specific polypeptide was seen at solubilization temperatures of 50°C and below, but was re- placed by a smeared aggregate at the top end of the gel when samples were solubilized a t 70 or 100°C (this broad aggregate can be seen in Fig. 2B in slices 1 through 6). The presence or absence of P-mercaptoethanol did not affect this pattern. The temperature-dependent state of aggregation of the lac per- mease was also noted by Teather et al. (26) and was not observed for either the lac /?-galactosidase or transacetylase.’

Analysis of the permease on slab gels (see Fig. 3) indicates that this polypeptide migrates as a broad band relative to the resolution of other proteins. Whether this represents hetero- genetiy in the size of the polypeptide (from M, = 35,000 to 29,OOO), several as yet unresolved classes of permease mole- cules, heterogeneous binding of SDS to the proteins, or an interaction of this polypeptide with lipid or some other com- ponent is yet to be determined.

Gene Products of the Y‘ Mutant lac Operon-In order to identify a polypeptide that might be the putative Yf permease precursor, the Yf mutant strain was analyzed using the double- label technique (Fig. 4). This revealed a new lac-specific protein in the soluble fraction of the Yf mutant migrating with

V. A. Fried, unpublished observations.

1 - I (

1.W

0. I

(

-0 . :

0.

ae Q

-0.

O.!

c

-0 . :

X - 0 8 n

1 e 1

I 1 I

C

1 I , 10 2 0 30 40

S l ice 0

FIG. 2. SDS-PAGE analysis of the double-label induced wild- type strain H3000. Only the differential percentage analysis is shown (A%). The gels were 9% acrylamide tubes cut into 2-mm slices. A , soluble proteins (lOO°C, 2 rnin). Total activity: 1.4 X lo6 dpm of ”H; 1.3 X 10’ dpm of I4C. B, membrane proteins (lOO°C, 2 rnin). Total activity: 1.3 X lo6 dpm of ‘H; 1.2 X IO5 dpm of 14C. C, membrane proteins (5OoC, 30 rnin); Total activity: 1.1 X lo6 dpm of 3H; 1.1 X lo5 dpm of I4C.

an apparent molecular weight of 87,000 (Fig. 4.4). This poly- peptide accounted for 0.8 to 1.5% of the labeled protein in the soluble fraction. The inset in Fig. 4A is the M, = 116,000 to 80,000 molecular weight region analyzed in SDS slab gels. The lac-specific soluble protein, clearly separated from P-galacto- sidase, co-migrated with other constitutively expressed 87,000- dalton polypeptides. The identity of the 87,000-dalton Yf polypeptide will be examined in the following section.

The new 87,000-dalton peptide was not present in the Yf membrane fraction. Instead, the membrane fraction contained a lac-specific polypeptide migrating in the same region of the gel as the wild type permease (Fig. 4B) . Like the wild type, this polypeptide aggregated when the sample was heated above 50°C. As noted above, a small amount of /?-galactosid- ase (116,000-dalton peak) is also found in the membrane fraction. To compare the wild type and Y‘mutant lac-specific membrane protein, double-label membrane preparations of the Y‘ and wild type strain were run on adjacent lanes of an SDS-polyacrylamide slab gel and gridded, and adjacent slices were counted. The double-label analysis of the “permease” region (Fig. 3) shows the wild type lac permease exhibiting a

248 A Precursor of the lacy f Permease

broad molecular weight distribution with a range of 34,000 to 29,000. The Yf mutant lac-specific membrane protein migrates with an apparent molecular weight of 28,000 and is clearly distinguishable from the wild type. Because of its presence as a lac-specific membrane protein and its unusual solubility properties in SDS, it is reasonable to suggest that this 28,000- dalton polypeptide is the yf permease, a mutant "protein.

Identity of the 87,000-dalton Y' Soluble Protein. The 87,000-dalton Y' Soluble Protein is a Novel lac-specific (in- ducible) Polypeptide Species-Further evidence that the 87,000-dalton peak in the soluble fraction of the Y' mutant represented a new species was obtained by two-dimensional gel analysis. In Fig. 5A is shown a fluorogram of a two- dimensional gel of ["Hlleucine-labeled soluble proteins from the uninduced Yf mutant. Fig. 5B is the same analysis for the induced Y' mutant. A spot at an apparent molecular weight of 87,000 (arrow) is present in the induced sample but not in the uninduced. This result supports the idea that the 87,000- dalton polypeptide is a new lac-specific protein and not an increase in the differential rate of synthesis of another consti- tutive 87,000-dalton polypeptide species.

The 87,000-dalton Y'Soluble Protein Is Not a Fragment of P-Galactosidase-Since the 87,000-dalton lac-specific soluble polypeptide in the Y' mutant is relatively large, it was con- ceivable that it was a fragment of the ZacZ gene product. As demonstrated below, this is unlikely, since it is not immuno- precipitated by antibody prepared against purified P-galacto- sidase.

IgG prepared against the P-galactosidase purified from wild type E. coli H3000 was used to form immunocomplexes with the double-labeled soluble fractions of both the wild type strain H3000 and the Yf mutant. Immunocomplexes were recovered with fixed S. aureus and analyzed by SDS-PAGE. As can be seen in Fig. 6A and 7A, the anti-P-galactosidase antibody precipitates only a single 116,000-dalton polypeptide in both strains; this represents the P-galactosidase subunit.

0.2

0 ! *

1 35K 29K

- FIG. 3. SDS-PAGE analysis of the wild type and Y' mutant

lac "protein. The leucine double-labeled membrane fractions of the wild type strain shown in Fig. 2 and the Y' mutant shown in Fig. 4 were run on adjacent lanes of an 11% acrylamide slab gel. The gel was f i s t stained with Coomassie blue to identify the molecular weight standards bracketing these lanes and then dried on filter paper. The lanes containing the labeled proteins were cut into slices and counted. The region of the gel about the permease (approximately 30,000 to 24,000 daltons) was cut into fine slices in order to achieve a high resolution. The double-label pattern was analyzed as described under "Experimental Procedures." A, wild type lac strain H3000; B, mutant.

2.5'

2 . 0 -

1.5-

1.0-

0.5-

10 I 2 0 30 40 5 0

Sl lce FIG. 4. SDS-PAGE analysis of the leucine double-label in-

duced Y' mutant strain. Experiment and analysis were as in the legend to Fig. 2 and the text. A, soluble proteins (IOO°C, 2 min). Znset shows the analysis of a slab gel (9% acrylamide) in the P-galactosidase- precursor region cut with much higher resolution. The scale units in the inset showing the actual distribution of activity are the same as on Fig. 1. Total activity: 1.7 X IO6 dpm of 3H; 1.5 X IO5 dpm of I4C. B, membrane proteins (5OoC, 30 min). Total activity: 1.1 X lo6 dpm of 3H; 1.0 X IO5 dpm of I4C.

Analysis of the material remaining after removal of the anti- P-galactosidase - antigen complexes indicated that the anti- body had quantitatively removed the /3-galactosidase from both wild type (Fig. 6C) and Y'mutant (Fig. 7B) extracts. No lac-specific polypeptides corresponding to the 116,000-dalton subunit could be observed in these fractions. On the other hand, the Y' 87,000-dalton soluble protein remained in the soluble fraction and had not been precipitated by anti$- galactosidase antibody.

Since the anti-/?-galactosidase antibody used in these ex- periments could precipitate amber fragments of P-galactosid- ase at least as small as 25,000 daltons? and since antibody in excess did not cross-react with the 87,000-dalton Y' soluble protein, it is unlikely that this lac-specific soluble peptide is a fragment of P-galactosidase.

The 87,000-dalton Y' Soluble Polypeptide Imnunoprecipi- tates with Anti-Thiogalactoside Transacetylase Antibody- If the 87,000-dalton polypeptide is the precursor of the 28,000-

V. A. Fried, manuscript in preparation.

A Precursor of the lacyf Permease 249

that the 87,000-dalton soluble polypeptide could contain both the lacy and lacA gene products. If so, it would be expected that the 87,000-dalton polypeptide could be immunoprecipi- tated by antibody raised to the purified thiogalactoside trans- acetylase. The following experiments demonstrate this specific immunoprecipitation.

In order to demonstrate the specificity of the anti-acetylase antibody, immunoprecipitates of induced wild type extracts

IF +

rn v) n

+

B .

FIG. 5. Two-dimensional analysis of Yc mutant soluble pro- teins. Induced and uninduced cultures of the Y'mutant were labeled with ["Hlleucine and analyzed by two-dimensional gel electrophoresis as described under "Experimental Procedures." Panel B is the in- duced Y', and Panel A is the uninduced Y' culture. The arroms indicate the position of the inducible lac-specific protein at an appar- ent molecular weight of 87,000. The large spot in the upper center of B is 8-galactosidase. (Only the upper portions of the gels are shown.)

5 l A p" d 5 l A p" d

t 1 1 1 1

-0.21

50 Slice

FIG. 7. SDS-PAGE analysis of the leucine double-labeled Y c mutant strain. The double-label material was that used in the experiment shown in Fig. 4. Panel A shows the SDS gel of material immunoprecipitated with anti-8-galactosidase antibody. Panel B shows a differential percentage analysis of the material not immuno- precipitable. 0, "H (induced); 0, "C (uninduced).

wt

87K- -. -

wt Yf

- -87K

0.2 - 8 0 h

a v "-7fl-U

-9.2 - 1

0 50 Slice

FIG. 6. SDS-PAGE analysis of the leucine double-labeled wild type strain H3000 immunoprecipitated with anti-8-galac- tosidase antibody. The double-labeled soluble fraction used in the experiment shown in Fig. 2 was immunoprecipitated with anti-8- galactosidase antibody as described under "Experimental I'roce- dures." Panel A shows the SDS tube gel (9%) of the material immu- noprecipitated by anti-P-galactosidase antibody. Panel B shows the SDS tube gel of the material not immunoprecipitable. Panel C is the differential percentage analysis of the data in Panel B. 0, .'H label (induced); 0. ''C (uninduced).

dalton Yr lac permease, then it should contain a polypeptide sequence coded for by a region beyond the classical l acy gene. Since the 87,000-dalton polypeptide does not cross-react with anti-P-galactosidase antibody, it is unlikely that a part of the P-galactosidase gene is included in this polypeptide. On the other hand, since the Y' mutant is lacA- (thiogalactoside transacetylase-negative), and since it has a lesion mapping at the distal end of the classical l acy gene (a), it seemed likely

2 5 K - Y ' ' 1

! - 2 5 K ! _ " i

A' A B FIG. 8. SDS-PAGE of anti-thiogalactoside transacetylase

immunoprecipitates. Anti-thiogalactoside transacetylase antibody was used to immunoprecipitate ["Hlleucine-labeled soluble extracts of a wild type lacZ'Y+A+ strain, H3000, and the lacl" mutant (lacZ*I"A-). The immunoprecipitates were run on an 11% acrylamide SDS slab gel. Labeled protein bands were detected by fluorography. Lanes A and E are the anti-acetylase antibody immunoprecipitates of the induced wild-t.ype lac strain H3000 and Y' mutant strain, respectively; equal volumes of soluble extracts containing equivalent amounts of label were used for immunoprecipitation. Lane A' repre- sents an experiment parallel to that shown in Lane A, except that 50 times the amount of extract was used in order to observe the wild type acetylase. wt, wild type.

250 A Precursor of the lacyf Permease

were examined by SDS-PAGE. A [3H]leucine-labeled extract of the IPTG-induced wild type lac strain H3000 was immu- noprecipitated with anti-acetylase antibody using S. aureus to recover immunocomplexes. The precipitated material rep- resented approximately 0.15% of the total soluble labeled proteins. This is similar to the level of thiogalactoside trans- acetylase as determined by assay of enzyme activity. The labeled material in the immunoprecipitates appears as a single band migrating with an apparent molecular weight of 25,000 (Fig. 8, Lane A’). This labeled material appears to be the thiogalactoside transacetylase subunit, since 1) it co-migrates with thiogalactoside transacetylase purified from the same strain (wild type acetylase has a subunit molecular weight of 25,000 (12)); 2) it is present only when the extract is induced for lac operon expression (no labeled precipitate is found in uninduced extracts); and 3) the unlabeled, purified acetylase competes for the immunoprecipitation of the labeled mate- rial.*

To determine whether the novel 87,000-dalton peptide of the Y‘ mutant contained transacetylase sequences, volumes containing equivalent amounts of labeled protein from the induced wild type and mutant cultures were precipitated with anti-thiogalactoside transacetylase antibody. Approximately 25 times as much label was immunoprecipitated from the induced Y‘ mutant extract as compared to the wild type. Analysis of these immunoprecipitates is shown in Fig. 8, Lanes A and B. No bands are visible in the immunoprecipitate of the wild type extract. This reflects the fact that the amount of acetylase is low in induced wild type strains (Lane A’ is this same experiment scaled up 50-fold). In contrast, the immunoprecipitate of the induced Y‘ extract (Lane B ) shows a major band at 87,000 daltons. The immunoprecipitated band represents approximately 1% of the total labeled soluble pro- tein in the Y‘ mutant; double-label analysis of comparable extracts showed that the 87,000-dalton peptide accounted for 1.5% of the total labeled soluble protein. Several other bands of lower molecular weight are observed in the immunoprecip- itate of the Yf mutant. The intensity of these bands varies in different experiments and their possible significance will be discussed later. The fact that the 87,000-dalton, lac-specific, soluble protein in the Y‘ mutant is immunoprecipitated by antibody specific for the lac thiogalactoside transacetylase is consistent with the notion that this novel polypeptide contains at least a part of the lacA gene product.

Kinetic Evidence for the 87,000-dalton yf Soluble Protein Being a Precursor of the 28,Gi”dalton Membrane Protein- While a biochemical relationship between the lacy‘ soluble protein and membrane protein has yet to be established, it is tempting to speculate that this novel soluble protein is the putative precursor of the lacy‘ permease. A necessary, but not sufficient, criterion for this relationship would be that label in the 87,000-dalton protein would chase into the mem- brane fraction with the slow kinetics observed for induction of yf transport activity. It would also be expected that this “chase” be significant only in the absence of further induction during the chase period. This is because the putative precur- sor, being a relatively stable species, would be diluted by de novo udabeied precursor during the chase period. If process- ing of the precursor were stochastic, which is likely for a soluble component, chase during continued induction would mask the processing of labeled material. Experiments, de- scribed below, are consistent with these predictions.

Pulse-chase experiments were performed with the Y‘ mu- tant using the double-label procedure and analysis of mem- brane and soluble proteins on SDS gels. In one set of experi- ments, the Y‘ mutant strain was “pulse-labeled” for 20 min and chased over a generation in the presence of excess cold

leucine with inducer still present. This experiment is shown in Fig. 9A. It can be seen that over one generation, no significant change was observed in the P-galactosidase, the 87,000-dalton soluble protein, or the 30,000-dalton membrane protein. In contrast, a pulse-chase experiment in which inducer was re- moved during the chase period (pulse-chase deinduced) is shown in Fig. 9B. During one generation of chase, the 87,000- dalton soluble protein decreased by 20%, while lac-specific material accumulated in the membrane, increasing by 60%. In real units, at the final time point indicated, about 700 dpm of 3H had appeared in the membrane, and 2,000 dpm had been lost from the soluble “precursor” peak. Since the precursor is nearly three times larger than the membrane protein form, these numbers are consistent with 1 mol of precursor con- verted to 1 mol of the membrane form. The fate of the other two-thirds of the precursor is unknown. These results are consistent with the unusual kinetics of induction and deinduc- tion of Y‘ permease activity (8) and are consistent with the idea that the 87,000-dalton soluble protein is a precursor of the Y‘ mutant “protein.

A

X o4 c 8 X

0.6,

0 100 2(

m i n of c h a s e

0

FIG. 9. Pulse-chase of lac-specific proteins in the Y‘ mutant. The Y‘ mutant was pulse double-labeled with leucine in the presence of inducer for 20 min and then chased in either the presence ( A ) or absence of inducer ( B ) (see under “Experimental Procedures”). Dou- ble-label samples were taken at the end of pulse (zero time) and at the indicated times during chase. The lac-specific proteins were analyzed as described in the legend to Fig. 4 and quantitated on differential percentage plots. The data are expressed as the percentage of lac-specific protein species at the beginning of the chase. 0, p- Galactosidase (116,000-dalton lac-specific protein peak); 0, Y‘ pre- cursor (87,000-dalton lac-specific protein peak); X, y permease (-30,000-dalton lac-specific protein peak).

A Precursor of the lacyf Permease 251

DISCUSSION

The experiments presented here identify a new lac-specific protein present in the ZacY'mutant. This protein fractionates as a soluble protein and has an apparent molecular weight on SDS gels of 87,000. This lac-specific soluble protein is a likely candidate for the putative precursor of the Yf permease. Evidence consistent with this hypothesis comes from pulse- chase experiments in which the slow kinetics of disappearance of the 87,000-dalton soluble protein is accompanied by a stoichiometric appearance of the 28,000-dalton lac-specific membrane protein.

The 28,000-dalton polypeptide found in the membrane frac- tion is presumably the Y' mutant permease ("protein), since it has a similar apparent molecular weight on SDS gels to the wild type lac permease and exhibits the same unusual aggre- gation in SDS at temperatures above 50°C. Attempts to label the Yf mutant permease with radioactive N-ethylmaleimide were unsuccessful. While this would have presented an alter- native method for detecting the Y' permease, the negative results were consistent with the observation that the Yf per- mease, activity was not inactivated by N-ethylmaleimide (8). Thus, while it is reasonable to believe that the lac-specific membrane protein present in the Y' mutant is the mutant permease, definitive proof will require structural analysis. Purification and characterization of the 87,000-dalton soluble polypeptide and 28,000-dalton membrane polypeptide are cur- rently in progress in order to establish a biochemical basis for the precursor-product relationship.

The l a c v mutant is a double mutant with lesions at the very proximal and distal ends of the lacy gene (8). It does not exhibit lac thiogalactoside transacetylase activity. Since the 87,000-dalton precursor is substantially larger than the lac M- protein, it is tempting to suggest that this precursor is a polyprotein containing both the lacy and lacA gene products. Possibly, the covalent association of the soluble lacA gene product with the "insoluble" "protein is responsible for the cytoplasmic localization of the precursor. The fact that anti- body specific to the lac thiogalactoside transacetylase will immunoprecipitate the 87,000-dalton precursor is consistent with this model.

It was noted that the anti-acetylase antibody immunopre- cipitants of the mutant contained several bands of lower molecular weight than the 87,000-dalton precursor band. While the intensity of these bands varies in different experi- ments, they are reproducible. This material may represent 1) polypeptides trapped nonspecifically in the immunoprecipi- tates; 2) proteolytic fragments of the precursor which cross- react with anti-acetylase antibody; or 3) polypeptides associ- ated with the precursor as specific complexes. If non-lac operon products are specifically associated with the precursor, they may represent part of the cellular systems involved in processing the precursor.

Examination of the DNA sequence of the wild type lacy-A region suggests an explanation for the origin of this chimera. The DNA sequence of the lacy gene region has been deter- mined from the COOH-terminal end of the lacZ gene through the NHz-terminal end of the lacA gene (9). The NHz-terminal end of the lacy gene starts with an AUG (Met) preceded by a Shine-Dalgarno sequence. The f i t and only translational terminator in the lacy gene is a single ochre codon at the 418 triplet position. The lacy gene product at this point is 417 amino acids long. The molecular weight of this primary gene product, as calculated from the amino acid composition pre- dicted from the DNA sequence, is approximately 47,000.

The lacA gene is in reading phase with the lacy gene and begins at triplet 438, as measured from the NH2-terminal end of the lacy gene. Since the lacy gene is terminated by a

single ochre codon, and since the lacA gene is in phase, a readthrough of this ochre would lead to a lacy-A fusion. the size of this product would be 74,000 daltons. This is similar in size to the 87,000-dalton soluble precursor form I observe in the Y' mutant. Thus, one explanation for my results would be that the Yf mutant has an altered termination signal and reads through to form the lacy-A chimera. This chimera is then slowly processed to give a defective "protein product, the Y ' permease. If correct, this suggests that the cell contains the machinery to process the precursor. This processing may involve a nonspecific cleavage at an exposed peptide junction between the l a c r and lacA gene products. It is also possible that the cleavage is due to a specific protease that recognizes some special feature of the precursor. In either case, the consequence of the cleavage is that the lacYf permease is produced as a functional membrane component. The fact that the lacA gene product cannot be found during the processing, either as an enzymatic activity or as a low molecular weight immunoprecipitable peptide, suggests that the COOH-termi- nal portion of the precursor is rapidly degraded after the initial cleavage event.

The presence of the ochre terminator at the end of the lacy gene indicates that a polyprotein precursor is not part of the normal pathway (9). The in vitro translation product of the wild type lacy gene appears to be identical to the 30,000- dalton lacy gene product found in vivo (27). However, the primary translation product of the lacy gene should be a 47,000-dalton polypeptide. The behavior of the lac permease on SDS gels is anomalous and the apparent molecular weight may be in substantial error. On the other hand, it is possible that the primary translation product of the lacy gene has been modified by proteolytic enzymes present in both the in vivo and in vitro systems. If true, this processing does not occur by removal of the NHZ-terminal end (e.g. removal of a signal peptide), since the NHZ-terminal amino acid sequence of the purified mature permease is identical to the DNA sequence at the translational initiation site (27). Thus, proc- essing, if it does occur, would require cleavage at the COOH- terminal end. Possibly, the proteolytic enzymes involved in processing the Y precursor may be involved in the biogenesis of the wild type permease.

Further study of the Yf precursor as an aqueous, soluble component, and its conversion to the mature Y ' permease as an integral membrane component should provide insight into one possible pathway for the biogenesis of biological mem- branes. It will be of interest to determine whether the covalent attachment of a soluble polypeptide (e.g. the lacA gene prod- uct) to a highly hydrophobic polypeptide (e.g. the lacy gene product) is in itself sufficient to determine the cellular locali- zation of the polyprotein in the aqueous phase. This soluble state could be a consequence of a major conformational rear- rangement of the polyprotein. It is also possible that a two- domain amphipathic polyprotein could specifically aggregate with itself or some other cellular component so as to remain an aqueous soluble complex. In either case, proteolytic modi- fication of the polyprotein would be necessary for the matu- ration of a stable membrane component. The proteolytic enzymes necessary for these events exist in E. coli, since the Y' precursor is successfully processed. Such processing mech- anisms may play a role in the biogenesis of other membrane proteins involving soluble precursors. This possibility remains to be explored.

Acknowledgment.+" wish to thank Dr. L. I. Rothfield for his critical reading of this manuscript, J. P. Longabaugh for helpful discussions, and D. Brock and L. Lehmann for excellent technical assistance.

252 A Precursor of the lacyf Permease

R E F E R E ~ ~ E S

1. Inouye, S., Wang, S., Sekizawa, J., and Inouye, M. (1977) Proc.

2. Randall, L. L., Hardy, S. J. S., and Josefsson, L.-G. (1978) Proc.

3. Rothman, J. E., and Lodish, H. F. (1977) Nature 269, 775-780 4. Lingappa, V. R., Katz, F. N., Lodish, H. F., and Blobel, G. (1978)

5. DiRienzo, J. M., and Inouye, M. (1979) Cell 17, 155-161 6. Poyton, R. O., and McKemmie, E. (1979) J. Biol. Chen. 254,

7. Poyton, R. O., and McKemmie, E. (1979) J. BioL Chem. 254,

8. Fried, V. A. (1977) J. Mol. Biol. 114,477-490 9. Buchel, D. E., Gronenborn, B., and Muller-Hili, B. (1980) Nature

10. Showe, M. K., and DeMoss, J. A. (1968) J. Bacteriol. 95, 1305-

11. Fowler, A. V., and Zabin, I. (1977) Proc. Natl. Aead. Sei. U. S. A.

12. Fried, V. A. (1980) J. Bacteriol. 143,506-509 13. Osborn, M. J., Gander, J. E., Parisi, E., and Carson, J. (1972) J.

Natl. Acad. Sci. U. S. A. 74, 1004-1008

Natl. Acad. Sci. U. S. A. 75, 1209-1212

J. Biol. Chem. 253,8667-8670

6763-6771

6772-6780

283,541-545

1313

74, 1507-1510

Biol. Chem. 247,3962-3972

(1951) J. Biol. Chern. 193,265-275 14. Lowry, 0. H., Rosebrough, N. J., F a r , A. L., and Randall, R. 3.

15. Fried, V. A., and Novick, A. (1973) J. Bacteriol. 114,239-248 16. Alpers, D. H., Appel, S. H., and Tomkins, G. M. (1965) J. Biol.

17. Laemmli, U. K. (1970) Nature (LondJ 227,680-685 18. Ames, G. F.-L. (1974) J. BioE. Chem. 249,634-644 19. OFarrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 20. Bonner, W. M., and Laskey, R. A. (1974) Eur. J. Biochem. 46,

21. Laskey, R. A., and Mills, A. D. (1975) Eur. J. Bioehem. 56,335-

22. Craven, G. R., Steers, E., Jr., and Anfinsen, C. B. (1965) J. Biol.

23. Zabin, I (1963) J. Biol. Chem. 238,3300-33~ 24. Kessler, S. W. (1975) J. Immunol. 115, 1617-1624 25. Jones, T. H. D., and Kennedy, E. P. (1969) J. Biol. Chem. 244,

26. Teather, R. M., Mulier-Hill, B., Abrutseh, U., Archele, G., and

27. Ehring, R., Beyreuther, K., Wright, J. K., and Overath, P. (1980)

Chem. 240,lO-13

83-88

34 1

Chem. 240,2468-2477

5981-5987

Overath, P. (1978) Mol. Gen. Genet. 159,239-248

Nature 283,537-540