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A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition

2002; Springer Nature; Volume: 21; Issue: 9 Linguagem: Inglês

10.1093/emboj/21.9.2107

1460-2075

Mikhail Bogdanov,

RNA and protein synthesis mechanisms

Article1 May 2002free access A polytopic membrane protein displays a reversible topology dependent on membrane lipid composition Mikhail Bogdanov Mikhail Bogdanov Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author Phillip N. Heacock Phillip N. Heacock Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author William Dowhan Corresponding Author William Dowhan Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author Mikhail Bogdanov Mikhail Bogdanov Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author Phillip N. Heacock Phillip N. Heacock Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author William Dowhan Corresponding Author William Dowhan Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA Search for more papers by this author Author Information Mikhail Bogdanov1, Phillip N. Heacock1 and William Dowhan 1 1Department of Biochemistry and Molecular Biology, Medical School, University of Texas-Houston, Houston, TX, 77225 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2002)21:2107-2116https://doi.org/10.1093/emboj/21.9.2107 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info To address the role of phospholipids in the topological organization of polytopic membrane proteins, the function and assembly of lactose permease (LacY) was studied in mutants of Escherichia coli lacking phosphatidylethanolamine (PE). PE is required for the proper conformation and active transport function of LacY. The N-terminal half of LacY assembled in PE-lacking cells adopts an inverted topology in which normally non-translocated domains are translocated and vice versa. Post-assembly synthesis of PE triggers a conformational change, resulting in a lipid-dependent recovery of normal conformation and topology of at least one LacY subdomain accompanied by restoration of active transport. These results demonstrate that membrane protein topology once attained can be changed in a reversible manner in response to alterations in phospholipid composition, and may be subject to post-assembly proofreading to correct misfolded structures. Introduction Although considerable progress has been made in understanding the assembly of multispanning membrane proteins (Bernstein, 2000; Dalbey et al., 2000), the precise molecular events involved in the folding of proteins into the membrane are not well defined. Investigation focusing on the role of amino acid sequence in governing the assembly of membrane proteins has predominated, while the role phospholipids play as structural determinants for the correct insertion, folding and topology of membrane proteins has been largely ignored. Therefore, to date there is little understanding of, or ability to predict, how membrane protein topogenesis occurs in a given phospholipid environment. Determinants of membrane protein topology and the mechanism of membrane protein insertion in Escherichia coli have been studied mainly for native proteins containing one or two transmembrane domains (TMs) (de Gier et al., 1998) and chimeric derivatives of a few polytopic membrane proteins (Gafvelin and von Heijne, 1994; Kim et al., 1994). Only one study focused on the interaction of positively charged cytoplasmic domains of membrane proteins with the headgroups of anionic phospholipids as a topological determinant (van Klompenburg et al., 1997). Can specific lipids influence the topological organization of membrane proteins? Is protein topology static or is it dynamic with respect to changes in membrane lipid composition? Is membrane protein sequence ‘written’ for a given membrane environment? To investigate the influence of lipids on topogenesis, we have focused on the lactose permease (LacY) of E.coli, a structural paradigm for the major facilitator superfamily (MFS) of secondary transport proteins (Saier et al., 1999). Members of this family are diverse in substrate specificity yet show significant homology in function, sequence and structure. These transporters are characterized by a 12-TM topology divided into two six-helix segments connected by a long cytoplasmic loop (Figure 1). Common to many sugar permeases, including LacY, is the very hydrophilic TM VII that is not predicted by membrane topology algorithms (Efremov and Vergoten, 1996) but has been verified biochemically (Calamia and Manoil, 1990; Wolin and Kaback, 1999). The mechanism by which the TMs are oriented and the connecting extra-membrane domains are folded is not understood. Figure 1.Secondary structural model of LacY with indicated modifications in the linear sequence. The figure is based on the most recent putative topology organization of LacY (Kaback et al., 2001). Rectangles indicate putative helical TMs numbered sequentially in roman numerals from the N-terminus (NH2) to the C-terminus (COOH). The hydrophilic loops connecting the TMs are numbered sequentially and their putative topological disposition in PE-containing cells is indicated by the prefix ‘C’ for cytoplasmic (IN) or ‘P’ for periplasmic (OUT). The position of single Cys replacements (see Table I) in a Cys-less derivative of LacY is indicated by an ‘X’, and the single-letter amino acid code of the replaced amino acid followed by the residue number. Download figure Download PowerPoint Phosphatidylethanolamine (PE) acts as a non-protein molecular chaperone in the folding of LacY by the programming of ‘conformational memory’ during assembly (Bogdanov et al., 1996, 1999; Bogdanov and Dowhan, 1998). This zwitterionic and major phospholipid of E.coli is required for proper assembly and full function of LacY. LacY couples the downhill movement of a proton with the uphill movement of substrate in a symport mechanism to drive active transport. However, LacY assembled in a mutant of E.coli lacking PE cannot accumulate substrate against a concentration gradient, but can still facilitate substrate transport. Bioenergetic properties of this mutant are not affected (Bogdanov and Dowhan, 1995). The loss of full function correlates with a structural alteration in the periplasmic domain P7, as indicated by loss of recognition by the conformation sensitive monoclonal antibody (mAb) 4B1 (Sun et al., 1996). PE is required either during de novo assembly in vivo or during refolding of partially denatured LacY in vitro, but is not required once ‘native structure’ has been attained. PE is not required for membrane insertion, but if added after membrane insertion it facilitates the final structural maturation of LacY as determined by conformation specific mAb4B1. Therefore, PE appears to facilitate the assembly of LacY by affecting the folding of non-native intermediates late in the maturation process. In order to establish the molecular basis for LacY dysfunction and misfolding in PE-lacking cells, we report a systematic comparative study of the transmembrane topology of LacY assembled in PE-containing or PE-lacking cells. In wild-type cells, membrane protein assembly occurs in the presence of abundant PE (75% of total phospholipid), making it difficult to discriminate between phospholipid-dependent and -independent assembly events. By using mutants lacking PE, a lipid-dependent assembly step for LacY was identified along the pathway to attainment of native structure. This lipid requirement differs from the general solvent effect of lipids and shows specificity for PE. Our results are consistent with a topological inversion of the first half of LacY through domain C6 when LacY is assembled in membranes lacking PE. The topological inversion of at least domain C6 can be reversed by the addition of PE after membrane insertion and is accompanied by a restoration of active transport function mediated by LacY. This is the first report of a dual membrane protein topology that is interchangeable post-insertionally in response to changes in phospholipid composition. These results implicate phospholipids as specific participants in determining membrane protein organization and suggest that regulation of membrane protein function can occur by topology ‘switching’ in response to changes in phospholipid environment. Results Rationale and methodology for determination of domain topology The topology of hydrophilic loops connecting TMs of LacY was established by the selective biotinylation [3- (N-maleimidylpropionyl) biocytin (MPB)] or alkylation [4-acetamido-4′-maleimidylstilbene-2,2′-disulfonic acid (AMS)] of single cysteines exposed either to the periplasmic face of whole cells or to the cytoplasmic face of inside-out membrane vesicles (IOV). The labeling strategy relied on the relative impermeability of MPB to the inner membrane of E.coli (Wada et al., 1999) and the full membrane impermeability of AMS (Long et al., 1998). MPB presumably readily passes through the pores of the outer membrane. Lack of reactivity with cysteines, either within the bilayer or on the interior surface of cells or IOV, was verified by first blocking water-accessible cysteines with membrane-impermeable AMS prior to addition of MPB. Cysteines protected from reactivity with MPB, from the outside of either whole cells or IOV, were exposed by permeabilization of the bilayer with toluene. Leader peptidase (Lep) has previously been shown to have the same orientation in the inner membrane of E.coli in both PE-containing and PE-lacking cells (Rietveld et al., 1995). Under our conditions, Lep in right-side-out membrane vesicles and IOV also had the same orientation in both cell types, and the IOV were sealed. The conditions for the sidedness-dependent modification of cysteines by MPB were established using whole cells and IOV containing overexpressed Lep (see Supplementary figure 1, available at The EMBO Journal Online). Orientation of LacY assembled in PE-containing and PE-lacking cells Previous results indicated a structural rearrangement in the region of the P7 domain of LacY, resulting in the loss of detection by a conformation-specific mAb (Bogdanov and Dowhan, 1999) whose epitope lies within this domain (Sun et al., 1996). To assess the effect of membrane phospholipid composition on the topological organization of LacY, single Cys replacement derivatives (Table I; Figure 1) of a Cys-less derivative of LacY were expressed from plasmids in a PE-containing (containing pDD72GM) or PE-lacking (lacking pDD72GM) strain (AL95) of E.coli that was null in the chromosomal copies of lacY and pssA. The replacements were in putative extramembrane domains connecting TMs, predicted by LacY–PhoA fusion analysis (Calamia and Manoil, 1990) and refined analyses of the whole protein (Venkatesan et al., 2000a, b, c). These derivatives display ≥60% of the normal active transport function of LacY (Frillingos et al., 1998). Control western blot analysis using LacY-specific antibody showed that all these derivatives were present in the membrane fraction at nearly wild-type levels of LacY, and that samples had similar amounts of LacY (see bottom panel of Figure 2D). No labeling of a species of the molecular weight of LacY was detected when a ΔlacY strain expressing Cys-less LacY was probed (Supplementary figure 2). The results presented below are representative of experiments performed twice or more. Figure 2.Determination of LacY topology in PE-containing and PE- lacking cells. Strain AL95 (null in chromosomal pssA and lacY) either with (+PE) or without (−PE) plasmid pDD72GM was used as the host for single Cys replacements of LacY in each P and C domain, as indicated in Figure 1. Unless otherwise noted, plasmid pLacY-F208 was used for analysis of domain C6. The data within each box come from the same autoradiogram, with lines to indicate splicing between lanes. (A) After growth and induction of LacY, whole cells were treated with MBP and the membranes subjected to immunoprecipitation, SDS–PAGE and western blotting using Avidin–HRP to detect biotin linked via the single Cys in each LacY derivative. (B) Where indicated, whole cells were treated with toluene prior to reaction with MBP followed by further analysis as in (A). (C) Cells expressing the indicated Cys derivatives were treated with AMS where indicated prior to treatment with MBP, and then analyzed as in (A). (D) Plasmids expressing single Cys derivatives at the indicated positions in domain C6 were expressed in PE-lacking cells. As indicated, some of the samples were treated with AMS prior to treatment with MPB, followed by analysis as described in (A). LacY derivatives were first visualized using Avidin–HRP (upper panel) followed by stripping of the solid support and visualization with mAb4B11 (lower panel) to quantify levels of total LacY. Download figure Download PowerPoint Table 1. Strains and plasmids used in this study Strains or plasmids Relevant characteristics Source or reference Strains W3899 pssA+ CGSCa AD90 pss93::kanR (derivative of W3899) DeChavigny et al. (1991) AD93 pss93::kanR recA srl::Tn10 (derivative of AD90) DeChavigny et al. (1991) AA9256 pss93::kanR ParaB-pssA+ ΔaraBA (derivative of W3899) Supplementary data AAL9256 pss93::kanR ParaB-pssA+ ΔaraBA lacY::Tn9 (derivative of W3899) Supplementary data AL95 pss93::kanR lacY::Tn9 (derivative of W3899) Supplementary data Plasmids pAH-PSS ′araC ParaB-pssA+′araD′ oriRKγ tetR ampR Supplementary data pDD72 pssA+ camR (temperature sensitive for replication) DeChavigny et al. (1991) pDD72GM pssA+ genR camS (temperature sensitive for replication) This work pT7-5/lacY OPlac-lacY ampR Bibi and Kaback (1990) pLacY-N ampR Cys-less lacY with indicated amino acid (N) replaced byCys, i.e. pLacY-G13 has a G13C replacement Frillingos et al. (1998) a Coli Genetic Stock Center. The predicted biotinylation patterns (Figure 2A, upper panel) were observed for single Cys derivatives of LacY expressed and probed in PE-containing cells, i.e. all derivatives with single cysteines in the periplasmic (P) domains were labeled, while those with single cysteines in the cytoplasmic (C) domains and the N-terminus were protected from labeling. Unless noted otherwise, all results for C6 were with the F208 replacement derivative. When cells were permeabilized with toluene prior to MPB treatment, a small amount of additional biotinylation occurred for the P1 domain, but extensive biotinylation of C domains occurred (Figure 2B, upper panel). These results are in agreement with the putative location of single cysteines in the current topology map of LacY (Figure 1). The results with the same single Cys derivatives probed in PE-lacking cells were dramatically different. The N-terminal, C2, C4 and C6 domains were all accessible to MPB from the outside of whole cells while the P1, P3, and P5 domains were all protected from reaction with MPB (Figure 2A, lower panel). The remainder of the domains from P7 to P11 behaved the same as in PE-containing cells. Consistent with these results, P1 and C8 were rendered accessible to MPB after toluene treatment, while there was no increase in the accessibility of C2, C4 or C6 (Figure 2B, lower panel). Cysteines accessible to MPB in PE-containing and PE-lacking whole cells were blocked from reaction with MPB if cells were first treated with AMS (Figure 2C). The same was true for three different single Cys derivatives, all in the C6 domain, that were expressed independently and accessible in PE-lacking cells (Figure 2D). This further establishes that MPB did not react with cysteines either sequestered in the membrane bilayer or in the lumen. To substantiate further the misorganization of LacY in PE-lacking cells, single Cys derivatives in IOV isolated from both cell types were probed. The labeling patterns for IOV without (Figure 3A) and with (Figure 3B) toluene treatment was a mirror image of the labeling pattern observed in whole cells. As with whole cells, pre-treatment with AMS blocked reaction of accessible domains with MPB (Figure 3C). The complementary and opposite labeling pattern observed with IOV strongly supports the major topological differences observed between PE-containing and PE-lacking cells. Figure 3.Determination of LacY topology in IOV prepared from PE-containing and PE-lacking cells. The same plasmid and cell combinations were used as described in Figure 2. IOV were prepared as described in Materials and methods from cells expressing the indicated Cys replacements, followed by treatment of the vesicles with MBP alone (A), with (+) or without (−) toluene prior to MPB treatment (B), or with (+) or without (−) AMS prior to MPB treatment (C). Download figure Download PowerPoint For both Lep (Supplementary figure 1) and LacY, putative inaccessible cysteines showed some labeling (ranging from 5 to 15%) even in the absence of toluene and there was some increase in labeling of putative accessible cysteines after toluene treatment (5–15%). This ‘leak-through’ labeling varied between experiments (compare C4, C6 and C8 for PE-containing cells in Figure 2A and B, and compare C2 and C6 for PE-lacking IOV in Figure 3A and B) and was observed for both PE-containing and PE-lacking cells. This labeling was probably due to partial penetration of the inner membrane by MPB. The increase in labeling of accessible cysteines after toluene treatment (P1 for PE-containing cells and C2 for PE-lacking cells in Figure 2B) was variable between experiments. However, in all cases where cysteines were not labeled or poorly labeled, addition of toluene dramatically increased labeling. Where labeling was strong, addition of toluene only moderately increased labeling. The latter was probably due to toluene-induced local changes in accessibility of exposed cysteines. The C-terminus of LacY remains in the correct orientation, regardless of the presence or absence of PE. A derivative of LacY was used with a Factor Xa protease site at the N-terminus of an 100-amino acid biotinylation domain attached as an extension to the C-terminus of LacY. This derivative was nearly completely degraded by Factor Xa protease with the same efficiency for IOV derived from either PE-containing (Zen et al., 1995) or PE-lacking cells (Supplementary figure 3). Several conclusions follow from these findings. From domain P7 to the C-terminus of LacY, the topological organization of LacY is the same when expressed in either PE-containing or PE-lacking cells. However, expression of LacY in PE-lacking cells results in global perturbations in which the N-terminus through domain C6 adopts an inverted topology, where normally non-translocated hydrophilic loops connecting TMs are translocated while normally translocated loops are not. Within the limits of the leak-through reactivity noted above, a uniform population of LacY is generated by assembly either in PE-containing or PE-lacking cells. A reversible topological switch triggered by a change in phospholipid composition Can the misorganization of LacY be corrected after assembly by the addition of PE? In vitro translation and assembly experiments indicated that LacY assembled in IOV lacking PE and lacking the structural determinants within domain P7 recognized by mAb4B1 could regain these structural determinants by post-assembly synthesis of PE within the vesicles. LacY is also recognized by mAb4B11, independent of PE, provided that LacY is integrated into the membrane (Bogdanov and Dowhan, 1998). This is presumably the result of the now established proper alignment, independent of PE, of the discontinuous epitope contributed by domains C8 and C10. In a strain with the chromosomal pssA gene under tight regulation of the araB promoter, the synthesis of PE can be induced by growth in the presence of arabinose or repressed by growth on glucose. The chromosomal or plasmid-borne copy of the lacY gene under control of its own promoter can be induced by the addition of isopropyl 1-thio-β-D-galactopyranoside (IPTG). Cells were grown first on glucose and IPTG in order to allow synthesis and membrane assembly of LacY in the near absence of PE. Then glucose and IPTG were removed, and cells were grown on arabinose to allow new synthesis of PE in the absence of new LacY synthesis. Cells were isolated and assayed for transport function and Cys accessibility, or membranes were isolated and subjected to western blot analysis with conformation-specific mAbs. Parallel cultures either expressing wild-type LacY or only a single Cys derivative were grown with 32PO4 to determine phospholipid composition at the times indicated. The results shown in Figure 4A were from cells carrying both a chromosomal and plasmid-borne copy of wild-type lacY. LacY overexpressed from the plasmid gene predominates. The level of PE is <3% of total phospholipid in cells grown in glucose (Figure 4A, lower panel, time 0). As demonstrated previously (Bogdanov et al., 1996), LacY assembled in the absence of PE (Figure 4A, upper panel, Glu +IPTG) was poorly recognized by mAb4B1 (conformation dependent), indicating a lack of proper conformation of the P7 domain. Recognition by mAb4B11 (membrane insertion dependent but PE independent) indicated the presence of ample LacY integrated into the membrane (Bogdanov and Dowhan, 1998). Switching to growth on arabinose in the absence of IPTG returned phospholipid composition to normal after 60 min (Figure 4A, lower panel). With restored PE levels, recognition of previously synthesized LacY by mAb4B1 was restored (Ara, −IPTG), indicating a regain of native conformation of the P7 domain. The intensity of the signal with mAb4B11 was reduced by ∼50%, with the same total membrane protein load as the sample from glucose grown cells. This is consistent with no new synthesis of LacY after the switch to arabinose but with a dilution of existing LacY with continued cell growth. Figure 4.Reversibility of LacY topology. In (A), strain AA9256/pT7-5/lacY (chromosomal pssA expression under arabinose regulation and lacY under IPTG regulation) was used. In (B) strain AAL9256/pLacY-F208 (loop C6) was used, and in (C) strain AAL9256/pLacY-F208 (loop C6) or AAL9256/pLacY-F250 (loop P7) was used (chromosomal pssA expression under arabinose regulation, chromosomal ΔlacY, and plasmid lacY under IPTG regulation with single Cys derivatives in either domain C6 or P7). Cells were first grown to an OD600 of 0.2 in the presence of glucose (Glu) and IPTG. Cells were washed twice by centrifugation to remove glucose and IPTG and then grown for 60 min in medium containing arabinose (Ara). (A) In the upper panel cell membranes were isolated after growth in either Glu plus IPTG or Ara minus IPTG (after first being grown in Glu plus IPTG), and subjected to SDS–PAGE and western blotting using the monoclonal antibodies indicated. Each pair of results represents 12.5 μg (left) or 25 μg (right) of membrane protein. In the lower panel, parallel cultures were first radiolabeled to constant specific activity with and then maintained in 32PO4 during growth, first in Glu plus IPTG followed by Ara minus IPTG. Cells were analyzed for their phospholipid (PG, phosphatidylglycerol; CL, cardiolipin) content (DeChavigny et al., 1991) at the times indicated after switching from Glu (time 0) to Ara. (B) Cells were grown first in Glu plus IPTG (dashed lines) followed by Ara minus IPTG (solid lines) as described above. LacY transport function using either uptake of lactose (filled squares) or TMG (crosses) to measure facilitated or active transport, respectively, was carried out after Glu and after Ara growth. Error bars for duplicate samples are indicated where significant. (C) Cells were analyzed either after growth in the presence of IPTG (+) plus glucose (−PE Glu) or after removing IPTG (+/−) and glucose, and grown for 60 min in the presence of arabinose (+PE Ara). Where indicted, cells were treated with toluene prior to treatment with MBP followed by analysis by western blotting as described in Figure 2. Parallel cultures were analyzed for phospholipid content and transport function. Download figure Download PowerPoint LacY expressed from pLacY-F208 (as well as from pLacY-F250 and pT7-5/lacY; not shown) and assembled in PE-lacking ΔlacY cells (Figure 4B) carried out facilitated transport of lactose (uptake and hydrolysis by β-galactosidase) but not active transport of the non-hydrolyzable substrate analog methyl-thio-β-D-galactopyranoside (TMG). After recovery of PE levels to normal after arabinose growth in the absence of IPTG, active transport was restored and the rate of facilitated transport per milligram of protein was reduced again, consistent with no new LacY synthesis. Only the basal level of LacY active transport (as in Figure 4B) was detected in cells grown in the presence of IPTG and glucose, or in the absence of IPTG with growth in either glucose or arabinose (not shown). In Figure 4C, strains with a chromosomal ΔlacY were used so there would be no interference with the analysis of plasmid-encoded single Cys derivatives of LacY. Treatment of intact cells expressing single Cys derivatives of LacY (+IPTG) with MPB showed that after growth on glucose (−PE, Glu), the C6 and P7 domains were biotinylated with and without toluene treatment, consistent with their periplasmic orientation. After removal of IPTG (+/−IPTG) and growth on arabinose (+PE, Ara), domain C6 was no longer accessible to MPB unless cells were first treated with toluene. Domain P7 remained accessible with and without toluene. The lack of increased reactivity of accessible cysteines after toluene treatment and the dependence of the accessibility of the C6 domain on toluene after return of PE to normal levels strongly supports a ‘sliding back’ of the C6 domain to the cytoplasmic side of the membrane. In order to establish that the changes in LacY biotinylation patterns observed after switching growth from glucose to arabinose are not due to instability of LacY or read-through expression of ‘new’ LacY in the absence of IPTG, the radiolabeling experiments described in Figure 5 were performed under the same growth conditions as described in Figure 4C. Cell lysis and isolation of membranes was carried out using NaOH treatment to obtain detectable levels of radiolabeled material from cells labeled in rich medium. LacY expressed and labeled during growth in the presence of glucose and IPTG (Figure 5A) is stable during a 60-min chase of radiolabel (by direct counts and exposure intensity) after switching to growth in the presence of arabinose and in the absence of IPTG. Therefore, the loss of MPB labeling of domain C6 after inducing the synthesis of PE cannot be due to degradation of LacY made in the absence of PE. Treatment with NaOH resulted in a significant amount of LacY dimer. In vitro biotinylated LacY is also displayed as both a monomer and dimer (Figure 5A, lane a) after NaOH treatment, as opposed to only a monomer (lane b), when using the cell lysis method outlined in Materials and methods. Furthermore, there is no detectable LacY expressed during growth in the presence of arabinose without IPTG (Figure 5B). Therefore, the toluene-dependent MPB labeling of domain C6 after induction of PE synthesis cannot be due to newly synthesized LacY. Figure 5.Stability and read-through expression of LacY. (A) Growth conditions for strain AAL9256/pLac-F208 (domain C6) and induction of LacY and PE synthesis were exactly as described in Figure 4C, except as follows. During growth in the presence of IPTG and glucose (minus PE plus LacY), the media was supplemented with 40 μCi/ml of [35S]methionine/cysteine during growth for 60 min. After removal of glucose, IPTG and radiolabel, cells were resuspended in LB medium containing unlabeled methionine/cysteine (0.05% each) and arabinose to induce PE synthesis (time 0) in the absence of LacY induction. At the times (min) indicated, equal aliquots were removed, cells were harvested by centrifugation, the pellets were rapidly frozen then thawed, resuspended in 250 μl of buffer A and lysed by addition of an equal volume of 0.4 M NaOH followed by incubation for 20 min on ice. The membranes were harvested by centrifugation, washed once with 4 M KCl, and washed once with Tris–HCl buffer pH 8.1. The pellets were solubilized at 37°C and subjected to immunoprecipitation as described in Materials and methods, and subjected to SDS–PAGE followed by imaging and quantification with a Beta Imager from Packard Ltd. Permease remaining at each time point was expressed as counts per minute (CPM), measured at the positions corresponding to either LacY monomers or dimers. Samples grown without radiolabel in glucose plus IPTG and labeled with MPB were prepared for immunoprecipitation and western blotting either by NaOH treatment as above (lane a) or as described in Materials and methods (lane b). (B) Cells were grown as described above, except radiolabel was added during growth in the presence of arabinose and in the absence of IPTG and methionine/ cysteine. Aliquots were removed at the times indicated after the addition of radiolabel and subjected to analysis of radiolabeled protein as above. Do