THEJOURNAL OF BIOLWICAL CHEMISTRY
Vol. 269,No. 11, Issue of March 18,pp. 8296-8302,1994 Printed in U.S.A.
Biochemical and Biophysical Identification of Cystic Fibrosis Transmembrane Conductance Regulator Chloride Channelsas Components of Endocytic Clathrin-coated Vesicles* (Received for publication,August 25, 1993, and in revised form, November 17, 1993)
Neil A. Bradbury*, Jonathan A. CohnO, Charles J. Venglarik, and Robert J. Bridges From the Department of Physiology & Biophysics and Gregory Fleming James Cystic Fibrosis Research Center, University of Alabama at Birmingham, Birmingham, Alabama 35294 and the §Department of Medicine, Duke University and Veterans Administration Medical Centers, Durham, North Carolina 27710
Cystic fibrosisresults from mutations in the geneen- low-conductance C1- channel (Drummet al., 1990; Anderson et coding the CFTR C1- channel. AlthoughCFTR occurs as al., 1991a, 1991b; Kartner et al., 1991). Moreover, reconstituan integral component ofthe plasma membrane, recent tion of purified CFTR into planar lipid bilayers resulted in the studies implicate CFTR in endocytic recycling and sug- appearance of a low-conductance linear C1- channel which gest that the protein may also exist in intracellular ve- could be activated by protein kinase A and ATP (Bear et al., sicular compartments.To test this, we analyzedCFTR in 1992) showing properties similar to those reported for epitheclathrin-coated vesicles (CCV) purified from cells con- lial cells expressing endogenous CFTR. The localization of stitutively expressing CFTR at high levels. CFTR immu- CFTR to the plasma membrane domainhas been demonstrated noreactivity was detected in CCV by immunoblot and by immunological and electrophysiological techniques (Kartner was identified as CFTR based on labeling of immunopre- et al., 1991; Anderson et al., 1991b; Tabcharani et al., 1991; cipitates with protein kinase A and by trypticphosBear and Reyes, 1992; Puchelle et al., 1992; Cohn et al., 1992), phopeptide mapping. Fusionof uncoated CCV with placonsistent with the known function of CFTR as anapical memnarlipidbilayersresultedin the incorporation of kinase-and ATP-activated C1- channel activity(7.8 pS at brane C1- channel. Although recent data from patch-clamp 20 “C; 11.9 pS at 37 “C), with a linear current-voltage re- studies suggest that plasma membrane-associated CFTR can lation under symmetrical conditions. Thus, functional be directly regulated via a phosphorylatioddephosphorylation CFTR occurs inCCV. Moreover, CFTRinteracts with the mechanism (Tabcharani et al., 1991; Berger et al., 1993), an plasma membrane specific adaptor complex duringen- implicit prerequisite for this simple model is the continued of CFTR C1- channels in the docytosis through clathrin-coated pits. Therefore, the presence of an appropriate number apical membrane necessary to sustain the required level of abundance ofCFTR in the plasma membrane may be stimulated C1- secretion. regulated by exocytic insertion and endocytic recycling, Acute recruitment of CFTR C1- channels into the plasma and these processes may provide an augmentation to protein kinaseA activation as a mechanism for regulatmembrane via exocytic insertion, and their removal via endoing CFTR C1 channels in the plasma membrane. cytic retrieval, into a recycling intracellular membrane vesicle pool could provide a mechanism for regulating the number of plasma membrane-associated C1- channels during a secretory Cystic fibrosis (CF),l themost common lethal genetic disease cycle. Similar regulatory mechanisms have alsobeen proposed of Caucasians, iscaused by mutations in the gene encoding the for the Glut4insulin-sensitive glucose transporter (Suzuki and cystic fibrosis transmembrane conductance regulator (CFTR) Kono, 1980; Zorzano et al., 19891, H+ pumps, Na+ channels, (Rommens et al., 1989; Riordan et al., 1989). Alterationsin the HCO,, and PO:- transporters (Stetson and Steinmetz, 1983; primary sequence of CFTR lead to a characteristic phenotype in Garty and Benos, 1988; Buanes et al., 1988; Kempson et al., CF cells, namely impaired transepithelial C1- secretion in re- 1990). The rate of plasma membrane turnover by endocytosis sponse to activation of the CAMP-mediated second messenger and exocytosis in secretory epithelial cells is acutely regulated cascade (Quinton, 1983,1990). It now is established that CFTR by agonists which stimulate C1- secretion (Bradbury etal., itself can function as a C1--selective anion channel. Thus, ex- 1992a; Bradbury and Bridges, 1992); moreover, CAMP-dependpression of CFTR cDNA in either epithelial or non-epithelial ent regulation of plasma membrane turnover is dependent of a proteinkinase A-activatable upon the presence of normal CFTR protein (Bradbury et al., cells results in the appearance 1992b). We have thus hypothesized that direct activation of channels by phosphorylation and indirect control by insertion * This work was supported in part by Cystic Fibrosis Foundation Grants F232, F270, R464, and 236,National Institutes of Health and retrieval of channels could augment the numberof active Grants DK45970, DK42017, and DK40701, and the Veterans Adminis- CFTR C1- channels in the plasma membrane. An important tration. The costs of publication of this article were defrayed in part by tenet of our hypothesis is that CFTR be present within intrathe payment of page charges. This article must therefore be hereby cellular membrane vesicles of the endocytic/exocyticmembrane marked “aduertisement”in accordance with 18 U.S.C.Section 1734 recycling pathway. Accordingly, determining whether CFTR is solely to indicate this fact. internalized and presentwithin endocytic vesicles is of impor$ Research Fellow of the Cystic Fibrosis Foundation. To whom correspondence and reprint requests should be addressed: Dept. of Physiol- tance. The aims of our present studiestherefore were to deterogy & Biophysics, University of Alabama at Birmingham, University mine if immunologically detectable and functionallyactive Station,Birmingham,AL 35294. Tel.: 205-934-6047; Fax: 205-975-6180; CFTR is present in endocytic vesicles. E.mai1:
[email protected]. The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis EXPERIMENTAL PROCEDURES transmembrane conductance regulator; FITC, fluorescein isothiocyaMaterials-Ficoll (type 400) (Pharmacia LKB BiotechnologyInc.) nate; CCV, clathrin-coated vesicles; PAGE, polyacrylamide gel electrophoresis; MES, 4-morpholineethanesulfonic acid; bis-Tris, 2-[bis(2-hy- and FITC-dextran (Sigma;average molecular weight40,000) were dialyzed extensively against water to remove low molecular weight condroxyethyl~aminol-2-~hydroxymethyl~-propane-1,3-diol.
8296
CFTR Is in Coated Vesicles
8297
taminants andlyophilized before use. FITC quenching antibodies were I1 was from Worthington Biochemifrom Molecular Probes. RNase type A cal. Lipids were obtained from Avanti Polar Lipids. Protein kinase was obtained from Promega. All other reagents were obtained from Sigma. All aqueous solutions were prepared inMilli-Q water and were filtered through a 0.45-pm Millipore filter. i + A b cTX-100 v 75. Cell Culture"T84 cell monolayers (Dharmsathaphorn et al., 1984) were grown on plastic culture dishes in a 1:l mixture of Dulbecco's 5%(v/v) bovine calf modified Eagle's medium and Ham's F-12 including serum. Cells were harvested uponbecoming confluent (5-7 days after plating). Coated Vesicle Prepara~ions-Preparation of clathrin-coated vesicles (CCV) was based on previously described methods (Pearse, 1982; Forgac et al., 1983).All procedures were camed out at 4 "C. Briefly, conI fluent monolayers of T84 cells were scrapedfrom the culture dish and 0 50 100 150 200 250 300 350 400 450 suspended i n isolation buffer (50 mM MES, pH 6.5,35 mM NaCI, 1 m w Tfme (seconds) EGTA, 0.5 mM MgC12, 3 mM NaN3) and protease inhibitors (2 mM dithiothreitol, 10 p~ leupeptin, 1 p~ pepstatin A, 0.5 mM benzamidine, 2 pg/ml soybean trypsin inhibitor, 40 pg/ml phenylmethylsulfonyl fluo' +Ab ' ! ride, all from 1 , 0 0 0 ~concentrate). Resuspended cells were sonicated (Rranson 1200 Bath sonicator) for 1min andhomogenized in a Douncetype homogenizer. Ribonuclease typeA (50 pg/ml) was added to a postm Clathrin Coated nuclear supernatant (3,000 x g,, x 15 min) t o disassemble polyriboFi 'I somes and, after 30 min at 4 "C, the preparation was centrifuged at 751 30,000 xg,, for 60 min to obtain a postmitochondrial supernatant. The postmitochondrial supernatant was then centrifuged at 113,000 x g,, for 60 min ainBeckman Ti45 rotor to pellet a crude microsomal fraction which was applied to a continuous gradient of20% (wiv) FicoI~90% (w/v) D,O-3% fwivi Ficol~l3.5% fwiv) DzO in isolation buffer. This e a dient was centrifuged to equilibrium in a Beckman SW28 rotor a t 90,000 x g,, for 16 h. A dense CCV fraction was identified by endocytosed fluorescent probes, a high protein:lipid ratio, electron microscopy, i . , . i , ~..LL"-l."d-.L.u and immunoblot analysis. 0 50 100 150 250 200 300 350 Protein Assays-Protein concentrationsweredetermined by the Time (seconds) method of Bradford (1976). FIG.1.Determination of latency of endocytosedFITC-dextran. Inzrnunoblot Analysis-Immunoblot analysis of CCV-associated Confluent monolayers of T84 cells were incubated with FITC-dextran CFTR was performed using well-characterized affinity-purified antibodies generated against a synthetic peptide corresponding to the C- (0.5 mg/ml) for 5 min at 37 "C, after which theywereextensively washed t o remove free and surface-boundmarker(Bradburyand terminal portion of CFTR (amino acids 1468-1480 of sequence KEETEEEVQDTRL) (Cohnet al., 1992). Monoclonal antibodies to a-adaptin Bridges, 1992). Fluorescence( E , 495 nm; E , 525 nmf was determined on an MSIII &-Scan Fluorimeter (PTI). 25 pg of protein from either (AC1-MI11were a generous giftfrom Dr. Margaret Robinson (Robinson, homogenate or CCV was added to a cuvette and made to 1 m1 with 1987). CCV were homogenized in 5-10 volumes of3% SDS at room isolation buffer. Fluorescence was determined in the absence or prestemperature and centrifuged to remove insoluble material. Proteins ence of a quenching antibody (Ab;20 pll added at the times indicated. were resolved on a 7% SDS-polyacrylamide gel and transferred to ni- Lysis of tresicles was performedby the addition of Triton X-100(TX-100; trocellulose membranes. Bindingof primary antibodies was determined a t a final concentration of 0.01% viv), at the time indicated. with a peroxidase-conjugatedsecondaryantibody and detected by chemiluminescence using an ECL detection kit (Amersham). Immunowith an audio PCM (2000T, AR Vetter, Rebensburg, PA), and stored on precipitationanddetection of "2P-labeled phospho-CFTR waspervideotape. Records were replayed, low-pass filtered(mom-Hite, model formed a s described (Cohn et al., 1992).Two-dimensional tryptic phos3200) a t 100 t o 400 Hz, and transferred with a TL-1 interface and phopeptide map analysis of immunoprecipitated proteins was pClamp software (Axon Instruments, Burlingame,CA) to a n IBM-comperformed by excising radiolabeled bands from SDS-PAGE gels, folpatible computera t a sample rateof 2 kHz for event duration analysis. lowed by overnight digestion with50 pg/ml trypsin. Tryptic digests were Event duration analysis was performed only on records containing a separated by electrophoresis in the first dimension and chromatography in thesecond dimension (Picciotto etal., 1992). Co-immunoprecipi- single C1- channel. Open and closed-time histograms were constructed with the use of a threshold setting of one-half of the open-channel tations were performedby incubating solubilized CCV with antibodies t o CFTR. Following precipitation with proteinA, proteins wereresolved current. Channel amplitude was measuredon records of 4-9 duration, filtered at 50 Hz, and acquired a t 1 kHz using a digital storageoscilloby SDS-PAGE, and immunoblotting was performed as described above. scope (Nicolet Model 310, Madison WI). Amplitudes were measured by Incorporation of CFTR into Planar Bilayers-Incorporation of Clconstructing amplitude histograms and fitting the results two to Gauschannels into planar lipid bilayers, current measurements, and data sian distributions using pClamp software (Axon Instruments, Foster analysiswereperformedessentially a s described by us previously (Bridges et al., 1989; Singh et ai., 1991).Planar lipid bilayers of defined City, CA). composition were made from bovine brain phosphatidylethanolamine RESULTS and phosphatidylserine a t a ratio of 7:3 (wiwi in n-decane a t a final concentration of 20 mgiml. Bilayers were painted with a fire-polished Isolation of Clathrin-coated Vesicles from Secretory Epithecapillary tube onto a 300-pm diameter aperture in a bilayer chamber lial Cells-Prior to isolation of CCV, T84 cells were incubated fabricated from polyvinyl chloride. The volumes of the cis and trans with FITC-dextran to label components of the endocytic pathby compartments were 400 pl, and both compartments were stirred way (Bradbury andBridges, 1992). Fractions obtained from the small magnetic stir bars. KC1 buffer solutions containing 10 mM bis-Tris density gradient centrifugation were monitored for high protein: propane (pH adjusted to 7.4 with HCI), 2 mM MgClz and 0.6 mM CaCl,, and 1mM EGTA were used for all experiments. The calculated calcium lipid ratios and FITC fluorescence; the largest signal for both activity was 1 VM (Chang et al., 1988). Once a bilayer of suitable ca- values coincided with CCV (data notshown). The percentage of pacitance (200-300 pF) was obtained, KC1 from a 3M stock solution was FITC-dextran within intactsealed vesicles was determined by added to the cis compartment to obtain a 300 mM cis to50 mM trans KC1 measuring the ability of a n anti-FITC antibody to quench the at room temperature (20-24 "C) gradient. Experiments were performed and at 37 "C. CCV were strippedof clathrin using purifiedbovine brain fluorescent signal. Addition of antibody resulted in quenching com- of 40-50% of the signalfrom the homogenate, whereas none of uncoating ATPase (Schlossmanet al., 1984) and added to the cis the fluorescent signal from the CCV fraction was quenched partment of the bilayer chamber. Channel activity was recorded unfiltered, digitized (16-bit, 40-kHz) (Fig. 1). To confirm that the remainingfluorescent signal was
5
1
1 + ~ dI
1
i+Tx-ioo
1
52
8
CFTR Is in Coated
8298
Vesicles
TAEILE I Marker enzvme analvsis of . isolated CCV fraction Enzyme activities Fraction
Homogenate
ccv
Alkaline phosphodiesterase (dasma membrane)"
Lactate dehydrogenase (cvtosol)"
Cytochrome c oxidase (mitochondria)"
*
2.76 f 0.16 (4) 981.59 -c 44.2 (4) 5492.25 55.7 (4) 9342 Not detectable (4) 0.19 0.01 (4) Not detectable (4)
=
a-Mannosidase
I1
(Golgi)"
Glucose-6phosphatase (endoplasmic reticulum)"
-c 169 (4) 0.686 f 0.04 (3) 211 * 3 (4) 0.006 f 0.001 (3)
&Hexosaminidase (lysosomes)*
9239 2 169 (4) 780 f 14 (4)
Marker enzyme activities for cytosol (lactate dehydrogenase), mitochondria (cytochrome c oxidase), and endoplasmic reticulum (glucose-6phosphatase) were determined spectrophotometrically at 37 "C according to published protocols (Stonie and Madden, 1990). Results are mean f S.E. for the number of preparations in parentheses and are expressed as milliunitdmg of protein. Enzyme assays for Golgi (a-mannosidase 11) and lysosomes (P-hexosaminidase) were determined fluorimetrically at room temperature. Lysosomal a-mannosidase activity was abolished by the addition of p-chloromercuriphenylsulfonicacid. Results are mean * S.E. for the number of preparations in parenthesesand are expressed as fluorescence unitdmg of protein.
FIG.2. Electron micrograph of clathrin-coated vesicle preparation.Coated vesicle fractions were applied to a copper grid coated with a carbon-stabilized Formvar film and left for 30-60 s. The excess solution was removed by blotting, and the sample was negatively stained with 3 drops of 1% uranyl acetate for 1 min and viewed by transmission electron microscopy. Bar = 100 nm.
from FITC-dextran withinendocytic vesicles, Triton X-100 was added to solubilize the vesicles. All remaining fluorescence was quencheduponaddition of detergent,consistentwiththe marker being trapped within CCV from the endocytic pathway (addition of Triton X-100 alone did not quench FITC-fluorescence, data not shown). We have previously shown that fluidphase markers are not trappedby vesiculation of the plasma membrane duringhomogenization of T84 cells (Bradbury et al., 1992a; Bradbury and Bridges, 1992). Marker enzyme analysiswas performed on fractions obtained during theisolation of CCV. Since CCV have no specifically associated enzyme activities,these assayswere performed to document the removal of contaminating membrane, and the results obtained were consistent with the removal of a t least 90% of contaminating organelles (Table I). Analysis of the apical membrane marker alkaline phosphatase showed that although enzymatic activity in the CCV fraction was undetectable in the absence of detergent, activity could be detected following Triton X-100 treatment. Thus, theCCV fraction was composed entirely of intact inside-out vesicles. This is consistent with thelocation of alkaline phosphataseon the outer surface of the apical plasma membrane, which corresponds to the inside of endocytic vesicles following endocytosis of the apical membrane. Thus, there was no contamination from outside-out plasma membrane vesicles. Electron micrographs (Fig. 2) of the coated vesicle prepara-
tion revealed structures with polygonal coating characteristic of CCV. The coated vesicles were of uniform size with >90% of all vesicles 95 nm in diameter, consistent withpreviously documented dimensions for CCV. CCV fractions were analyzed by immunoblot using a panelof monoclonal antibodies directed against known components of CCV (Fig. 3). As suggested by the electron micrographs, the 180-kDa clathrin heavy chain wasdetected confirming that the vesicles obtained were indeed clathrin-coated. Monoclonal antibodies against plasma membranespecific wadaptin detected a band of 100 kDa (Robinson, 1987). The lower 56-kDa band detected by the monoclonal antibody is most probably an Nterminal proteolytic fragment of e-adaptin containing the recognized epitope.2 In addition, immunoprecipitation of solubilized CCV with CFTR antibodies resulted in the coprecipitation of the plasma membrane adaptor protein e-adaptin (Fig. 4). Clathrin-coated Vesicles from Secretory Epithelial Cells Contain Immunologically Detectable CFTR-Western blot analysis of CCV derived from epithelial and non-epithelial tissues was performed using an antibody directed against the C-terminal portion of CFTR. CCV CFTR appeared as a band of 165-170 kDa, consistent with a mature fully glycosylated protein (Fig. 5). This protein co-migrated with the predominant signal deM. Robinson, personal communication.
1.
CFTR Is in Coated
Vesicles
2.
kDa
8299
116 -
200 kDa
200 116 91 66
-
-
clathrin heavy chain a-adaptin “I
-
66 97
45 45
ft
a-adaptin 4
-
anti-
NonImmune
CFTR
precipitating antibody
FIG.3. Immunoblot analysis of clathrin-coated vesicles from
T84 cells. CCV (20 pg) were resolved on 10% acrylamide SDS-PAGE
and electrophoretically transferred to nitrocellulose. Binding of primary FIG.4. Co-immunoprecipitation of ClWR and a-adaptin. CCV antibodies was detected using a horseradish peroxidase-conjugated secwere solubilized in buffer (50 mM HEPES, pH 7.3, 1% Triton X-lOO,lO% ondary antibody and luminol enhanced chemiluminescence (ECL; Ama t 4 “C in the ersham). Lane 1, clathrin heavy chain; lune 2, a-adaptin. The positions glycerol, and protease inhibitors) and incubated overnight presence or absence of monoclonal antibodies against CFTR. Samples of molecular size markers are indicated in kilodaltons. were precipitated with protein A and washed extensively. Electrophoresis, transfer to nitrocellulose, and protein immunoblotting with antitected in human pancreas and colon using the same antibody bodies against cr-adaptin were performed a s described under “Experimental Procedures.” Lune 1 shows a-adaptin co-immunoprecipitated (Cohn et al., 1992). CFTR was also detected in CCV from rat with anti-CFTR antibodies.Lane 2, nonimmune rabbit serum was used colonic enterocytes, an epithelial tissue known to express as a control for anti-CFTR antibodies in theimmunoprecipitation.
CFTR (Fiedler et al., 1992), but not in CCV from rat brain, a tissue which does not express CFTR (Fig. 5) (Fiedler et al., 1992). These results confirm that thepresence of CFTR is not a generalized characteristic of CCV, but isspecific for CCV from C1- secretory epithelial cells. In contrast to the single CFTR band observed in CCV, lower molecular weight forms of CFTR were also observed in the T84 cell lysate, corresponding to core-glycosylated CFTR (Cheng et al., 1990) (data not shown). Immunoprecipitations were performed to characterize further CCV-derived CFTR. The a-1468 antibody specifically precipitated a protein kinase substrateof 165-170 kDa from both T84 cell lysates andpurified CCV (Fig. 6A ). Although two other labeled bands were observed following immunoprecipitation from cell lysates, these bands were also precipitated in the absence of primary antibody. These lower bands are absent from CCV fractions, and theonly detected 32P-labeled phosphorylation product is mature CFTR. No immunoprecipitation of CFTR was observed in theabsence of primary antibody. Shown in Fig. 6, B and C , are two-dimensional tryptic phosphopeptide maps of the immunoprecipitated bandfrom T84 cell lysates and CCV, respectively. The peptides labeled 1to 5 are each identical with phosphopeptides shown to occur in the R domainof CFTR (Cohn et al., 1992; Picciotto et al., 1992). These results definitively identify the 165-170-kDa band in Fig. 5A as CFTR. Clathrin-coated Vesicles from Secretory Epithelial Cells Contain Functionally Active CFTR-Current traces of a CCV-derived CFTR C1- channel incorporated from the cis side of a phosphatidylethano1amine:phosphatidylserineplanar lipid bilayer before and after theaddition of protein kinase A(100 nM) and ATP (1mM) to thecis bath areshown in Fig. 7. The applied potential was-30 mV (cis to trans) with a 30050 mM (cisdrans) KC1 gradient. Omission of protein kinase A and ATP from either of an active the cis or thetrans bath resulted in the appearance channel in only 1 out of 20 experiments (Table 11). The orientation of CFTR in stripped CCV is such that the cytoplasmic domains of the CFTR C1- channel will be exposed to the bulk media, whereas the extracellulardomains of CFTR will be located within the membranevesicle. Thus, addition of stripped vesicles to the cis chamber of the bilayer setup will result in incorporation of CFTR C1- channels such that thecytoplasmic domains of CFTR will be exposed in the cis bath, and the
1
2
3
4
5
6
205 kDa
-+I 116 kDa -
97 kDa -
66 kDa
-
FIG.5 . Immunoblot analysis of coated vesicle CFTR from epithelial and non-epithelial cells. CCV fractions from T84, rat colonic enterocyte, and rat brain were resolved on 7% acrylamide SDS-PAGE Lanes 1 3 are 5 pg, and electrophoretically transferred nitrocellulose. to 2 pg, and 1 pg, respectively, of T84 CCV protein. Lane 4 is 10 pg of protein from rat brain CCV. Lanes 5 and 6 are 5 pg and 1 pg, respectively, of protein from rat colonic enterocytes. The positions of molecular size markers are indicated in kilodaltons.Arrow indicates CFTR.
extracellular domains of CFTR will be exposed in the trans bath. Addition of protein kinase A and ATP to thecis bath, but not the trans bath, supported C1- channel activity (Table 11) consistentwith the predicted orientation of coated vesicle CFTR. Although addition of trans protein kinase A and ATP failed to activate CFTR C1- channels, subsequent addition of protein kinase A and ATP to the cis bath supported channel activity confirming that CFTR C1- channels hadbeen incorporated. All subsequent experimental data were obtained following addition of protein kinase A and ATP to the cis bath. An additional important requirement for the successful incorporation and activation of CFTR C1- channels was the necessity to uncoat the CCV. In 20 control experiments, CFTR C1-
CFTR Is in Coated
8300
Vesicles
A
CONTROL 0C-
205
B
C
I
97
0.5 pA
66
I4 s
0
0
FIG.6. Immunoblot and phosphopeptidemap analysis of coated vesicle CFTR. A, coated vesicle CFTR was immunoprecipi-
FIG.7. Current traces of clathrin-coatedvesicle-derivedCFTR C1- channel. Coated vesicles were stripped of clathrin using purified ATP-dependent uncoatase and fused with planar lipid bilayers a s described under "Experimental Procedures." Upper trace is from the bilayer before the additionof protein kinase A and ATP. Lower traces are channel activity 20 minutes after the additionof protein kinase A(100 nM) and ATP (1 mM) to the cis bath. Conditions:300 to 50 mM KCI, -45 mV cis to trans. o indicates channel open state; c indicates channel closed state.
tated using antibody a-1468 and phosphorylated a s described (Cohn et al., 1992). Proteins were solubilized in buffercontaining 1%Triton X-100,1% deoxycholate, 0.1%SDS, and protease inhibitors. Solubilized proteins were incubated with a-1468 overnight a t 4 "C and precipitated with protein A-agarose beads. Labeling with protein kinase A was performed with 10 units of kinase and 50 pCi of [y-"P]ATP. PhosphoryTABLE I1 lated samples wereresolved by SDS-PAGE, and the gels were dried and Frequency of CFTR channel activity by cis or trans addition of exposed to x-rayfilm. Lane 1,cell lysate precipitated witha-1468 (+anprotein kinase A and ATP tibody); lane2, cell lysate precipitated withouta-1468 (-antibody); lune Stripped vesicles were added to the cis bath of the bilayer chamber. 3, CCV precipitated with a-1468 (+antibody); lune4, CCV precipitated without a-1468 (-antibody). B , two-dimensional tryptic phosphopeptide Values shown are for the numberof experiments whenCFTR channels maps of coated vesicle CFTR were performed by excising gel slices from were observedlthe number of experiments performed. When protein the 165-170-kDa band from the experiment inA. Bands were washed kinase A andATP were omitted, the bilayer wasmonitored for a t least and digested overnight with 50 pg/ml trypsin. Tryptic digests were 90 min before discontinuing the experiment. Channel activity was obATP to the cis separated by electrophoresis in the first dimension a t 400 V in 10% served 10-30 min after addition of protein kinase A and acetic acid, 1% pyridine, pH 3.5,and by chromatography in the second bath. No activity was observed when protein kinase A and ATP were dimension usingpyridine:l-butano1:water:aceticacid (10:15:13:3v/v). 0 added to the trans bath. is the origin. Left, positive; right, negative. Phosphopeptides 1-4 were (+)-Proteinkinase (-)-Protein kinase Side of protein kinase previously identified a s components of the R-domain of CFTR (Cohn et NATP NATP NATP addition al., 1992), and each corresponds to a predominant phosphopeptide de1/20 18121 cis bath tected in digests of CCV-associated CFTR. C , two-dimensional tryptic 1/20 0120 trans bath phosphopeptide maps of T84 cell lysate CFTR were performed a s described in B.
channel activity was obtained only twice when using coated CCV. Together with the requirement for protein kinase A and ATP in the cis bath, these results support theconclusion that the observed CFTR C1- channel activity wasderived from CCV (and not a contaminating vesicle population). CCV-derived CFTR C1- channel activity was determined a t several holding potentials(Fig. 8A). Current amplitudes at these potentials were determined by amplitude histogram analysis fitted to Gaussian distributions (Fig. 8B). A plot of the current-voltage relationship (Fig. 8C) of the CFTR C1- channel under asymmetric conditions displayed Goldman rectification, with aconductance of 7.8 pS a t 20 "C. When measured a t 37 "C, channel conductance increased to 11.9 pS. Under symmetrical conditions, the current-voltage relation was linear (data not shown). The anion versus cation selectivity was estimatedfrom the reversal potentials of the current-voltage curves (shownin Fig. 8C) using the Goldman-Hodgkin-Katz equation and was shown to be at least15:l. Halide selectivity experiments showed a greater permeability of the CFTR C1- channel to Brover C1-of 1.4:l and a lower permeability to I-. It proved difficult to assign an I- permeability since I- appeared to cause a fast-type block of the channel, a finding also reported for CFTR C1- channels detected in patch clamp experiments (Tabcharani et al., 1991). The mean open probability of the protein kinase A-stimulated channel was0.55. Event duration analysis yielded a single open time constant (7,) of 360 ms and one dominant closed time constant(7,)of 330 ms. Fluctuation anal-
ysis of single-channel records, obtained under the described data collection configuration, yielded a power density spectrum that was fitted by a single Lorentzian function with a corner frequency of 0.91 Hz (data not shown). This value is inexcellent agreement with the expected value of 0.92 Hz derived from the open and closed time, where 2 d Cequals the sum of 7;' plus T~-'.The biophysical characteristics of CCV-derived CFTR are for plasmamembrane thus consistentwiththosereported CFTR in both native and overexpressing heterologous systems, using the patch clamp technique (Haws et al., 1992h3 DISCUSSION
The goal of our studieswas to determineif CFTR was present in CCV. We have isolated CCV from T84 cells, a secretory epithelial cell line which constitutively expresses CFTR. Several criteria were used for characterizing the CCV fraction obtained. Firstly, CCV fractions had a highprotein:lipid ratio, the basis on which the density gradient isolation was achieved. Secondly, immunoblot analysis of the CCV fraction revealed the presence of clathrin heavy chain and the endocytic-specific adaptor protein a-adaptin. Finally, electron microscopic examination of the CCV fraction revealed a homogeneous population of uniformly sized (95 nm) vesicles with bristledcage structures characteristic of CCV. Five lines of evidence argue for the presence of CFTR within C. J. Venglarik, B. D. Schultz, R. A. Frizzell, and R. J. Bridges, submitted for publication.
8301
CFTR Is in Coated Vesicles A
C
B 300:50 KC1 2,
0.1
C
0 mV
a
/
w
2
C0-
: -40
Lr,
\
I
-
/
-20
x
-30 mV
E
Ai=-0.42 pA
a
rn
C-
2
iL
0-0.6 -0.4 -0.2 0.0
x
-45 m V C-
E
300:50 KC1
w
A i = - 0 . 5 2 pA
u
e
I (PA)
LL
0-
-0.6-0.4-0.2 0.0 Amplitude (PA)
FIG.8.Currenttraces, amplitude histograms, and current-voltagerelationship of coated vesicle-derivedCFTR C1- channels. CFTR was activated by protein kinase A and ATP in the cis bath as shown in Fig. 6, and currents were recorded at various potentials. Parts A and B show representative 18-9 current records and corresponding amplitude histograms. Part C shows the mean current (+S.D.) of three experiments performed at 20 "C (open circles) or 37 "C (filled circles) as a function of the command potential. We expect the currentsto rectify due to the ionic asymmetry (300:50 KCl, cis:truns), and, therefore,the data were fit to the Goldman-Hodgkin-Katz equation(solid lines). The conductance(7.8 pS at 20 "C; 11.9pS at 37 "C) and reversal potential (37 mV at 20 "C;35 mV at 37 "C) were estimated by simple linear regression. Based on the reversal potential, the C1:K selectivity was calculated to be 20:l (20 "C) or 15:l (37 "C). the isolated CCV. Firstly, thepredominantbandin CCV detected by immunoblot using thea-1468 antibody was a protein of 165-170 kDa in agreementwith the predicted mass of CFTR. Secondly, the immunoprecipitated protein was a substrate for protein kinase A , CFTR contains multiple protein kinase A et al., 1989). phosphorylationconsensussequences(Riordan Thirdly, two-dimensional tryptic phosphopeptide map analysis identifies 5 peptides which were identical with those shown for CFTR R-domain (Cohn et al., 1992; Picciotto et al., 1992). Fourthly, a close in vivo association betweenCFTR and a-adaptin, as demonstrated by co-precipitation experiments, places CFTR in endocytically derived CCV (Robinson, 1992). Finally, the CFTR C1- channel was functionally incorporated into planar lipid bilayers following removal of the clathrin coat from CCV. The properties of CCV-derived CFTR C1- channels in bilayerswere essentially identical with thosereported for plasmamembrane CFTR observed in patchclamp studies. Thus, channel activity was regulated by protein kinase A and ATP and displayed a linear current-voltage relationship under symmetrical ionic conditions. Kinetic analysis of CCV-derived CFTR in planar lipid bilayers yielded results consistent with those obtained from plasma membrane CFTR in multichannel pat~hes.~ Several studies have attempted to document a subcellular localization of CFTR. For example, Puchelle et al. (1992) using confocal immunofluorescene microscopy, and Marino and Webster (1993) using immunogold electron microscopy have shown CFTR staining in subapical membrane vesicles, as well as in 0-form invaginations of the plasma membrane. Although the precise nature of the vesicles was not determined, they are nonetheless consistent with an endosomal CCV. Heterologous expression of CFTR in CHO cells has suggested that CFTR may be present in a n endosomal compartment. However, since no subcellularfractionation or characterization was performed (Lukacs et al., 1992), it is not clear whether endosomes alone contained CFTR or whether CFTR was also present in other subcellular organelles. In addition, it is possible that overexpression of CFTR in a heterologous expression system may lead to targeting not seen in a natively expressing epithelial tissue. The results presented in the present paper provide evidence for
orthe definitive localization of CFTR toanintracellular ganelle; i.e. a plasma membrane-derived clathrin-coated vesicle. The observation that functionally active CFTR is present in a role for CFTR C1CCV raises thequestion of whether there is channels in intracellular membrane vesicles. C1- conductance in the limiting membranes of intracellular organelles is required for electroneutral acidification of the organelle interior. Further, it has been suggestedthat gatingof the organellarC1channel, and hence acidification, can be brought about by a reversible protein kinase A-dependent phosphorylation (Bae and Verkman, 1990; Mulberg et al., 1991). Comparison of the rate and final pH of intracellular organelle acidification from normal and CF airwayepithelial cells suggests that acidification is abnormal in CF cells, presumably resulting from impaired C1--dependent acidification (Barasch et al., 1991). Acidification studies with CCV derived from C1- secreting epithelial cells constitutively expressing CFTR may be useful in determining the potential role of CFTR in endosomal acidification. The presence of CFTR in CCV is consistent with our hypothesis that plasma membrane CFTR levels could beacutely regulated via exocytic insertion and endocytic retrieval. However, while the resultsreported here support thishypothesis, further levels studies arerequired to establish that plasma membrane of CFTR are in fact acutely regulated via a n endocytic/exocytic mechanism. There aretwo membrane compartmentsof the cell from which CCV form: the plasma membrane and the transGolgi network. At present, we do not know what proportion of CFTR is in plasma membrane (endocyticl-derived versus Golgi (exocytic)-derived CCV. However, the isolation of a CCV fraction containing FITC-dextran and a-adaptin indicates that at least a portionof the isolated CCV in our preparation isderived from the plasma membrane by endocytosis. In contrast to other trafficked proteins such as the Glut4 transporter, CFTR is a low abundance protein, making quantitative assessment of the plasma membrane and subcellular organellar levels of CFTR during a secretory cycle very diEcult. It is interesting to note, however, that recent studies have shown that disruptionof the microtubular network inT84 cells inhibits CAMPbut not Ca2+-dependent C1- secretion from T84
CFTR Is Vesin iclCeosated
8302
cells (Fulleret al., 1994). If such regulated traffickingof CFTR Bradbury, N. A., and Bridges, R. J. (1992)Biochem. Biophys. Res. Commun. 184, 1173-1180 occurs in response to secretagogues, then there are several Bradbury, N. A., Jilling, T., Kirk, K. L., and Bridges, R. J . (1992a)Am. J . Physiol. examples from other transport proteins that suggest possible 262, C752X759 molecularmechanisms.Efficientinternalization of plasma Bradbury, N. A., Jilling, T., Berta, G., Sorscher, E. J., Bridges, R. J., and Kirk, K. L. (199213) Science 256, 530-533 membrane proteins is associated with clustering in clathrinBradford, M. M. (1976) Anal. Biochem. 72, 248-254 coated pits on the cell surface (Courtoy et al., 1991). Indeed, Bridges, R. J., Worrell R. T., Frizzell, R.A., and Benos, D. J. (1989)Am. J . Physiol. 256, C902-C912 internalization of CFTR may be brought about by clustering in Buanes, T., Grotmol, T., Landsverk, T., and Raeder, M. G. (1988)Gastroenterology that CFTR will clathrin-coatedpits;however,itisunlikely 95,417-424 interact directly with clathrin. Instead, clustering of CFTR in Canfield, W. M., Johnson, K. F., Ye, R. E., Gregory, W., and Kornfield, S. (1991)J . Biol. Chem. 266, 5682-5688 clathrin-coated pits may be mediated by its binding with the Chang, D., Hsieh, P. S., and Dawson, D. C. (1988) Comput. B i d . Med. 18,351-366 a-adaptin of t h e HA-2 adaptor complex (Ahle and Ungewickell,Chen, W. J., Goldstein, J . L., and Brown, M. S., 11990) J. Biol. Chem. 256, 31163123 1989; Keen a n d Beck, 1989; Beltzer and Speiss, 1991; Pearse, S. H., Gregory, R. J., Marshall, J., Paul, S., Souza, D.W., White, G. A,, 1985). Indeed, Sorkin and Carpenter (1993) have shown that Cheng, O'Riordan, C. R., and Smith, A. E. (1990) Cell 63, 827-834 activation of the epidermal growth factor receptor upon binding Cohn, J.A,, Nairn, A. C., Marino, C. R., Melhus, O., and Kole, J . (19921 Proc. Natl. Acad. Sci. U. S. A. 89, 2340-2344 epidermal growth factor alters its ability to bind a-adaptin, and Collawn, J . F., Stangel, M., Kuhn, L. A,, Esekogwu, V , Jing, S., Trowbridge, I. S., hence provides a mechanism for efficiently internalizing actiand Tainer, J.A. 11990) Cell 63, 1061-1072 Courtoy, P. J., Quintart, J., Limet, J. N., De Roe, C., and Baudhun, P, (1991) in vated epidermal growth factor receptors. Several short amino Endocytosis (Pastan, I., andWillingham,M. C., edslpp. 163-188, Plenum acid sequences (e.g. NPXY a n d YXRF) have been associated Publishing Corp., New York with the clustering and efficient endocytic internalizationof a Dharmsathaphorn, K., McRoberts, J . A., Mandel, K. G., Tisdale, L. D., and Masui, H. !1984)Am. J. Physiol. 246, G204-G208 number of plasma membrane proteins (Chenet al., 1990; ColM. L., Pope, H.A,, Cliff, W. H., Rommens, J. M., Marvin,S. A,. Tsui L.-C., lawn et al., 1990; Canfield et al., 1991). These internalization Drumm, Collins, F. S., Frizzell, R. A,, and Wilson, J . M. (1990) Cell 62, 1227-1233 signals arelocalized to the cytoplasmic domains of internalized Fiedler, M. A., Nemecz, Z. K., and Shull, G. E. (1992) Am.J. Ph.ysiol. 262, L779proteins (including the low density lipoproteins and transferrin L784 Forgac, M., Cantley, L., Wiedenmann, B., Altsiel, L., and Branton, D. (19831 Proc. receptors) and frequently containa tyrosine or other aromatic Natl. Acad. Sci. U. S. A. 80, 1300-1303 Fuller, C. M., Bridges, R. J., and Benos, D. J . (1994)Am. J. Physiol., in press amino acid as part of a predicted tight turn. Recent studies Garty, and Benos, D.J . 119881 Physiol. Reu. 68,309-373 have also suggestedthat a phenylalanine residueclose to t h e N Haws, H., C., Krouse, M. E., Xia, Y.. Gruenert, D. C., and Wine, J . !19921 Am. J . terminus of the Glut4 transporter is required for its efficient Physiol. 263, L692-L707 internalization (Piper et al., 1993). Indeed, there are several Kartner, N., Hanrahan, J. W., Jensen, J.T., Naismith, A. L., Sun, S., Ackerley, C . A,, Reyes, E. F., Tsui, L.-C., Rommens, J . M., Bear, C. E., and Riordan, J. R. phenylalanine residues in the N-terminal cytoplasmic domain (1991) Cell 64, 681-691 of CFTR. Since the regulation of plasma membrane turnover by Keen, J. H., and Beck, K. A. (19891 Biochem. Biophys. Res. Commun. 158, 17-23 Kempson, S. A,, Helrnle, C., Abraham, M. I., and Murer, H. (1990lAm. J. Physiol. CFTR is dependent upon activation of t h e CAMP-mediated sec258, F1336-F1344 ond messenger cascade (Bradbury et al., 1992b1, there exists Lukacs, G. L., Chang, X.-B., Kartner, N., Rotstein, 0. D., Riordan, J. R., and Grinstein, S. (19921 J . B i d . Chem. 267, 14568-14572 the possibility that such an internalization sequence may be Marino, C. R., and Webster, P. (19931 Clin. Res. 41, 219A cryptic in the phosphorylatedactivated form of CFTR, possibly Mulberg,A. E., Tulk, B. M., and Forgac, M. 119911 J. Biol. Chem. 266,20590-20593 leading toa reduced affinity of a-adaptin for CFTR.If so, inhi- Pearse.~,B. M. F. (1982) Proc. Natl. Acad. Sci. U . S. A. 79. 451-455 bition of CFTR endocytic retrieval would providea mechanism Pearse, B. M. F. (19851 EMBO J 4,2457-2460 Picciotto, M. R., Cohn, J. A,, Bertuzzi, G., Greengard, P., and Nairn,A. C. (19921J . for retaining phosphorylated-active CFTR C1- channels in the Biol. Chem. 267, 12742-12752 Piper, R. C., Tai, C., Kulsza, P., Pang, S., Warnock, D., Baenziger, J., Slot, J . W., apical membrane following stimulation of C1- secretion. ~~
Acknowledgments-We thank Mai Huynh, John Clark, Jolanta Kole, and Kip Smith for excellent technical assistance. We also acknowledge AndrewKaz for immunoprecipitationsandtwo-dimensional phosphopeptide map analyses. Electron microscopy was performed by Eugene Arms of the Comprehensive Cancer Core Facility at theUniversity ofAlabama a t Birmingham. We also thank Dr. Kevin L. Kirk for helpful discussions. We would also like to thank M. Robinson for the generous gift of monoclonal antibodies. REFERENCES Anderson, M. P., Berger, H. A,, Rich, D. P., Gregory, R. J., Smith,A. E., and Welsh, M. J (1991a) Cell 67. 775-784 Anderson, M. P., Rich, D. P., Gregory, R. J., Smith,A. E., and Welsh, M. J. (1991bJ Science 2 5 1 , 6 7 9 4 9 1 Ahle, S., and Ungewickell, E. 11989) J . B i d . Chem. 264, 20089-20093 Bae, H:R., and Verkman, A. S. (19901 Nature 348, 637-639 Barasch, J.,Kiss, B., Prince, A., Saiman, L., Gruenert, D., and AI-Awqati, Q. !1991 J Nature 352, 70-73 Bear, C. E., and Reyes, E. F. (1992)Am. J. Physiol. 262, C251-C256 Bear, C. E., Li, C., Kartner, N., Bridges, R. J., Jensen,T. J., Ramjeesingh, M., and Riordan, J . R. (1992) Cell 68, 809-818 Beltzer, J . P., and Speiss, M. (19911 EMBO J. 10, 3735-3742 Berger, H.A,, Travis, S. M., and Welsh, M. J.(1993)J. Biol. Chem. 268,2037-2047
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