Vesicular Stomatitis Virus Glycoprotein Contains a Dominant ...

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THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular’ Biology, Inc

268, No. 5, Issue of February 15, pp. 3313-3320,1993 Printed in U.S.A.

Vesicular Stomatitis Virus Glycoprotein Contains a Dominant Cytoplasmic Basolateral Sorting Signal Critically Dependent upon a Tyrosine* (Received for publication, July 27, 1992)

D’Nette C. Thomas$, Colleen B. Brewer, and MichaelG . Rothg From the Department of Biochemistry, Uniuersity of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235-9038

To investigate the contribution of the cytoplasmic domain of the vesicular stomatitis virus G glycoprotein toits basolateral expression in polarized epithelial cells, chimeric proteins containing the external and transmembrane domains of an apically targeted protein, the influenza virus hemagglutinin (HA), and either the G cytoplasmic domain or an unrelated cytoplasmic sequence, were introduced into Madin-Darby canine kidney (MDCK) cells. Additionof the cytoplasmic tail of G to a truncated HA resulted in delivery of greater than 95% of the chimeric protein to the basolateral cell surface, indicating that the G cytoplasmic domain contains a dominant basolateral sortingsignal. A similar chimera, containing the cytoplasmic tail of herpes simplex I glycoprotein gC, was not sorted basolaterally. Deletion of the cytoplasmic tail from G protein itself decreased the fidelityof sorting to the basolateral surface, but not the extent to which the protein reached the plasma membrane.Mutation of cytoplasmic tyrosine 501 of G caused an identical loss of basolateral targeting, suggesting that the tyrosine, or the sequence surrounding it, is required for efficient basolateral transport of G. Mutation of tyrosine 501 hadno effect on internalization of G, which was much slower than that of endocytic receptors. Thus, VSV G protein contains an efficient cytoplasmic basolateral targeting signal that is not an efficient internalization signal.

Matlin (1989), Bomsel and Mostov (1991), Simons and Wandinger-Ness (1991), Hopkins (1991), and Mostov et d . (1992)). The most thoroughly investigated model for epithelial polarity is the Madin-Darby canine kidney (MDCK)’ continuous cell line. When infected with enveloped viruses, MDCK cells sort the viral membraneglycoproteins to either the apical or basolateral surfaces and certain viralglycoproteins have been quite useful in establishing the pathways followed by apically and basolaterally targeted proteins in these cells. Thus, the vesicular stomatitis glycoprotein G (VSV G), which is transported basolaterally, and the influenza virus hemagglutinin (HA), found predominantly at the apical surface, have been shown to comigrate through the exocytic pathway until they reach the trans-Golgi network where they are sorted (Rindler et al., 1984; Fuller et al., 1985; Wandinger-Ness et a t , 1990). Transport of these proteins from the trans-Golgi network is rapid and appears to be direct (Misek et al., 1984; Matlin and Simons, 1984; Pfeiffer et al., 1985; Rindler et al. 1985; Lisanti et al. 1989a).More than 95% of VSV G is sortedtothe basolateral surface of virus-infected MDCK cells, whereas polarity of HA is less strict, ranging from70 to 99% apical in MDCK cells infected with influenza virus. The viral proteins are similar to endogenous cell glycoproteins in this respect, as individual proteins exhibit quite different degrees of apical polarityinMDCK cells, whereas basolateralproteinsare usually sorted moreunidirectionally. The identity of the sorting determinants responsible for polarized delivery of proteins to either theapical or basolateral membranes of these cells is unknown. Some earlier work, based largely upon site-directed mutaThe plasma membrane of an epithelial cell typically contains two distinctmembranedomainsseparated by tight genesis of viral glycoproteins, suggested thatinformation junctions and is both functionally and structurally polarized. important for sorting transmembrane proteins to either the T h e apical membrane of these cells faces the external envi- apical or basolateral surfaces residedin their external domains ronment and isspecialized forthe processes of secretion and/ (Roman and Garoff, 1986; Mostov et al., 1986; McQueen et or absorption, while the basolateral membrane communicatesal., 1987; Roth et al., 1987; Compton et al., 1989; Lisanti et types of dominant with the internal tissue and has a composition more charac- al., 1989b). However, more recently two teristic of nonpolarized cells, such as fibroblasts (Simons and intramembrane or cytoplasmic sorting signals have been idenFuller, 1985; Rodriguez-Boulan andNelson, 1989; Nelson, tified. A glycosylphosphatidylinositol anchor specifies sorting 1989). Transmembrane proteins that have a polarized distri- to the apical surface in MDCK cells (Lisanti et al., 1989a; Brown et al., 1989) and several apparentlydifferentshort bution on the surface of these cells must therefore contain information which specifies preferential localization to oneof cytoplasmic sequences can specify targeting to the basolateral surface (Brewer and Roth, 1991; Casanova et al., 1991; Hunthe two membranedomains (for reviews see Caplanand ziker et al., 1991; Yokode et al., 1992). For some proteins, such * This research was supported in partby Grant GM37547 from the as VSV G (Puddington et al., 1986; Brown et al., 1989; McNational Institutes of Health. The costs of publication of this article Queen et al., 1987; Compton et al., 1989) and polyimmunog-

were defrayed in part by the payment of page charges. This article must thereforebe hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. $ Supported by Training Grant GM08203 from the National Institutes of Health. I An Established Investigator of the American Heart Association. TOwhom correspondence should be addressed: Tel.: 214-688-3276; Fax: 214-688-8856; Bitnet: ROTHaUTSW.

The abbreviations used are: MDCK cells, Madin-Darby canine kidney cell; VSV G, vesicular stomatitis virus G protein; HA, influenza virus hemagglutinin; gC, herpes simplex virus gC protein; PBS, phosphate-buffered saline; DMEM, Dulbecco’s modified Eagle’s medium; BSA, bovine serum albumin; PAGE, polyacrylamide gel electrophoresis.

3313

3314

SignalTargeting Basolateral Dominant

lobulin receptor (Mostov et al., 1986), sequences important brook et al., 1989) with reagents from Promega Corp. (Madison) or for sorting have been reported to lie in both the cytoplasmic GIBCO/Bethesda Research Laboratories (Gaithersburg). cDNAs not previously described were sequenced with Sequenase (United States and the externaldomains. BiochemicalCorp.,Cleveland, OH) according to instructions from However, complicating the analysisof experiments inwhich themanufacturer.Reagentsnot otherwiseidentified were from exogenous proteins are introduced into epithelial cells is the Sigma. possibility that some proteins may preferentially reach one of Preparation and Maintenance of Transfected Clonal Cell Linesthe two surface domains without interactingwit.h the cellular Clonal MDCK cell lines expressing wild type or mutant HA or G sorting machinery at all, that is, by default. Some exogenous proteins were preparedand maint.ainedaccording to established secretory proteins have been shown to be secreted in approx- protocols (Brewer and Roth, 1991). Each cell line formed an electrically tight monolayer and properlylocalized methionine transporters imately equal amounts from both sides of transfected MDCK a t t,he basolateral surface, as measured by a standard methionine cells grown in monolayers (Kondor-Koch et al., 1985; Gon- uptakeassay (Brewer andRoth, 1991; see Table I). For sorting zalez et al., 1987) suggesting that in those cells bidirectional experiments, cells were plated a t high density on Costar Transwell secretion ist.he default for secretory proteins lacking a sorting filters(Costar Corp., Boston) and grown for 5 dayspriortothe was measured in several a default experiment. For each protein studied, sorting signal. No similarexperimenthasdemonstrated pathway for transmembrane surface proteins, and thislack of independently derivedclonal cell lines to avoid any possibility of sorting differences due to properties of a cell line rather'than the understanding of the destinationof a membrane prot,ein lack- protein. ing a functional sorting signal has complicated the interpreAssays for Surface Arrival of HA or G Proteins-Cell monolayers tation of experiments designed bo identify such signals. onTranswellfilters were washedseveral timeswithphosphateRecently it has been shown that conversion of cysteine 543 buffered saline (PBS (GIBCO)) containing 1 mM Mg2+and 0.1 mM t o tyrosine in the shortcytoplasmic tail of HA not only creates Ca2+, and thenwere incubated 1 h a t 37 "C in media lacking methioa sequence specifying internalization through coated pits (La- nine and cysteine. Trans3'S-label (ICN, Irvine, CA) was diluted to a zarovits and Roth, 1988) but also results in essentially com- concentration of 2.5 mCi/ml in this same media, and the cells were labeled for 30 min from the basolateralside by placing the Transwell plete basolateral targeting of that protein inMDCKcells filters in 140 p1 of labeling solution on a piece of parafilm. The cells (Brewer and Roth,1991). The simplest interpretation of these were then incubated a t 37 "C for 15 min in normal media to allow observations is that a single sequence is recognized for both recentlysynthesizedproteinsto fold before the monolayers were sorting events. Similar observations have been madefora shifted to 20 "C for 2 h to allow the labeled protein to accumulate in lysosomal membrane protein and the Fc receptor (Hunziker the trans-Golgi network. Cells expressing HA proteins were then et al., 1991), whereas sequences specifyinginternalization and shifted to 37 "C in media containing 10 rg/ml of trypsin on one side basolateral sorting are different for the polyimmunoglobulin of the monolayer and 20 pg/rnl soybean trypsin inhibitor on the other side of the filter. Controls had trypsinon both sides of the monolayer receptor (Casanova et al., 1991; Okamoto et al., 1992) and low or no trypsin at all. This incubation was continued for 1.5 h to allow density lipoprotein receptors (Hunziker et al., 1991; Yokode proteins released from the Golgi to proceed to the cell surface. After et al., 1992). Like all of these proteins, the VSV G protein is this chase, the cells were incubated an additional 15 min a t 37 "C targeted withhighefficiency to the basolateral surface of with media containing 100 rg/ml soybean trypsin inhibitor on b0t.h MDCK cells (Rodriguez-Boulan and Pendergast, 1980; Pfeif- sides of the cell. The cells were cooled on ice in this media and then lysed in 2.5 ml of Nonidet P-40 lysis buffer (50 mM Tris HCI, pH 8.0, fer et al., 1985), contains one cytoplasmic tyrosine, and is internalized through coated pits (Pesonen et al., 1984; Gruen- 1%Nonidet P-40, 0.1% SDS, 0.1 unit aprotinin (Boehringer, Mannheim) containing 100 pg/ml soybean trypsin inhibitor). The lysates berg and Howell, 1987). were scraped from the filters after 15 min and then spun at12,000 X T o investigate the possibility that the basolateral transport g for 30 min to pellet nuclei and cytoskeleton. 1.6 ml of the supernaof G also depends upon a cytoplasmic sequence capable of tant was then transferred to a 5-ml polypropylene centrifuge tube specifying localizationto coated pits, a series of clonal MDCK celllines were preparedthatexpresseither wild type or TABLE I genetically altered VSV G proteins or a variety of chimeric or Measurements of polarity of clonal MDCK cell lines truncated HA proteins. The cytoplasmic domain of VSV G The number of measurements of methionineincorporationare protein was found to contain dominant basolateral sorting shown inparenthesis.Wheremultiplemeasurements were made, information that was critically dependent upon the presence averages are shown. Individual measurements varied less t,han 5% of the unique cytoplasmic tyrosine. However, this sequence from the average. Fraction o f methionine functioned poorly, if at all, for internalization. The signifiProtein transport from Cell lines a. cm2 cance of these findings for apical andbasolateralsorting expressed basolateral surface mechanisms, and for the concept of sorting by "default," are HA C30 517 0.95 (2) discussed. MATERIALSANDMETHODS

Construction of Chimeric and Truncated Genes, Site-directed MLLtagenesis, and Subcloning-Construction of cDNAsencoding the HHG and HHgC chimerahave been previously reported (Lazarovits et al., 1990). T o make theG'""- proteins, stop codonswere introduced by site-directed mutagenesis (ZoHer and Smith, 1987) a t codons 485 and 489 to yield proteins with either 1 or 5cytoplasmicresidues. Gtai"4mhas only one cytoplasmic arginine and G'a'1-488has a cytoplasmic sequence of RVGIH. GY- was constructed by mutating codon 501 (coding for tyrosine) of the G tail to code for serine (Lazarovits, 1988). HA'""- was constructed using the site-directed mutagenesis protocol of Kunkel (1985). Codon 539 was mutated to a stop codon, and codon 538 was simultaneously mutated to code for lysine. These genes were subcloned into the expression vector pCB6 (Brewer and Roth, 1991) under control of the cytomegalovirus immediate-early promoter for expression in MDCK cells, or into an SV40 expression vector (Doyle et al., 1985), for the experiments in CV-1 cells. All subcloning was performed according to established protocols (Sam-

C31

987

0.96 (2)

CA1 C8

1330 437 277

0.97 (1) 0.96 (1) 0.95 (1)

c4

1504 813

0.94 (3) 0.94 ( 3 )

~ ~ h i l -

C8 C13 C40

390 324 357

0.96 (1) 0.92 (1) 0.96 (1)

G

C17 CI8

1025 1410

0.96 (4) 0.95 (3)

GY-

CI2 C17

912 1213 400 505 376

0.96 (3) 0.96 (3) 0.92 (2) 0.94 (2) 0.93 (2)

HHG

C14

HHgC

c1

GtAi1-484

c3

Cxtail-4R8

C28 C18

SignalTargeting Basolateral Dominant

3315

CV-1 cells were maintained in DMEM containing 10% Serum Plus containing1.5 ml of NET/Gel (50 mM Tris-HC1, pH 8.0, 1 mM Cells were infected insuspensionwith EDTA, 150 mM NaC1, 0.25% gelatin,0.05% NonidetP-40, 0.01% (Hasleton,Lenexa,KN). NaN3) + 3% bovine serum albumin (BSA),150 pl of a 10% suspension recombinant SV40 virus stocks (Doyle et al., 1985) containing the of protein A-Sepharose (Pharmacia LKBBiotechnology Inc.) diluted wild type G or GY- gene.The infected cells were plated at subconfluin NET/Gel, and 1 pl of anti-HA rabbit serum. Binding was carried ence on 60-mm tissue culture dishes, and the virus was allowed to out overnight at 4 "C with gentle shaking. The precipitates were multiply for 30 h under normal growth conditions prior to the experiment. The cells were washed once in DMEM lacking methionine washed sequentially in NET/Gel containing 0.5 M NaC1, NET/Gel medium for 15-30 min. The containing 0.1% SDS, and then finally in 10 mM Tris-HC1, pH 8.0, and cysteine and then prestarved in this cells were labeled for 30 min in starvation medium containing 100 interspersed with washes in plain NET/Gel. Precipitates were SUSpended in sample buffer (Laemmli, 1970) and analyzed by electro- pCi/ml Trans"'S-label. The labeled proteins were chased to the surphoresis on 12.5% polyacrylamide gels. The gels were either prepared face with normal DMEM for 1 h a t 37 "C. After cooling on ice, the cells were rinsed three times in ice-cold PBS containing Ca'+ and for fluorography and analyzed by laserscanningdensitometry (Brewer and Roth, 1991) or were fixed (40% methanol:lO% acetic Mg2+ and incubated twice for 30 min each in a solution containing acid50% H20), dried, and analyzed by phosphor imaging (Molecular 0.5 mg/ml NHS-SS-biotin (PierceBiochemical Co.). Unreacted NHSSS-biotin was removed by two washes of DMEM containing1%BSA. Dynamics Model 400E PhosphorImager, Sunnyvale, CA). Control samples were set aside at this step for measurements of the The amountof HA appearing on theapical surface was calculated from paired monolayers as follows. For samples treated with trypsin amount of35S-labeledG protein that had reacted with NHS-SSon either the apical or basolateral side, the percentage of cleaved biotin. These samples were never reduced. A second set of samples protein recovered as HA1 and HA2 was corrected for the level of was retained at 4 "C as controlsfor the extent to which biotin could endogenous cleavage measured in a parallel sample without trypsin. be removed from VSV G proteins at thecell surface. On the remaining This correction assumedthat endogenous cleavage did not occur samples, the temperaturewas raised to 37 "C withwarm DMEM, and preferentially at either the apical or basolateral surface. The amount proteins were allowed to be internalized for intervals of 2,4, 8, 15, or 30 min. After internalization, cells were returned toice and thebiotin of trypsin-specific cleavage at the apical surface was expressed as a remaining on the surface was removed withglutathione reducing percentage of total protein reaching both surfaces during the chase, solution (50 mM glutathione,90 mM NaC1, 1 mMMgC12, 0.1 mM which was the sumof trypsin-accessible HA in each setof monolayers CaC12,60 mM NaOH, 10% fetal bovine serum, pH 8.6) which cleaves that were compared. In some experiments, MDCK cells were infected reducing solution with influenza virus as previously described (Brewer and Roth, 1991) the disulfide bond in the biotinylating reagent. The and were subjected to the same analysis asdescribed for continuous was applied twice for 20 min each, and free glutathione was then MDCK cell lines to determine the polarity of surface arrival of the reacted with 5 mg/ml iodoacetamide in PBS containing 1% BSA. Cells were lysed in 0.6 ml of Nonidet P-40lysis buffer,and thelysates viral HA. As a control for any possible contribution of transcytosis to assays were centrifuged for 10 min a t 12,000 X g. The supernatant from this for delivery of HA to thecell surface, in each experiment an additionalcentrifugation (0.55 ml) was transferred to another tube for immumonolayer was included that had trypsin added to bothsides of the noprecipitation with 0.5 ml NET/Gel, 100 pI of a 10% suspension of filter. Since transcytosed proteins would become exposed to trypsin protein A-Sepharose, and 1 ~1 of anti-VSV serum. Immunoprecipiat both surface domains during transport, such proteins would be tations were incubated overnight a t 4 "C with gentle shaking. The Sepharose pellets were washed as described above and boiled for 10 scored twice in assays in which trypsin was added only to the apical or basolateral sides of paired monolayers. Thus, if transcytosis was min in 20 p1of 10% SDS in H20 to release the bound proteins. A a n important contributor to delivery of HA chimera to the plasma fraction of this supernatant was saved and analyzed by PAGE to membrane, the sum of cleaved proteins in paired monolayers would determine the total amount of labeled VSV G in each lysate. The be greater than that recovered from asinglemonolayer in which remaining supernatant was incubated overnight with avidin-agarose trypsin was added to both sides. However, the percentageof the total to bind biotinylated G, which represented G that had become inacprotein cleaved in the control samples was never less than the sum cessible to glutathione during the incubation of cells a t 37 "C. The calculated by adding percentagescleaved on the apicalside of mono- agarose was collected by centrifugation, washed as above, and analayers to thatcleaved on the basolateral side of the othermonolayers lyzed by PAGE. After fluorography, the proteins were quantified by in apaired set. No significanttranscytosis wasobservedfor any densitometry. protein. For most experiments, duplicate or triplicate samples were taken Antibody capture assaysfor surface arrivalof VSV G proteins were for each intervalof internalization measured. To control for variation performed on MDCK monolayers subjected to an identical protocol in the extentof 35Slabeling among samples, the amount of G protein of prestarving, labeling, and 20 "C treatment as described above. recovered with avidin a t each time pointwas normalized to the total After the 20 "C block, the cells were then shifted to 37 "C for 30, 60, radioactive G recovered from the same sample. The average of these o r 90 min to allow the G proteins to begin to proceed to the surface normalizedvalues were then compared to the averagenormalized in media containing 1%BSA as ablocking agent fornonspecific value for samples that had notbeen treated with glutathione, which binding of antibodies. The cells were then incubatedfor an additional represented the population of G proteinsbiotinylated at the cell interval (usually 1 h) at 37 "C with anti-VSV G serum added to the surface at the start of the experiment. Values for internalization of apical or the basal media. The cells were cooled on ice in the same G proteins were expressed as an initial rateusing the rate of change media to allow antibodies to continue binding to surface proteins for of internalized protein between 0 and 8 min. Since we did not know a n additional 30 min. The monolayers were washed four times with the extent to which recycling protein influenced internalization at cold media containing 1%BSA and lysed in 2.5 ml of Nonidet P-40 later time points, we could not calculate an endocytic rate constant lysis buffer. These lysates were scraped from the filters, centrifuged, from our curves. and immunoprecipitatedwithprotein A-Sepharose as described above. After the overnight binding, the supernatant was then subRESULTS jected to another round of precipitation with additional anti-VSV The testfor a dominant targeting signal requires the transserum to recover VSV G proteins that had not hound antibodies at the cell surface. Both sets of immunoprecipitates were washed and fer of a sequence thought to contain thatsignal to a reporter analyzed by electrophoresis and fluorography or phosphor imaging as protein and the demonstration that the chimeric reporter has described above. The degree to which VSV G proteins were delivered acquired the traffic pattern of the "donor" protein. To deterin a vectorial manner was determined by first calculating for a pair mine whether G protein contains a dominant cytoplasmic of monolayers amount of radioactive G protein which had reached signal specifying delivery to the basolateralsurface, we introeach surface domain, corrected for the difference in totalradioactive G protein between the samples in the pair. The quantity of the duced into MDCK cells DNA encoding a chimericHA, named protein thatwas detected at thebasolateral surfacewas then reported HHG (Lazarovits et al., 1990), in which sequences encoding as a percent of that which had arrived at both cell surfaces during the HA cytoplasmic domain had been replaced with those of the chase. G protein. As a necessary control for distinguishing theeffect Biotin Internalization Assay-VSV G proteins at the cell surface of adding the G sequences from the effect of subtracting HA were biotinylateda t 4 "C througha linkage sensitive to reduction and their rate of internalization was assessed by determining the rate at sequences, we constructed a gene encoding a form of HA that 37 "C a t which the biotinylated proteinsbecame inaccessible to sub- contained only 3 cytoplasmic residues (HAtai"). To control sequent treatment with glutathione at 4 "C. For these experiments, for an effect of replacing HA cytoplasmic sequences that was

3316

Dominant Basolateral Targeting Signal

not specific to the G sequences, DNA was introduced into trypsin on the apical and basolateral surfaces, respectively. MDCK cells that encoded a secondchimericHA, HHgC The ratio of the percent cleaved (a/b) indicated that HAtai” (Lazarovits et al., 1990), in which a sequence encoding the was predominantly (although not exclusively) apical. Lanes c herpes simplexvirus gC cytoplasmic domain replaced the and d are controls in which trypsin was either present on analogous region of HA. Both HHgC and HHG have been both sides of the monolayer ( c ) or not present at all ( d ) . shown to form correctly folded, trimeric proteins at the same Therefore the cleavage in lane c is a measure of total HA’””rate, and to the same extent, as wild-type HA (Lazarovits et arriving at both surfaces, while that in lane d is a measure of al., 1990). Thus, neither protein has a detectable change in endogenous cleavage that occurred in the absence of any the external domain. added trypsin. Lanes e-h are the immunoprecipitatesfrom an TO investigateapossiblerolein sorting for the unique identical experimentperformed with a clonalcell line expresstyrosine 501 of the G protein cytoplasmic domain, the codon ing HHG that demonstrate that this protein was delivered for this residue was changed to specify serine (Lazarovits, predominantly to the basolateral surface. As expected, the 1988). The protein expressed from this mutated cDNA was mobility of the uncleaved protein and of the HA2 tryptic named GY-. It was expected that, if the unique tyrosine was fragment of HHG were lower than those of HAtai” due to the as critically important fora basolateralsortingsignalas larger size of cytoplasmic domain in thechimera. The level of tyrosines have been shown to be important forspecifying cleavage of HHG seen when trypsin was present in theapical localization to coated pits, mutation of the tyrosine would media ( e )was similar to that seen whenno trypsinwas present essentiallyinactivatethe signal. If so, loss of theentire at all ( h )suggesting that this was simply due to endogenous cytoplasmic domainshouldnotfurtherimpairbasolateral proteases. However, the cleavage seen when trypsin was prestargeting. To test. this hypothesis, two truncated genes for G ent on the basolateral side of the cell ( f ) was similar to the protein were also constructed. The cytoplasmic sequences of amount seen when trypsin was present on both sides ( g ) all of these proteins is shown in Fig. 1. For each of these suggesting thatnearly all of the cleavage seenin lane g proteins, several independently derived, clonal, polarized occurred at the basolateral side of the cell. Table I1 presents MDCK cell lines were established (TableI) as described under this data quantitatively for these as well as other clonal cell “MaterialsandMethods,”andthe degree to which each linesexpressing thesameconstructs. Onaverage,95% of protein was transported directly to the apical or basolateral HHG was delivered to the basolateral surface and 76% of surface was determined (Tables I1 and 111).For each protein, HA””- was delivered to theapical surface,demonstrating that the independently derived, clonal cell lines gave essentially the G cytoplasmic sequences had a dominant effect in targetthe same results in all assays conducted, and results from ing HA to the basolateral plasmamembrane. The fractionof several cell lines were pooled and averaged for each of the HAtail- transported apically in these experiments was similar experiments reported below. to thatpreviously reported using this assay for HA transport Addition of the Cytoplasmic Tail of VSV G to a Truncated inMDCK cellsinfected with influenza virus(Matlinand Simons, 1984). Thus, removing the HA tail did not increase H A Results in a Basolaterally Targeted Protein-Measurement of the degree of polarized transport of various forms of the proportionof HA transported basolaterally. Intheseexperiments,trypsin was present continuously HA were made using the assayoriginally described by Matlin and Simons (1984). HAS labeled with a brief pulse of radio- during the 90-min chase a t 37 “C and is capable of cleaving active amino acids were trapped in the trans-Golgi network essentially all of the HA at the cell surface in 2 min (Brewer by a chase at 20 “C and thenreleased for 90 min at 37 “C in and Roth, 1991). Since virtually no cleavage of HHG was medium containing trypsin on one side of cells in tight mono- detected at the apical surface, these experiments eliminate layers, with trypsin inhibitor present on theopposite side. In the possibility that basolateral expression of HHG is influenced by transcytosis from the apical surface. (See“Materials theseexperiments HAS were cleaved by theextracellular trypsin into two disulfide-linked fragments, HA1 and HA2, and Methods” for description of an additional control for that were quantitatively recovered by immunoprecipitation of transcytosis of all chimeric and mutant HA proteins).In cell lysates. HA, HA1,and HA2 were resolved by SDS-PAGE addition, in experiments in which the interval of chase at under reducing conditions and quantified either by phosphor 37 “C was varied between 0 and 90 min, the proportion of imaging or by fluorography and densitometry. Polarity was surface HHG expressed at the basolateral domain and the determined by comparing the extent that trypsin on one side proportion of surface HAtai1-expressed at the apical membrane did not vary, indicating that results after 90 min of of the cell cleaved HA into HA1 and HA2 with the extent that trypsin on the other side of the cell cleaved HA. Fig. 2 chase are not influenced by removal of HHG or HAta”- propresents the results of two such experiments. Lanes a-d are teins from one of the two surface domains and their subsequent transcytosisor delivery to some other cellular compartproteins precipitatedfrom a clonalcell line expressing HA””-. Lanes a and b show proteins from cells that were exposed to ment, such aslysosomes (data not shown).

HA HAha-

ILAIYATVAGSLSLSIMMAGIS

G GYGha4

SSIASFFFIIGLIIGLFL SSIASFFFIIGLIIGLFL SSIASFFFIIGLIIGLFL

G’“ .

SSIASFFFIIGLIIGLFLS V G I E

VGIHLC~QIYl’DIEMNRLGK

VGIHLCIKLKHTKKRQISTDIEMNRLGK

FIG. 1. Carboxyl-terminal sequences for wild-type and mutant proteins are shown in single letter code. In the first set, the complete ectodomain of HA, and in thesecond set, thecomplete ectodomain of G , is joined to thesequences shown. The uertical line separates the transmembrane sequences from those predicted to be in the cytoplasm. The transmembrane-cytoplasmic junction for the HAS is that predicted by a hydropathy algorithm (Kyte andDoolittle, 1982) to be t.he minimum extent of the transmembrane domain.

Dominant SignalTargeting Basolateral TABLE I1 Sorting of HA and chimera HAS were pulse-labeled and chased to the cell surface in medium containing trypsin on either the apical or basolateral sides of the cell. The percent of the protein reaching the apical surface was calculated by dividing the fraction of protein encountering trypsin onthe apical side of the cell by the fraction encountering trypsin present on both sides X 100%. The average of the number of measurements shown, with the standard deviation, is presented. Values from different clonal cell lines were grouped because individual clones yielded similar results in assays for rate, extent, andpolarity of expression.

3317

a conformation necessary for proper targeting to the apical surface. The protein might then be sent to the basolateral surface by default. Such an effect would necessarily be subtle, since the biosynthesis and structureof the HHGchimera has been compared to thatof wild-type HA in a numberof assays and found to be essentially identical (Lazarovitset al., 1990). Nevertheless, MDCK cell lines were constructed expressing a second, well folded chimeric protein, HHgC, containing the lumenal and transmembrane domain ofHA and the cytoplasmic tail of gC (Fig. 1).The 11 amino acids of the gC tail Protein Percent TotalNumber number of of bear no similarityto anyproposed basolateral targetingsignal measurements apical cell lines or known internalization signal and therefore serve as an HA 66 k 11 4 2 appropriate foreign sequence control. HA””76 + 5 5 3 HHgC was sorted inefficiently to the apical surface (65%) HHG 5+4 6 4 but was similar to wild-type HA (66%) in that regard (Table HHgC 65 + 5 7 2 11). Thus, foreign cytoplasmic sequences do not inevitably direct HA efficiently to the basolateral surface. This implies TABLE I11 that in HHG the G cytoplasmic sequences must have played Sorting of G proteins inMDCK cell lines a more specific role. G proteins were pulse-labeled and chased to the cell surface. The Deletion of the Cytoplasmic Tail from the VSV G Protein fraction of radioactive G proteins that had arrived at the apical or Results in Loss of Basolateral Targeting-Since the VSV G basolateral cell surface was measured onseparate samples and protein has been reported to contain sorting information in summed to give the labeled surface population. The percentage of this surface population that was detected at thebasolateral surface is the extracytoplasmic domain, it was of interest to determine which the basolateral signal in the G cytoplasmic shown (see Fig. 3 for an example of this type of experiment). As in the extent to Table 11, the variation reported is thestandard deviation of all domain contributed to the intracellular sorting of G protein measurements. Values from different clonal lines were averaged be- in MDCK cells. The polarity of surface arrival of two truncause individual clones yielded similar results for rate of transport to cated VSV G proteins lacking most or all of their cytoplasmic the cell surface and amount and Dolaritv of Drotein exmessed. ) was assessed by capturing G tails (Fig. 1, Gtail“rla,G”i’-488 Percent Total number of Number of protein with anti-VSV antibody as it arrived at the plasma basolateral measurements cell lines” membrane after a brief pulse and chase protocol similar to G wild type 99 f 1 3 2 that used with HA (see “Materials and Methods”). After an Gmii67 & 7 3 3 interval sufficient for radiolabeled proteins to come into conGY64 f 10 3 2 tact with the antibodies present in either the apical or the ’Of the threecell lines studied for GUiI-,two expressed G”i”484and basal media, the cells were cooled on ice, unbound antibody one expressed Gmii-4w.The value shown is the average for both proteins, because they did not differ significantly in biosynthetic rate was washed away, and thecells were lysed. The protein which bound antibody as a consequence of reaching a cell surface or polarity. exposed to antiserumwas then removed from the cell lysates by precipitation with protein A-Sepharose. The remaining nA‘allnnG samples was recovered by radioactive G proteininthese adding additional anti-VSV serum to the lysate and again apical + + + - + - trypsin precipitating with protein A-Sepharose. The immunoprecipibasolateral - ++ + + Present tates were analyzed by SDS-PAGE and quantified by phosHA phor imaging or fluorography and densitometry (Table 111). HA1 Almost all wild-type G protein that bound antibody at the plasma membrane was detected at the basolateral surface. HA2 Truncation of G resulted in loss of this tight polarity, with only 67% delivered to the basolateral surface. This result indicated that the tail of the protein was necessary for its a b c d e f g h exclusive basolateral targeting. Taken together, the pattern FIG.2. Polarity of arrival of HA””- and HHG at the apical of sorting of G””- and HHGindicated that theG cytoplasmic or basolateral surfaces of MDCK cells. HAS were pulse-labeled and chased for 90 min in the presence of trypsin on oneor both sides sequences were sufficient to specify efficient sorting of a of cell monolayers before immunoprecipitation and analysis by protein into thebasolateral transport pathway and necessary PAGE. When trypsin was applied to a single side of the filter, soybean for the normal sorting of G protein. trypsin inhibitor was always present on the opposite side. The posiRemoval of Y501 from VSV G Also Results in Loss of tions of uncleaved HA, and of the subunits produced by trypsin cleavage, HA1 and HA2, are shown on a typical fluorogram. u and e, Basolateral Targeting-Cytoplasmic tyrosines have been identrypsin was present only in the apical medium; b and /, trypsin was tified as important for some, but not all, basolateral sorting present in the basolateral medium; c and g, trypsin was added to both signals. The cytoplasmic tail ofVSV G contains a single sides of the cells; d and h, no trypsin was added. tyrosine, at position 501. T o determine if this tyrosine contributed to thebasolateral targeting activityof the G tail, the Addition of the Cytoplasmic Tail of Herpes Simplex Glyco- sorting of GY- , a protein in which tyrosine 501 was changed protein gC to the Transmembrane and Lumenal Domains of to a serine, was measured by the antibody capture assay. Fig. H A Does Not Result in Basolateral Targeting of the New 3A shows the amount of wild-type G protein (a-d) and GYChimeric Protein-One explanation for the ability of the G (e-h) that could be captured by anti-VSV antibody at either cytoplasmic sequences to direct HA to thebasolateral surface the apical (a, e) or basolateral (byf ) surface of MDCK cell might bethat anyforeign cytoplasmic sequence would prevent monolayers. As expected, G showed strong basolateral polarthe transmembrane or lumenal domain of HA from adopting ity; however, GY- had apparently lost this tight basal target-

-

-

-

Dominant SignalTargeting Basolateral

3318

the two proteins were expressed using an SV40 virus expres. sion vector in CV-1 cells, and the rates of internalization wert determined during the first 8 min after a shift from 4 to 37 "C G G"The resultsof these experiments are summarized in TableIV aplcal + + + + The initial rate of internalization of wild-type VSV G was - + -+ -+ basolateral - + observed to be5-25-fold slower than the rates common11 7reported for cell surface receptors with cytoplasmic internalG ization signals dependent upon tyrosine. In addition, there was no significant decrease in the rate of VSV G internalization when the cytoplasmic tyrosine was changed to serine. ace bdf g h Since previous experiments measuring internalization of G repreclpltallon protein hadimplanted the protein into theplasma membrane 6 wlth BddlIlOnal antl-G by fusing virions to the cell a t low pH (Matlin et al., 1983; Pesonen et al., 1984; Gruenberg and Howell, 1987), we investigated the possibility that theconformational changeinduced 0 B in G protein a t low pH might activate an internalization signal that is crypticin the pH 7.0 form of the protein. However, the slow rate of internalization of G or GY-was not ac bd e l g h changed when CV-1 cells expressing those proteins were FIG. 3. The effectof mutation of tyrosine501 on the polarized expression of G protein. A, polarity of G and GY- measured subjected to the protocol used previously to fuse vesicular by antibody capture a t MDCK cell surfaces. Proteins were pulse- stomatitis virions to theplasma membrane, an incubation in labeled and chased to the cell surface for 90 min with anti-G serum medium at pH6.3 for 30 min followed bypH 4.9 for 30 s (data present in the medium on either the apical (a and e ) or basolateral not shown). These observations indicate that tyrosine 501 ( b and f ) sides of the monolayer. Cells were then lysed, precipitated does not play an essential role in the internalization of VSV with protein A-Sepharose, and analyzed by gel electrophoresis and fluorography. Lanes c, d, g, and h are controls demonstrating that G, and, in fact, that this protein does not contain a high anti-G serum added to the medium does not adhere to MDCK cell affinity coated pit localization signal at all. Therefore, the surfaces and bind G protein afterlysis. For these samples, a lysate of cytoplasmic basolateral targeting sequence in G protein canuntransfected MDCK cells that had been treated with anti-G serum not also function as anefficient internalization signal. in the apical (c and g) or basolateral ( d and h) medium was mixed Sorting of HA in Continuous MDCK Cell Lines-Reports of with a lysate from cells expressing G protein that had been labeled the apical fraction ofHA expressed transiently in MDCK with 35Samino acids. The mixed lysates were precipitated with protein A-Sepharose. B , controls for variation in levels of G protein expres- cells through infection with influenza virus (Rodriguez-Bousion. Each lysate precipitated with surface-bound anti-G ( A ) was lanand Pendergast, 1980; Misek et al.,1984; Matlin and precipitated again with additional anti-G ( B ) , and the amount re- Simons, 1984) or with vaccinia virus vectors (Stephens etal., covered in both precipitations was usedto determine the totallabeled 1986) have varied from 70 to 90% apical. In our experiments G protein in the sample. investigating the polarity of initial transport of chimeric HAS expressed by permanent cell lines, we observed that apical ing. The polarity of GY- (64% basal, Table 111) is not signifi- delivery ofHA wild-type and chimeras was consistently at cantly different from that ofGmi", suggesting that the se- the low end of the range previously reported for virus-infected quence containing Y501 is essential tothe basolateral cells, but similar to that of Matlin and Simons (1984), who targeting activity present in the G cytoplasmic sequence. used a similar experimental protocol. To determine whether A possible contribution of transcytosis to sorting of G HAS expressed by cell lines were sorted less efficiently than proteins was investigated by comparing the resultsof experi- under conditions of virus infection, where expression levels ments performed as described above in which the intervalof are orders of magnitude higher, we infected our cell lines chase varied from 30 to 60 min. The ratio of apical to baso- expressing the A/Japan HA with X31 influenza virus, which lateral G or GY-did not change over this interval, excluding produces an antigenically distinct HA. The rate and extent of the possibility that a significant fraction of G or GY-arrived X31 HA arriving at theapical or basolateral surfacewas then at its final destination by transcytosis. measured by accessibility to extracellular trypsin using the Y501 Is Not Part of a High Affinity Coated Pit Localization pulse-chase protocol described for experiments with continuSignal-In certain cases, the cytoplasmic feature responsible ous cell lines. The polarity of expression of wild-type A/Japan for efficient localization to coated pits may be very similar or HA in continuous cell lines (66 f 11%apical, Table 11) was identical to thatresponsible for basolateral targeting. VSV G more variable, but notsignificantly different,than expression protein is known to be internalized and hasbeen shown to be localized to coated pits (Matlin et al., 1983). To determine if TABLE IV tyrosine 501 in the G protein is also part of a coated pit Initial rates of internalization of G and G'" localization signal, the initial rates of internalization of wildThe values reported are the averages, with standard deviations, type G and GY- weremeasured in MDCK cells using a biotin from five different experiments inwhich the timecourse of internalinternalization assay (Graeve et al., 1989; see Methods). The ization of G proteins that had been biotinylated at thecell surface at "C wasmeasured for the first 30 min after shift to 37 "C.The initial internalization rates of G and GY-were similar, and very slow, 4rates reported are theslopes of the internalization curves containing in both cell types. However, this slow internalization meant 3 colinear points between 0 and 8 min, a period when recycling that during the period when recycling of the labeled surface proteins could have minimal effect on the internalization rate. population was negligible, only a few percent of the proteins Internalization Number of Protein rate' measurements were internalized, and the signal-to-noise ratio of proteins expressed in MDCK cells was too low to allow us to detect 2.2 f 0.9 5 G wild type 1.9 f 0.7 5 GYsignificant differences in initial internalizationrates of G and GY-. Therefore, since tyrosine-based internalization signals 'Rates are the percent of the biotinylated surface population appear tobe functional in both epithelial cells and fibroblasts, internalized per min. A

preclpltatlm wllh surface-bound anll-G

Dominant Basolateral Targeting Signal

3319

of the X31HA (76 f 9% apical)by viruses infecting the same protein transmembrane and cytoplasmic domains have been reported to be sorted apically in MDCK cells (the cell lines. HlGlprotein, McQueen et al., 1986) or MA104 rhesus monkey DISCUSSION kidney cells (the HGG protein, Roth et al., 1987), in apparent cytoplasmic domain of We present two observations that strongly suggest that the conflict with our observations that the VSV G protein contains a dominant basolateral sorting signal. cytoplasmic tail of the VSV G protein contains basolateral However, it is difficult to compare sorting in different epithesortinginformation. Deletion of the cytoplasmicsequence from G abrogates exclusive basolateral transport, and transfer lial cells, since transport pathways appear to be used differently by cells of different tissueorigin (reviewed in Mostov et of this sequence to a differentprotein,HHG,resultsin efficient basolateral targeting of the resulting chimera. This al., 1992). Although their origin is not exactly known, MA104 effect on the polarityof HHG is due tospecific sequences in cells at least superficially resemble cells of proximal tubule cytoplasmic domain, thatof herpes (Roth et al., 1987), more than they do the distal tubule cells the G tail, as an unrelated simplex glycoprotein gC, does not produce the same effect. thought to be the origin of MDCK cells. Another difficulty Since both the HHG and HHgC proteins have been shown to for comparing the sorting of the HGG protein expressed in be structurally indistinguishable from HA (Lazarovits et al., MA104 cells with that of HHG expressed in MDCK cells is that the former protein was found to be structurally quite 1990), it is highly unlikely that the Gsequences in HHG influence sorting by altering the structure elsewhere in the different, and much less well folded (Lazarovits et al., 1990), subject of this than HA or the HHG protein that is the protein. current report. For instance, greater than 90% of HA and Previous investigations of the location of sorting information in G protein have produced contradictory results. Brown HHG become resistant to degradation by trypsin by 60 min after synthesis, whereas none of HGG becomes trypsin-reand colleagues (1989) have documented the predominantly basolateral transport of a chimeric protein in which the G sistant (Lazarovits et al., 1990).At the cell surface, HGG transmembrane and cytoplasmic sequences were fused to the reacts with both a monoclonal antibody that binds only triexternal domain of an apical glycosylphosphatidylinositol- meric HA and witha rabbit antiserummade against denatured linkedprotein,placentalalkalinephosphatase. An earlier and acylated HA that recognizes only epitopes that are not et al., 1987) exposed on trimeric, cell surface HA wild type (Roth et al., study from thesamelaboratory(Puddington reported that replacementof the G cytoplasmic domain with 1986). Since thechimeric H l G l protein studied by McQueen that of HA resulted in a chimeric protein thatwas essentially and colleagues in MDCK cells was the product of a recombiunpolarized in MDCK cells. These results were interpreted nant cDNA quite similarto the cDNA for HGG, it is possible that theG cytoplasmic sequences inboth proteinswere folded cautiously, since it was not clear to what extent the structures of the chimeric proteins were altered, anda hypothesis current incorrectly and inactivefor sorting. In some proteins the cytoplasmic sequence responsible for at that timeproposed that transport to the basolateral surface occurred by default; therefore, itwas not clear to what extent basolateral targeting may be very similar or identical to that responsible for coated pit localization (Brewerand Roth,1991; the Gcytoplasmicsequences contained basolateral sorting information. However, in view of the results reported here, it Hunziker et al., 1991; Le Bivic et al., 1991).Aromatic residues, in particular tyrosines, have been shown to be critical for is likely that basolateral delivery of the placental alkaline phosphatase-(= chimerainvolved recognition of a sorting sig- many internalization signals and a few basolateral targeting nal in the G cytoplasmic domain, and the loss of polarity signals. The results presented here suggest that the sequence observed for the GHA chimera was due to loss of this signal. surrounding the uniquecytoplasmic tyrosine of G protein, or McQueen and colleagues (1987) andComptonand col- the tyrosine itself, is essential to the basolateral targeting leagues (1989) investigated sorting of rather similar chimeric activity found in that domain,since change of this aminoacid G proteins inwhich thetransmembraneand cytoplasmic to serine reduced the polarity of transport of the resulting removal of the entire sequences of G were replaced with thoseof HA. In bothcases, mutant, GY-, to the same extent as chimeric G proteins were detected predominantlyat the baso- cytoplasmic domain. VSV G protein is known to be internallateral surfaces of MDCK cells, either aftera pulse and chase ized and has been visualized in coated pits (Matlin et al., (Compton et al., 1989) or by iodination of the steady-state 1983). However, the slow rate of internalization observed for surface population (McQueen et al., 1987). We also observed G and GY- indicated that G does not have a high affinity that G protein containing only one or five cytoplasmic amino internalization signal. Under conditions in which G protein acids showed a weak preference (67%) for transport to the was inserted into the plasma membrane by normal cellular basolateral surfaceinotherwise well polarized MDCK cell biosynthesis, G was internalized 5-25-fold more slowly than lines. It is possible that G protein contains information in proteins with well characterized, highly efficient internalizaboth external andcytoplasmic domains thatallow it to inter- tion signals. act with the cellular apparatus responsiblefor basolateral The low internalization rate we observed for wild-type G sorting. However, sequences of theexternaldomainthat was somewhat surprising in light of previous data which influence sorting might do so indirectly, for instance, if they suggested that this protein was internalized quickly (Matlin allow attachment to asecond protein that itself carries a et al., 1983; Pesonen et al., 1984; Gruenberg andHowell, 1987). genuine sortingsignal. VSV G protein functions to attach the Each of these earlier internalization experiments was pervirus to MDCK cells by binding a cellular receptor (as yet formedby binding VSV to the apicalsurface of cells and undefined) that is presentexclusively on the basolateral sur- incubating briefly at a low pH to promotefusion of the viral face (Fuller et al., 1985). One way that external sequences of envelope andplasmamembrane.Thedisappearance of G G protein mightinfluence sorting ofa portion of newly protein from the cell surface was then measured. VSV G has synthesized G protein would be by bindingtothisvirus been shown to undergo a conformational change at low pH receptor in the exocytic pathway prior to reaching the com(Doms et al., 1987); however, we have eliminated thepossibilpartment for sorting the latter to the basolateral surface. ity that this pH-induced conformational change activates an In experiments complementary to those above,chimeric otherwise cryptic internalization signal. It is possible that in VSV G earlier experiments the apparentlyrapid internalization of G proteinscontainingtheHAexternaldomainand

Dominant SignalTargeting Basolateral

3320

was caused by incomplete disaggregation of the viral envelope a large after fusionwith the cell membrane,resultingin increase in avidity for coated pits. The observation that certain proteins exhibita weak preference for transport to either the apical or the basolateral surface has important implications for the concept of unsorted, or “default” transport inMDCK cells. Both wild-type HA, which is 66% apical in continuous MDCK cell lines, and Gtd- protein, which is 65% basolateral in the same cell type, might be arriving at the plasma membrane with little or no interaction with MDCK intracellular sorting mechanisms,or one might be partially sorted, or both might be. Given our current lack of knowledge of sorting mechanisms, it is currently not possible to distinguish weak interactions with the hypothetical cellular sorting apparatus from other kinds of weak interactions, which could be either attractive or repulsive, thatwould serve to bias “unsorted or “default” transport toward one of the two membrane domains. Suchbiased transport might be different for different proteins,based upon their individualchemical properties. Although potentiallyindependent of a specialized sorting machinery, this type of transport would not appear completely unsorted, and conceivably might seem reasonably well polarized. As an example of the problem, we observed that 76% of the HAtai” proteinis expressed at the apical cell surface. Is this due to a “biased” default transport or to weak recognition by cellular components dedicated to sorting apical membrane proteins? Our ability to distinguish “biased” transport from “sorted transport will require identification of molecules responsible for sorting. Until then, the conceptof “default” transport is not very useful. Acknowledgments-The VSV G cDNA was originally provided by Dr. J. K. Rose (Yale University). REFERENCES Bomsel, M., and Mostov, K. (1991) Curr. Opin. Cell Biol. 3, 647-653 Brewer, C. B., and Roth, M. G. (1991) J. Cell Biol. 114,413-421 Brown, D. A., Crise, B., andRose, J. K. (1989) Science 2 4 5 , 1499-1501 Caplan, M., and Matlin, K. S. (1989) Functional Epithelial Cells in Culture (Matlin, K., and Valentich, J., eds) pp.71-127, Alan R. Liss Inc., New York Casanova, J. E., Apodaca, G., and Mostov, K. E. (1991) Cell 66,65-75 Compton, T., Ivanov, I. E. Gottlieb,,T., Rindler,M., Adesnik, M., and Sabatini, D. D. (1989) Proc. Natl. Acad. Scz. U. S. A. 86,4112-4116 Doms, R. W., Keller, D. S., Helenius, A,, and Balch, W. E. (1987) J. Cell Biol. 1 0 6 , 1957-1969 Dovle. C.. Roth M. G.. Sambrook J.. and Gething M:J. (1985) J. Cell Bid. 100, 704-714 Fuller, S. D., Bravo, R., and Simons, K. (1985) EMBO J. 4 , 297-307 Gonzalez, A,, Rizzolo, L., Rindler, M., Adesnick,M.,Sabatini, D. D., and

Gottlieb, T. (1987) Proc. Natl. Acad. Sci. U. S. A. 8 4 , 3738-3742 Gottlieb, T. A., Gonzalez, A,, Rizzolo, L., Rindler, M. J., Adesnik, M., and Sabatini, D. D. (1986) J. Cell Bid. 1 0 2 , 1242-1255 Graeve, L., Drickamer, K., and Rodriguez-Boulan, E. (1989) J. Cell Biol. 1 0 9 , 2809-2816 Greenberg, J., and Howell, K. E. (1987) Proc. Natl. Acad. Sci. U.S.A. 84,57585762 Hopkins, C. R. (1991) Cell 66,827-829 Hunziker, W., Harter, C., Matter, K., and Mellman I. (1991) Cell 66,907-920 Kondor-Koch, C., Bravo, R., Fuller, S., Cutler, D.,’and Garoff, H. (1985) Cell 43,297-306 Kunkel, T.A. (1985) Proc. Natl. Acad. Sci. U. S. A. 8 2 , 488-492 Kyte, J., and Doolittle, R. F. (1982) J. Mol. Biol. 1 5 7 , 105-132 Laemmli, U. K. (1970) Nature 227,680-685 Lazarovits, J. (1988) Mutations That Direct the Influenza VirusHemagglutinin into the Endocytic Pathway. PhD dissertation, University of Texas Southwestern Medical School, Dallas Lazarovits, J., and Roth, M. (1988) Cell 5 3 , 743-752 Lazarovits, J. Shia, S.-P., Ktlstakis, N., Lee, M.-S., Bird, C., and Roth, M. (1990) J. Biol. Chem. 267,4760-4767 Le Bivic, A,, Sambuy, Y., Patzak, A,, Patil,N.,Chao, M., andRodriquezBoulan, E. (1991) J. CellBiol. 115,607-618 Lisanti, M. P., Le Bivic, A,, Sargiacomo, M., and Rodriguez-Boulan, E.(1989a) J. Cell Biol. 1 0 9 , 2117-2127 Lisanti, M. P., Caras, I. W., Davitz, M. A., and Rodriguez-Boulan, E. (1989b) J. Cell Biol. 109, 2145-2156 Matlin, K. S., and Simons, K. (1984) J. Cell Biol. 9 9 , 2131-2139 Matlin, K., Bainton, D. F., Pesonen, M., Louvard, D., Genty, N., and Simons, K. (1983) J. Cell Biol. 97,627-637 McQueen, N. L., Nayak, D. P., Stephens, E. B., and Compans, R. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,9318-9322 McQueen, N. L., Nayak, D. P., Stephens, E. B., and Compans, R. W. (1987) J. Biol. Chem. 2 6 2 , 16233-16240 Misek, D. E., Bard E., and Rodriguez-Boulan,E. (1984) Cell39,537-546 Mostov, K. E., de Bruyn Kops, A,, and Deitcher, D.L. (1986) Cell. 4 7 , 359364 Mostov, K., Apodaca, G., Aroeti, B., and Okamoto, C. (1992) J. Cell Biol. 116, 577-582 ” _

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