The EMBO Journal vol.14 no.11 pp.2424-2435, 1995

Two independent targeting signals in thecytoplasmic domain determine trans-Golgi networklocalization and endosomal trafficking of theproprotein convertase furin

Wolfram Schafer, Annemarie Stroh,Susanne Berghofer, Johanna Seiler,Martin Vey, Marie-Luise Kruse',Horst Franz Kern1,Hans-Dieter Kienk andWolfgang Garten2Institut fur Virologie der Philipps-Universitit Marburg,Robert-Koch-Strasse 17, and 'Institut fur Zytobiologie undZytopathologie der Philipps-Universitat Marburg,Robert-Koch-Strasse 5, 35037 Marburg, Germany

2Corresponding author

Communicated by H.-D.Klenk

Furin, a subtilisin-like eukaryotic endoprotease, isresponsible for proteolytic cleavage of cellular andviral proteins transported via the constitutive secretorypathway. Cleavage occurs at the C-terminus of basicamino acid sequences, such as R-X-K/R-R and R-X-X-R. Furin was found predominantly in the trans-Golginetwork (TGN), but also in clathrin-coated vesiclesdispatched from the TGN, on the plasma membraneas an integral membrane protein and in the mediumas an anchorless enzyme. When furin was vectoriallyexpressed in normal rat kidney (NRK) cells it accumu-lated in the TGN similarly to the endogenous glyco-protein TGN38, often used as a TGN marker protein.The signals determining TGN targeting of furin wereinvestigated by mutational analysis of the cytoplasmictail of furin and by using the hemagglutinin (HA) offowl plague virus, a protein with cell surface destina-tion, as a reporter molecule, in which membraneanchor and cytoplasmic tail were replaced by therespective domains of furin. The membrane-spanningdomain of furin grafted to HA does not localize thechimeric molecule to the TGN, whereas the cytoplasmicdomain does. Results obtained on furin mutants withsubstitutions and deletions of amino acids in the cyto-plasmic tail indicate that wild-type furin is concen-trated in the TGN by a mechanism involving twoindependent targeting signals, which consist of theacidic peptide CPSDSEEDEG783 and the tetrapeptideYKGL765. The acidic signal in the cytoplasmic domainof a HA-furin chimera is necessary and sufficient tolocalize the reporter molecule to the TGN, whereasYKGL is a determinant for targeting to the endosomes.The data support the concept that the acidic signal,which is the dominant one, retains furin in the TGN,whereas the YKGL motif acts as a retrieval signal forfurin that has escaped to the cell surface.Key words: furin/TGN38/TGN targeting signal/trans-Golgi network

IntroductionProteins synthesized in the rough endoplasmic reticulum(ER) of eukaryotic cells use the exocytic pathway fortransport to their final destinations. Proteins lacking specialsorting signals are vectorially transported along the entireroute from the ER through the cis-Golgi network, thecis-, medial- and trans-Golgi, and the trans-Golgi network(TGN) to the plasma membrane (reviewed by Griffithsand Simons, 1986; Pfeffer and Rothman, 1987). Otherproteins have targeting signals for incorporation intospecific organelles of the exocytic pathway. Thus, eachcompartment is equipped with characteristic residentproteins, some of which are enzymes involved in the post-translational modification of the transit proteins. Thetargeting information is either present as a positive sortingsignal, such as the mannose-6-phosphate modificationof glycoproteins destined to lysosomes (reviewed byKomfeld, 1987), or as passive signals, some of which areknown as retention or retrieval signals for soluble ormembrane-bound proteins of the ER (Rothman, 1987;Pelham, 1989; Jackson et al., 1993). Recently, signals forretention of membrane proteins in the Golgi (reviewed byHurtley, 1992) and in the TGN (Luzio et al., 1990; Boset al., 1993; Humphrey et al., 1993; Wong and Hong,1993; Ponnambalam et al., 1994) have also been identified.The Golgi complex plays a crucial role in the processing

and sorting of luminal and membrane-bound proteins, andsome of its main responsibilities are the modifications ofthe oligosaccharide portions of glycoproteins (Griffithsand Simons, 1986; Mellman and Simons, 1992). Hence,the sequential steps of the oligosaccharide synthesis reflectthe topography of the modifying enzymes which reside incertain sections of the Golgi complex. Another post-translational modification in the exocytic pathway of cellsis the conversion of biologically inactive proproteins intobiologically active proteins and peptides that takes placein the TGN or after the exit of proteins from the TGN,but before their delivery to the plasma membrane (Klenket al., 1981; de Curtis and Simons, 1988; Sossin et al.,1990). Many membrane-bound and secretory proteins,such as prohormones, proproteins of neuropeptides, growthfactors, coagulation factors, cell receptors, cell adhesionmolecules and a great number of spike glycoproteins ofenveloped viruses are among these precursor molecules(reviewed by Barr, 1991; Klenk et al., 1993). Most of theviral glycoproteins need to be cleaved, in order to mediatethe fusion of viral envelopes with cellular membranes,which is a prerequisite for infection and may influencethe spreading and pathogenicity of viruses (reviewed byKlenk and Rott, 1988). The C-terminal cleavage at anarginine residue of a dibasic or multibasic site is commonto most of those proproteins. The proteases responsiblefor the cleavage at arginine- and lysine-rich sequences

22©Oxford University Press2424

TGN localization and trafficking of furin

were not well understood until recently. It was known thatthe activating enzyme of the fowl plague virus (FPV)hemagglutinin (HA) is calcium dependent and has aneutral pH optimum (Klenk et al., 1984), and that it canbe inhibited by specific peptidylchloroalkylketones (Gartenet al., 1989). These enzymes are expressed in mammalian,avian and invertebrate cells (Kuroda et al., 1989). Thefirst mammalian enzyme processing proproteins at multi-basic sites was human furin which resembles the Kex2protease of the yeast Saccharomyces cerevisiae activatingsubstrates at paired basic residues, i.e. Arg-Arg or Lys-Arg (Fuller et al., 1989; van de Ven et al., 1990).Subsequently, an increasing number of homologous DNAsequences was found in higher eukaryotic cells, and thenew family of subtilisin-like endoproteases now comprisesthe convertases PCI/PC3, PC2, PC4, PACE 4 and furin(reviewed by Seidah et al., 1991; Steiner et al., 1992), aswell as isoforms of Dfurl and Dfur2 genes (Roebroeket al., 1992) and PC5/PC6 (Lusson et al., 1993; Nakagawaet al., 1993a,b).

Furin selectively cleaves proproteins at the C-terminusof the amino acid sequence R-X-K/R-R (Hosaka et al.,1991; Molloy et al., 1992; Vey et al., 1992) or, lesscommonly, at the motifs R-X-X-R (Molloy et al., 1992) orR-X-X-X-K/R-R (Watanabe et al., 1993). Furin substratesinclude nerve growth factor (Bresnahan et al., 1990), vonWillebrand factor (Wise et al., 1990), insulin receptor(Robertson et al., 1993), anthrax toxin (Klimpel et al.,1992) and several viral glycoproteins, such as HA of avianpathogenic influenza viruses (Stieneke-Grober et al., 1992;Walker et al., 1994), gp 160 of HIV (Hallenberger et al.,1992), fusion (F) protein of Newcastle disease viruses(Gotoh et al., 1992; Moehring et al., 1993), F protein ofhuman parainfluenza virus type 3 (Ortmann et al., 1994),PE2 of Sindbis virus (Moehring et al., 1993) and gBprotein of human cytomegalovirus (Vey et al., 1995).Furin has been assumed to be a processing endoproteaseof the constitutive secretory pathway (reviewed by Barr,1991; Steiner et al., 1992). Immunocytological observa-tions suggested that furin is present mainly in the Golgiregion (Bresnahan et al., 1990; Misumi et al., 1991; Oda,1992). More recently, evidence has been obtained that itresides in the TGN (Bosshart et al., 1994; Molloy et al.,1994;.

In this study, we have analyzed the subcellular localiza-tion of furin and the targeting signals determining itstransport routes. The trafficking signals have beenelucidated by site-directed mutagenesis of furin and byconstructing chimeras of furin and HA. We will show thattwo independent signals exist in the cytoplasmic domainof furin that determine its intracellular localization: anacidic peptide responsible for TGN retention and the motifYKGL that appears to be involved in endosomal retrievalof furin escaped to the cell surface.

ResultsSubcellular compartmentalization of furinFurin has been partially purified from Madin-Darby bovinekidney (MDBK) cells and identified as the activatingendoprotease of FPV HA (Stieneke-Grober et al., 1992),but the endogenous protease is not detectable in thesecells by immunofluorescence or other morphological tech-

Fig. 1. Localization of furin and TGN38 analyzed by confocalimmunofluorescence. NRK cells infected with recombinant vacciniavirus VV:bfur were fixed with paraformaldehyde and permeabilized.Cycloheximide (A-F) and brefeldin A (D, E and F) were added to themedium 2 h and I h, respectively, before cells were fixed forimmunostaining. Furin is shown by labeling with Texas Red (A, D),TGN38 by FITC (B, E) and superimpositions of furin and TGN38 areshown in yellow (C, F). The scale bar represents 10 tm.

niques. To identify the compartment where furin mainlyresides, immunocytological experiments were thereforecarried out on furin expressed from cloned bovine cDNA.Since TGN38 is a well established endogenous markerprotein for the TGN in normal rat kidney (NRK) cells(Luzio et al., 1990), we chose this cell line for our studies.NRK cells were infected with furin recombinant vacciniavirus and analyzed by indirect immunofluorescence 7.5 hafter infection, when the ultrastructure of the Golgi com-plex was still intact. To focus on mature furin, proteinbiosynthesis was inhibited by cycloheximide 2 h beforecells were analyzed. Under these conditions, virtually allnewly synthesized furin should have been chased out ofthe ER. The cells were fixed, permeabilized and preparedfor indirect double immunofluorescence analysis withantibodies specific for furin and TGN38, respectively.Figure 1 shows that furin and TGN38 are concentrated inareas close to the nucleus that are characteristic for the

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W.Schafer et al.

Golgi region. Superimposition of furin (red) and TGN38(green) images revealed that the distribution of furin andTGN38 is very similar, but not totally identical (FigurelA-C). In order to address the question of whether furinis localized throughout the Golgi or in the TGN, we usedthe fungal metabolite brefeldin A (BFA). This drug hasprofound effects on the secretory pathway in mammaliancells in which it induces a rapid redistribution of proteinsof the early and medial Golgi to the ER and a collapse ofthe TGN in the vicinity of the microtubule organizationcenter (Pelham, 1991; Reaves and Banting, 1992). Furinand TGN38 are similarly distributed within NRK cellsafter 1 h of treatment with BFA, and both proteins appearwithin a concentrated patch of immunofluorescence. Thisregion is most likely juxtaposed to the microtubuleorganization center, as shown before with TGN38 byReaves and Banting (1992) (Figure 1D-F). From thesefindings, we conclude that furin is predominantly concen-trated in the TGN and behaves like TGN38 after the BFA-induced collapse of the TGN.

In order to obtain detailed information on furin localiza-tion at the ultrastructural level, we analyzed furin expressedby recombinant vaccinia virus and endogenous TGN38 inNRK cells by pre-embedding immunoperoxidase electronmicroscopy. At 7.5 h after infection with vaccinia virus,the distribution of furin is predominantly seen in thetubulo-vesicular part of the TGN. Figure 2A showsimmunoperoxidase staining for furin restricted to the TGNin several different cross-sections of the Golgi complexthroughout a single cell. Already at low magnification,clathrin-coated buds and clathrin-coated vesicles areprominent within the furin-labeled areas. Figure 2B showsa higher magnification of clathrin-coated vesicles typicalfor the TGN which seem to accumulate furin-specificreaction product. Figure 2C and D show immunoreactiveproduct specific for furin (C) and TGN38 (D) throughoutsingle Golgi stacks. The distribution of TGN38 is markedby peroxidase staining on the trans-most cistemae of theGolgi complex and within a tubular network distal fromthe trans-cisternae most probably representing the TGN(Figure 2D). This pattern was seen in infected and un-infected cells as well (data not shown). The localizationof furin by the same staining method is slightly differentfrom TGN38 distribution with respect to the trans-mostGolgi cistemae, which appear devoid of furin-specificlabel (Figure 2C). Thus, although mostly overlappingwhen analyzed by immunoelectron microscopy as well asimmunofluorescence, the distribution patterns of furin andTGN38 are not completely identical.

Mutations affecting the targeting of furinThe furin-related protease Kex2 was shown to be localizedin cellular organelles equivalent to the TGN of highereukaryotic cells (Redding et al., 1991), and tyrosineresidues in the cytoplasmic domain of Kex2 proved tobe critical for this localization (Wilcox et al., 1992).Furthermore, the correct localization of TGN38 has alsobeen reported to depend upon the cytoplasmic domain(Luzio et al., 1990), in which the peptides SDYQRL orYQRL seem to play a major role (Bos et al., 1993;Humphrey et al., 1993; Wong and Hong, 1993). A similarsequence, YKGL, is present within the cytoplasmic tailof furin. Hence, it was reasonable to assume that the TGN

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Fig. 2. Localization of furin and TGN38 analyzed by immunoelectronmicroscopy. NRK cells were infected with vaccinia virus VV:bfur,fixed, permeabilized, stained by immunoperoxidase reaction andprepared for electron microscopy using the pre-embedding technique.(A) An overview of a single furin-labeled cell at low magnification.Immunostained cross-sections of the TGN and immunostainedclathrin-coated buds are indicated. (B) Furin-labeled clathrin-coatedbuds and furin-labeled clathrin-coated vesicles at X53 000magnification. (C) TGN labeling specific for furin. (D) The trans-cisternae of Golgi and TGN stained for TGN38. Cell compartmentsare marked as follows: Golgi stacks (GC), trans-Golgi network(arrow), clathrin-coated buds (closed triangles), cathrin-coated vesicles(arrowheads) nucleus (n), and vaccinia virus-induced inclusion body(IB). The scale bars represent I tm.

localization of furin might be mediated by this tyrosinesignal. We examined the validity of this concept bygenerating several furin mutants with substitutions ofsingle amino acids, a deletion of several amino acids,truncations of the cytoplasmic amino acid sequences, andadoption of the TGN localization signal of TGN38 asindicated in Figure 3. The constructs were expressedin NRK cells using recombinant vaccinia viruses andpSG5:bfur vectors, and the mutated furin molecules wereanalysed as follows.To examine the intra- and extracellular distribution of

wild-type furin and furin mutants, cells were metabolicallylabeled with [35S]methionine, the intact cells were bio-tinylated and the entire furin of the lysed cells and thecell supematants was immunoprecipitated. The immuno-precipitated furin of the cell lysates was subjected to asecond precipitation with streptavidin-agarose, in orderto separate biotinylated furin at the cell surface from non-biotinylated material. Cellular furin depleted of surface

2426

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TGN localization and trafficking of furin

Mutational analysis of the targeting signals in the cytoplasmic tail of furin

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Fig. 3. Schematic representation of the cytoplasmic domains of wild-type furin and furin mutants. The C-terminal amino acid sequences of wild-typefurin and mutated furin are shown in one letter code. The open bar symbolizes the C-terminal part of the putative membrane anchor. The tyrosine-containing motive is underlined by filled dots, the acidic amino acids within the C-terminal peptide are marked by dashes. Substituted amino acidsare marked by arrowheads. Transport of furin to the Golgi complex and cell surface was detected by immunofluorescence analysis (cf. Figures 5-7).The percentage of furin at the cell surface was determined as the quotient of biotinylated streptavidin-precipitable furin and total cell bound furin.Surface biotinylated furin was compared with mature furin as seen in Figure 4A and B.

furin is shown in Figure 4A, and the biotinylated fractionsrepresenting surface furin are shown in Figure 4B. Usingthis procedure, small amounts of wild-type furin were

detected at the cell surface that were not seen whenintracellular transport of furin was blocked by BFA.These results are compatible with the view that, underphysiological conditions, furin appears at the plasmamembrane, presumably in a transitory fashion. This alsoseems to be the case with the point mutants in which onlytyrosine and leucine residues were replaced. In contrast,the deletion mutants appeared on the cell surface in largeamounts. However, the biotinylation experiments revealedalso that there were still differences among the lattermutants (Figure 3). Interestingly, mutant d766-797 was

present at the surface in lower amounts than mutant d765-797, although both differ from each other only by Leu765.When this leucine was replaced by alanine in the YKGLmotif of the non-truncated cytoplasmic domain, as it isthe case with mutant L765A, furin accumulated mainlyintracellularly with little surface expression. These resultsindicate that the YKGL motif is involved in the intra-

cellular localization, but that additional amino acids inthe C-terminal sequence beyond this motif play a more

important role.We then analyzed secretion of furin (Figure 4C). After

a 2 h chase period, >50% of labeled wild-type furinappeared in the supernatants of cell cultures in solubleform. Secreted furin was not observed when intracellulartransport was blocked by BFA. These results are com-

patible with the concept that furin is converted in theTGN into a soluble form by C-terminal truncation (Veyet al., 1994). Figure 4C shows also that the point mutantswere as efficiently converted into the soluble form andsecreted into the media as was the wild-type. In contrast,the deletion mutants d743-797, d765-797, and d766-797,that were expressed in substantial amounts at the cellsurface, were poorly secreted. The data presented so farshow that cytoplasmic tail mutants, like wild-type furin,are secreted by truncation, unless they accumulate at theplasma membrane in membrane-bound form. It is alsoclear from these data that all mutants analyzed here are

able to pass through the entire exocytic transport route.

2427

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Fig. 4. Distribution of vaccinia virus-expressed wild-type and mutatedfurin in cell culture. NRK cells infected by VV:bfur wild-type andmutants (cf. Figure 3) were labeled with I35Slmethionine 3.5 h post-infection for 3 h, followed by a 2 h chase with non-radioactivemethionine in the presence of cycloheximide. Cells were biotinylatedat the surface and then lysed by RIPA buffer. The whole cellular furinimmunoprecipitated by furin-specific antiserum was subjected to aprecipitation by streptavidin-agarose, and the precipitates were

analyzed by SDS-PAGE. (A) Immunoprecipitates depleted frombiotinylated furin represent intracellular furin. The upper bandsrepresent the transport-competent form, the lower bands the immatureform of furin. (B) Streptavidin-immunoprecipitated fractions representfurin at the cell surface. (C) Soluble furin released from the cells wasimmunoprecipitated from cell supernatants and analyzed by SDS-PAGE. The fluorography of (B) was overexposed (3X relatively to A)to make the wild-type furin visible. Wild-type furin expression in thepresence of brefeldin A is shown as negative control for surfacebiotinylation and furin release.

The cellular distribution of mutant and wild-type furinwas also compared by immunofluorescence microscopyof intact and permeabilized NRK cells infected with thecorresponding recombinant vaccinia viruses (Figure 5).Wild-type furin and mutants with amino acid substitutionsin Tyr753 and/or the YKGL motif show only low or

background signals on the cell surface (Figure 5A, C andE) and the characteristic Golgi immunostaining (Figure SB,D and F). Single tyrosine substitutions in the cytoplasmicdomain showed the same immunofluorescence pattems asmutant Y753/762A (data not shown). The picture changedwhen the cytoplasmic domain was completely eliminated.Furin was distributed all over the cell surface, and thestrict Golgi staining pattern was lost in permeabilizedcells (Figure 5G and H). This indicates that single aminoacid substitutions in the tyrosine motif have no visibleeffect on furin targeting.

Another set of mutants was analyzed in NRK cells byimmunofluorescence experiments using the expressionplasmid pSG5 in combination with the T7 polymerasevaccinia expression system which also revealed the co-

localization of wild-type furin with TGN38 (Figure 6A,B and C). To indicate the TGN localization in eachtransfected cell, double immunofluorescence analyses withantibodies against furin and TGN38 were performed. Furinof mutant d765-797 possessing only the imperfect putativetargeting motif YKG appeared for the most part on the

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Fig. 5. Analysis of TGN targeting signals of furin. NRK cells wereinfected by recombinant vaccinia viruses expressing wild-type furinand furin mutants (A-H) as described in Figure 3 or by the vacciniawild-type (WR strain) as control (I and J). The cells were either leftunpermeabilized for surface staining (A, C, E, G and I) or labeledafter permeabilization with saponin (B, D, F, H and J). Furin wasvisualized by affinity-purified rabbit immunoglobulins against furin-and Texas Red-conjugated anti-rabbit immunoglobulins from donkey.The scale bar represents 20 tm.

cell surface (Figure 6D), as was the case with furin lackingits cytoplasmic tail (Figure 5G). In permeabilized cells,mutated furin was present throughout the whole cell body,mostly in discrete spots which presumably are endosomal/lysosomal structures. Mutant d766-797, containing thecomplete motif YKGL at its C-terminus, was significantlyless expressed at the cell surface compared with mutantd765-797 and was found within the cell, partially withinGolgi/TGN and in spots corresponding to the endosomal/lysosomal system (Figure 6G, H). These observationscorrelate with the results of the biotinylation experiments(Figure 4A). In contrast to the point mutants Y753/762A and L765A shown in Figure 5, single amino acidsubstitutions combined with a tail truncation, as present

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Fig. 6. Analysis of the tyrosine-containing retrieval signal in thecytoplasmic domain of furin. NRK cells were transfected bypSG5:bfur-wt (A, B and C), pSG5:bfur-d765-797 (D, E and F),pSG5:bfur-d766-797 (G, H and I), pSG5:bfur-L765A/d766-797 (J, Kand L) and pSG5:bfur-d763-797/QRL (M, N and 0) subsequent toinfection with 17 polymerase recombinant vaccinia virus. Expressionof furin and TGN38 was studied by immunofluorescence analysis8.5 h after vaccinia infection. Cycloheximide was added 2 h beforefixation of cells. Furin was visualized by Texas Red and TGN38 byFITC. The scale bar represents 20 gm.

in mutant L765A/d766-797, almost completely abolishedTGN localization (Figure 6J, K). Similar results were

obtained when Tyr762 was replaced by alanine (data notshown). Since it was reported that YQRL is necessaryand sufficient for TGN localization of truncated chimericTGN38 (Bos et al., 1993; Humphrey et al., 1993), we

investigated truncated furin with YQRL exchanged forYKGL. Immunofluorescence analysis of mutant d763-797/QRL revealed that furin was not localized at the cellsurface (Figure 6M) but preferentially in the Golgi region(Figure 6N). Since single amino acid exchanges in theYKGL motif of full-length furin have no visible effect onTGN targeting (Figure 5), we addressed the question ofwhether major changes or the complete elimination of thismotif affect the targeting as long as the residual C-terminus is intact. Therefore, we analyzed two mutantswith double amino acid exchanges or elimination ofYKGL, mutant Y762A/L765A and mutant d762-765,respectively, for their TGN localization. Surprisingly, bothmutants are distributed in patterns nearly identical to

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Fig. 7. Analysis of the acidic peptide in the cytoplasmic domain offurin. Furin mutants Y762/L765A (A, B), d762-765 (C, D), d777-797(E, F), d790-797 (G, H) and double deletion mutant AS (I, J) were

expressed in NRK cells. The cells were permeabilized andimmunostained as described in Figure 6. The scale bar represents20 gm.

TGN38 (Figure 7A-D). These observations indicate thatanother domain in the cytoplasmic tail C-terminal ofYKGL is critical for TGN localization. To narrow downthe second targeting signal, additional mutants with C-terminal truncations were analyzed. Mutant d790-797showed the same localization as wild-type furin, whereasmutant d777-797, which had lost the C-terminal part ofthe acidic peptide region, had an altered distribution. Furinwas visible not only in the Golgi, but also all over thecytoplasm in discrete spots (Figure 7E-H). Furin of mutantd777-797 also appeared at the cell surface (data notshown). On the other hand, a double deletion mutant(mutant AS), in which the peptides in positions 762-773and 784-797 were lost, co-localized with TGN38 (Figure71, J). These observations show that the second targetingsignal of furin is located in the peptide 774-784. Thus, it

2429

Surface Permeabilized TGN38

W.Schafer et al.

does not comprise the entire acidic region, but only its C-terminal part.As summarized in Figure 3, the results obtained with

the mutants indicate that furin has two signals in itscytoplasmic domain that are responsible for TGN/endosomal localization: the YKGL motif in position 762-765 and a peptide in position 774-784 that containsclustered acidic residues. For this reason, we will refer tothe latter domain as acidic TGN signal. If both signalsare lost, a large proportion of furin is found at the cellsurface, as is the case with mutants d743-797, d765-797and L765A/d766-797. If only the YKGL motif is present,as is the case with mutants d766-797 and d777-797, thedistribution of furin still differs from wild-type, but is notidentical with that of the mutants d743-797 or d765-797.When the YKGL motif is substituted by YQRL, furinaccumulates almost totally in the TGN, indicating thehigher stringency of the TGN38 signal. When YKGL offull-length furin is altered by single amino acid substitu-tions, as shown with the mutants L765A, Y753/762A andY762/L765A, or when it is completely eliminated, as isthe case with mutant d762-765, there is no effect on TGNlocalization. While mutant d790-797 is localized strictlyin the TGN, mutant d777-797, with the acidic regiondisrupted, is found not only in the TGN but also inendosomes. In addition, mutant AS, with major deletionsin the YKGL- and the C-terminal region but containingthe acidic decapeptide CPSDSEEDEG, is still in the TGN.These data indicate the existence of two independentsignals that determine the localization of furin, theCPSDSEEDEG peptide, that appears to be a specific TGNtargeting signal, and the YKGL motif, that appears to beinvolved in endosomal targeting.

Targeting of influenza HA-furin chimerasAlthough furin mutant d743-797 appears at the plasmamembrane, a fraction is still present within the cell (Figure5G, H). It was therefore of interest to find out whetherthe information for TGN localization resides exclusivelyin the cytoplasmic tail or at least partially also in themembrane-spanning domain. For this purpose, we haveused the influenza virus HA, normally transported to theplasma membrane, as a reporter molecule to which themembrane-spanning or the cytoplasmic domain of furinhave been grafted (Figure 8a). The experiments werecarried out in NRK cells that showed stable expression ofHA-furin chimeras. HA, as expected, showed cell surfaceexpression (Figure 8b, A, B). HFH, the HA constructcontaining the furin membrane anchor and the cytoplasmictail of HA, was present on the plasma membrane andin discrete spots, which were identified as endosomes/lysosomes by fluorescein isothiocyanate (FITC)-dextranuptake, and were distinct from the TGN38 pattern (Figure8b C-F). Using the same plasmid in combination withthe T7 vaccinia transient expression system, a higherproportion of HFH was expressed on the cell surface, andHFH did not co-localize with furin within the NRK cells(data not shown). In contrast, stable expression of theHHF chimera, containing the ecto- and transmembranedomain of influenza HA and the cytoplasmic tail of furin,showed co-localization with TGN38 (Figure 8b G, H).This indicates that the information for TGN targeting isconfined to the cytoplasmic domain of furin. The mem-

brane domain of furin does not contain a specific TGNsignal, but leads to an accumulation of HFH on the cellsurface and in the endosomal/lysosomal system.

Since HA was found to be a suitable reporter moleculefor the TGN targeting function of the cytoplasmic tail offurin, it was also used to further prove the concept thattwo distinct signals are present in this domain. Therefore,chimeras containing the peptide YKGL and the cyto-plasmic portion of the mutant AS with the acidic motif,respectively (Figure 8a) have been analyzed in NRK cellsusing the T7 expression system. Immunofluorescenceanalysis of HA-YKGL showed that the peptide YKGLdirects HA mainly to the endosomes which could belabeled by FITC-dextran (Figure 8b I, J), whereas HA-AS, containing the acidic TGN signal CPSDSEEDEG,co-localized with furin (Figure 8b K, L). It has to benoted, that the furin peptide 743-761, which is also a partof the HA-AS chimera, does not contain a TGN signal,because furin mutants d765-797 and L765A/d766-797containing this sequence have lost the TGN localization(Figure 3). In conclusion, the sequence motif YKGLcontributes to an intracellular localization of HA. Theacidic TGN signal is necessary to localize the influenzaHA in the TGN. These observations confirm the conceptalready derived from the studies on the furin mutants thatthe acidic sequence is the major TGN targeting signal,whereas the YKGL motif has only a supporting function.

DiscussionTrafficking of furinOur data show that furin is transported along the exocyticpathway and accumulates in the TGN. It thus resemblesTGN38 which is often used as a marker protein for thiscompartment. However, when we compared the localiza-tion of both proteins by using immunoperoxidase electronmicroscopy, some differences between furin and TGN38became apparent. Furin was restricted to the TGN, whereasTGN38 was also found in trans-Golgi cistemae, a compart-ment which has been shown to be morphologically distinctfrom the TGN (Griffiths et al., 1989; Rambourg andClermont, 1990; Ladinsky and Howell, 1992).We also present evidence for the export of furin from

the TGN. This is indicated by our immunoelectron micro-scopic data which show that furin is present in clathrin-coated vesicles budding from the TGN. Furthermore, wecould, for the first time, detect membrane-bound furin atthe cell surface directly by biotinylation. The presence offurin at the plasma membrane was also suggested bystudies in which antibody uptake (Molloy et al., 1994)and activation of the anthrax toxin protective antigen(Klimpel et al., 1992) have been analyzed. Finally, dataderived from studies on processing of bacterial toxins(Moehring et al., 1993; Tsuneoka et al., 1993) and oninhibitory effects of chloroquine and ammonium chloride(Chapman and Munro, 1994) suggest that furin is able toenter the endocytotic transport route. Whether transportof furin to the plasma membrane and into endosomes isobligatory before it accumulates in the TGN is not known.In any case, intracellular traffic of furin involves recyclingfrom the plasma membrane and endosomes into the TGNas is also believed to be the case with TGN38 (Ladinski andHowell, 1992; Jones et al., 1993; Luzio and Banting, 1993).

2430

TGN localization and trafficking of furin

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Fig. 8. Analysis of influenza hemagglutinin/furin chimeras.(a) Schemes of the hemagglutinin (HA) of fowl plague virus and ofHA chimeras containing the transmembrane domain of furin (HFH),the cytoplasmic domain of furin (HHF) and partial amino acidsequences derived from the cytoplasmic domain of furin. The aminoacid residues flanking the furin fragments are indicated.(b) Recombinant pSG5 constructs were stably expressed (A-H) ortransiently expressed (I-L) in NRK cells. At 2 h prior to fixation andpermeabilization of the cells, cycloheximide was added to the medium.Immunofluorescence labeling was performed as described in Materialsand methods using an antiserum from rabbit specific for HA visualizedby Texas Red (A, C, G), the monoclonal antibody 2A1 1-H6 specificfor HA visualized by rhodamine (E, I) and by fluorescein (K), theaffinity-purified antibodies specific for TGN38 from guinea pigvisualized by fluorescein (B, D, H), and the affinity-purified antibodiesspecific for furin (L). FITC-dextran specifies the endosomes/lysosomes(F, J). The scale bars represent 20 gm.

Unlike TGN38, furin is present not only in membrane-bound form, but is also secreted as a soluble protein afterproteolytic removal of the membrane anchor in the TGN.Since the information for TGN targeting is located in thecytoplasmic domain of furin, it is clear that the solubleprotein is unable to recycle from the plasma membrane.This observation is compatible with the view thatsolubilization is primarily a mechanism for the down-regulation of intracellular furin (Vey et al., 1994). Thereis a discrepancy in the extent of secreted furin in our

studies and in that by Bosshart and co-workers (Bosshartet al., 1994). The reason for this discrepancy is unknown.One possible explanation for this could be due to the factthat different cell types and different expression systems

were used, which may result in differential levels of furinexpression and/or enzyme activity responsible for thesecretion. Although soluble furin has been found to becatalytically active on artificial substrates (Vidricaire et al.,1993), its biological role as a secretory protease remainsto be elucidated.

TGN targeting signalsWhereas sialyltransferase (Munro, 1991; Wong et al.,1992), galactosyltransferase (Nilsson et al., 1991; Teasdaleet al., 1992), and corona virus M glycoprotein (Machamerand Rose, 1987; Weisz et al., 1993) are localized in theGolgi complex by retention signal information mainlyinherent in their transmembrane domains, the evidence

2431

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obtained from the few proteins analyzed so far indicatesthat the critical information for TGN targeting is at leastpartially in the cytoplasmic domain. Since soluble mutantsof furin lacking the cytoplasmic and transmembranedomains were secreted (Hatsuzawa et al., 1992; Molloyet al., 1992; Rehemtulla et al., 1992), it could be assumedthat the information for TGN localization is present inone or both of these domains. We show now that removalof the cytoplasmic tail alone is sufficient to shift furinaccumulation from the TGN to the plasma membrane.This is clearly indicated by the results obtained with thefurin deletion mutants L765/d766-797, d765-797 andd743-797. In the chimera HHF, on the other hand, thecytoplasmic tail of furin resulted in TGN localization ofthe influenza virus HA. We therefore concluded that theinformation for TGN targeting of furin resides exclusivelyin the cytoplasmic tail. Our data resemble in this respectthese obtained by Bosshart and co-workers (1994),although we never observed the almost exclusive lyso-somal accumulation of tail-less furin reported in that study.The most important result of the present work, however,is the observation that furin has two independent targetingsignals in its cytoplasmic domain, the YKGL765 motif andthe C-terminal acidic peptide CPSDSEEDEG783.

Initial signal information was available from mutatedKex2 protease, which has been shown to be dischargedinto yeast vacuoles when a tyrosine in the cytoplasmicdomain was changed (Fuller et al., 1989; Wilcox et al.,1992). Further information that a structural motif, specifiedby a short linear array of amino acids in the cytoplasmicdomain, is important for TGN residence of highereukaryotic proteins came from investigations of TGN38(Luzio et al., 1990; Bos et al., 1993; Humphrey et al., 1993;Wong and Hong, 1993). Mutated cytoplasmic domains ofTGN38 that contain the sequences YQRL (Bos et al.,1993; Humphrey et al., 1993) or SXYQRL (Wong andHong, 1993) are sufficient to localize TGN38 chimeraproteins in the TGN. The sequence YKGL is conservedin human (van den Ouweland et al., 1990), rat (Misumiet al., 1990), mouse (Hatsuzawa et al., 1990) and bovine(Vey et al., 1994) furin, and is closely related to thepeptide YKGI in Xenopus furin (Korner et al., 1991). Wehave identified here the tyrosine motif as a targeting signalof furin, and its presence supports the concept of furinrecycling between the cell surface and the TGN. Tyrosinemotifs exist in a series of integral membrane proteins,among them receptor molecules that are rapidly internal-ized from clathrin-coated pits of the plasma membranealong the endocytic pathway. Such internalizing signalsare located in the cytoplasmic domain, 20-30 amino acidsapart from the membrane anchor, containing 4-6 aminoacids, frequently an N-terminal tyrosine, an aromatic orbulky hydrophobic residue, located three residues apartand, preferentially, a positively charged amino acidinbetween (reviewed by Trowbridge et al., 1993). Singleamino acid substitutions within the tyrosine-containingmotif and in the surrounding residues influence not onlythe internalization efficiency, but also the correct targeting,as shown for the transferrin receptor, the mannose-6-phosphate (MPR) receptor and the low density lipoproteinreceptor (LDLR) (reviewed by Trowbridge et al., 1993).The modulation of the tyrosine-containing motif might bethe explanation of why YKGL is less efficient than YQRL

(Figure 4). It was interesting to see that the HA-YKGLchimera appeared to be localized predominantly in endo-somes. This observation suggests that furin passes throughendosomes when recycling, and that the YKGL motifcarries the information for this transport step.

The concept that furin has an additional targeting signalis derived from the observation that mutants that stillcontain the YKGL motif, but lack the acidic part of thecytoplasmic tail, accumulate at least partially at the plasmamembrane and in endosomes. Moreover, TGN accumula-tion can occur even in the absence of the YKGL motif,as is the case with mutants d762-765 and AS. Thus, it isclear that both signals act independently of each other.The acidic signal comprising the amino acids betweenCys774 and Gly783 is necessary and sufficient for TGNlocalization. TGN38 does not possess such a negativelycharged peptide within the cytoplasmic domain and thismay therefore account for the differences in localizationof these two TGN proteins, as shown in Figure 2. Littleis known about clustered acidic amino acids used assorting signals. Accumulations of acidic amino acids inthe cytoplasmic domain have also been found with severalinternalized proteins, among them the transferrin receptor(Collawn et al., 1990), the LDLR (Matter et al., 1992),epidermal growth factor receptor (Chen et al., 1989), thelysosomal acid phosphatase (Lehmann et al., 1992) andthe MPR (Schulze-Garg, et al., 1993). Interestingly, theacidic domains of the cytoplasmic tail of the calcium-independent MPR are involved in binding of the Golgi-specific adaptor AP- I which is closely associated with theexit of MPR from the TGN (Le Borgne et al., 1993). Itis therefore tempting to speculate that AP-1 might alsoplay a role in furin targeting.

Evidence for a third type of TGN sorting element wasrecently provided by studies on TGN38. The localizationof TGN38 is mediated by two non-overlapping signals, thetyrosine-containing motif YQRL and an as yet undefinedsignal within the membrane domain (Ponnamballam et al.,1994). Since TGN38 was partly found in trans-Golgicisternae (Figure 2), differences between targeting signalsof furin and TGN38 were not unexpected, and the resultsobtained in the present study on chimeras HHF and HFHexclude, indeed, a TGN targeting signal located in thetransmembrane domain of furin. However, the immuno-fluorescence pattern observed with the chimera HFH(Figure 8b E) is compatible with the existence of anendosomal/lysosomal targeting signal in the transmem-brane domain of furin, as suggested by Bosshart and co-workers (1994).The possible roles of the tyrosine-containing and the

acidic TGN signals and their interactions with the sortingmachinery of the cell remain to be determined. Sincetyrosine-containing signals have been found on manyinternalized proteins, it is reasonable to assume, however,that the YKGL motif is a retrieval signal for furin thathas escaped to the plasma membrane, and that the acidicsequence is responsible for retention of furin in the TGN.

Materials and methodsGeneration and expression of recombinant genesThe bovine furin gene cloned in the recombinant phage M13mpl9:bfur(Vey et al., 1994; nucleotide sequence has been submitted to the EMBL

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TGN localization and trafficking of furin

Data Bank with the accession number X75956) and the HA gene ofFPV influenza virus A/FPV/Rostock/34 (H7NI) cloned in pUC18-HA(Roberts et al., 1993) were used for recombinant DNA techniques andmutagenesis. The plasmid pSG5:bfur was constructed as follows. ASitiaI site was inserted between the EcoRl and BgllI cleavage siteswithin the multiple cloning site of the original vector pSG5 (Greenet al., 1988), in which a DNA fragment comprising the full-lengthbovine furin excised from the recombinant phage M13mpl9:bfur wasinserted. pSG5:HA was constructed by excision of the HA gene frompUC18-HA by HioidIll and insertion of the HA gene into BglII-EcoRI sites of pSG5 using HindIII-BgII and Hinzdll-EcoRI linkers,respectively. Recombinants of the plasmid pSG5 containing the HA-furin chimeras HFH and HHF, respectively, were produced by arecombinant PCR technique (Higuchi et al., 1988) using the pUC18-HA and the M13mpl9:bfur in combination with the oligonucleotidesHHFI -PCR and HHF2-PCR for HHF and with HA 1713/HFH l-PCR andHA- 1317/HFH2-PCR for HFH. The full-length constructs HHF andHFH were obtained as follows: after digestion of the PCR fragmentsand pUC I 8-HA with the restriction endonucleases AspI and HinzdlIl, theDNA fragments were purified by agarose gel electrophoresis. HFHneeded an additional Avill digestion within the gene fragment correspond-ing to the furin-specific transmembrane domain. The tailored hybridPCR fragments and pUC18-HA vector were ligated to the desired HHFand HFH chimeras and cloned in pUC18. HFH and HHF were thentransferred into the BglIl site of pSG5 yielding pSG5:HFH andpSG5:HHF, respectively. By analogy with HHF, the double mutant HA-AS of the HA-furin chimera was constructed by a recombinant PCRtechnique and oligonucleotide-directed mutagenesis. PCR fragments ofHA-furin were obtained from pUC18-HA and M13mpl9:bfur using theoligonucleotides HA/Fl-PCR, HA/F2-PCR, HA-1713R and HA-1317.The full-length HA gene connected to the gene corresponding to thecytoplasmic domain of furin was cloned in pUC 18 and pSG5 as describedfor HHF yielding pUC18-HA/F and pSG5:HA/F. The recombinantDNA of M13mpr19:bfur, pSG5:bfur, pSG5:HA and pSG5:HA/F wasmutagenized by using the oligonucleotides as listed below and thein vitro mutagenesis kit (muta-gene phagmid, Bio-Rad Laboratories,Munich, Germany), based on a method described by Kunkel (1985).The sequences of all the recombinant constructs were confirmed by thedideoxy chain termination method.

For expression, NRK cells were infected with the recombinant vacciniavirus vTF7-3 containing the T7 RNA polymerase gene (Fuerst et al.,1986) and transfected (Bucci et al., 1992) with the pSG5-DNA of thedesired construct. Expression of the constructs was performed withthe vectors pSG5:bfur, pSG5:HFH, pSG5:HHF, pSG5:HA-YKGL andpSG5:HA-AS containing the promotor for T7 RNA polymerase, thefurin gene (wild-type and mutants) and the HA-furin chimeras. Forstable expression, NRK cells were co-transfected with pSG5 constructsand the neomycin resistance-conferring plasmid, pIG- 1, at a ratio of10:1. Geneticin-resistant cell clones were selected by addition of 1.5 mggeneticin per ml medium (Sigma, Deisenhofen, Germany). The selectedcell clones were screened for expression of HA-furin constructs byimmunofluorescence. HA-positive cell clones were subcloned andpropagated as stable expressing cells.

Recombinant vaccinia viruses of wild-type furin and furin mutantswere generated as described before using the pSCl 1 vector (Macketet al., 1984; Chakrabarti et al., 1985; Roberts et al., 1993; Vey et al.,1994). Virus stocks were grown in CV-I cells and diluted in Dulbecco'smodified Eagle medium (DMEM) without fetal calf serum (FCS). NRKcells which were grown in DMEM, supplemented with 0.45% glucose,5%7 FCS and 100 g,g/ml gentamycin, were infected with recombinantvaccinia viruses coding for wild-type furin, VV:bfur, mutated furin orthe VV:HAwt (Roberts et al., 1993), each at a multiplicity of infection(m.o.i.) of 10. The virus inoculum was replaced by DMEM without FCSI h after infection.

Oligonucleotides used for recombinant DNA techniques were asfollows: 5'CTACAAGGGAGCACCCCCTGAA3' (L765A), 5'GGGT-GAAAGTGGCCACCATGGAC3' (Y753A), 5 'CTCATCTCCGCCAA-GGGACTA3' (Y762A), 5'CGCGGCCTCATCTCCCCACCTGAAG-CCTGGCAG3' (d762-765), 5'GGCGAGAGGACCGCCTAAATCAA-AGACCAG3' (d790-797), 5'GAGTGCCCGTCCTAATCAGAGGAG-GAC3' (d777-797), 5'CTCATCTCCGC+CAAGGGAGCACCCCC-TGAA3' (Y762/L765A), 5'TACAAGGGACTATAACCTGAAGCC-TGG3' (d766-797), 5'ATCTCCTACAAGGGAGCATAACCTGAA-GCCTGGCAG3' (L765A/d766-797), 5'CTACAAGGGATAACCCCC-TGAA3' (d765-797), 5 'CAGCTGCGCTAGGGCTTCAGC3 ' (d743-797), 5'CTCATCTCCTACCAAAGACTATAACCTGAAGCCTGGC-AGG3' (d763-797/QRL), 5'ATGGACCGCGGCCTCATCTCCTGC-

CCGTCCGACTCAGAG3' (d762-773) (for mutant AS), 5'GAGG-ACGAGGGCTGAGGCGAGAGGACC3' (d784-797) (for mutant AS),5'AACGGAAACATGCGGTCCACTATTTCTCGCTCGGGCTTCAG-CTTCCGAGGGGTG3' (HA/Fl -PCR), 5'CACCCCTCGGAAGCTG-AAGCCCGAGCGAGAAATAGTGGACCGCATGTTTCCGTT3' (HA/F2-PCR), 5'CTATAAGITTGGAAA3' (HA-YKGL), 5'GTATGGT-CTTACAATGCTGAA3' (HA- 1317), 5'ACGTACAAGCTTAGAT-CTTTATATACAAATAGTGCACCCATGTTTCCGTT3' (HA- 171 3R),5'CTGGTCCTGCAGCTGCGCAAGAACGGAAACATGCGGTCAC-TATTTTATA3' (HFH 1 -PCR), 5' CCGCATGTTTCCGTTCTTGCGC-AGCTGCAGGACCAG3' (HFH2-PCR), 5'CTTGTTTTCATATGTGTG-TCGGGCTTCAGCTTCCGA3' (HHFl -PCR) and 5'TCGGAACTGAA-GCCCGACACACATATGAAAACAAG3' (HHF2-PCR).

Bacterial expression of and raising antisera against thebovine furin subtilisin domain and TGN38For the preparation of antiserum against bovine furin, a cDNA fragmentcoding for amino acids 146-374 of bovine furin was amplified by PCRfrom pBluescript II+/bfur containing the full-length cDNA of bovinefurin (Vey et al., 1994). In order to facilitate the subsequent cloningstep, the oligonucleotides used as PCR primers were flanked with BglIIrestriction sites. The furin DNA PCR fragment was digested with BglII,ligated into the BamHI restriction site of the Escherichia coli expressionvector pQEl 1 (DIAGEN, Hilden, Germany), and the plasmid wastransfected into the Ecoli M 15 [pREP] strain. The furin fragmentcontaining a hexameric histidine tag was purified by affinity chromoto-graphy according to the protocol provided by DIAGEN (Hilden,Germany). The purified furin fragment, as judged from SDS-PAGE, wasdialyzed against PBS and used in 200 ,ug portions for the immunization ofrabbits by four injections at 4-weekly intervals.

For the production of antibodies against TGN38, the ,B-galactosidasefusion protein gene of TGN38 was bacterially expressed (Brake et al.,1990). E.coli cells were lysed with SDS sample buffer. The fusionprotein was separated by SDS-PAGE, stained by Coomassie brilliantblue, excised from the gel, homogenized in PBS and used for immuniza-tion of guinea pigs. The guinea pigs were bled after four injections at2-weekly intervals.The sera were tested by enzyme-linked immunosorbent assay (ELISA),

Western blotting, immunoprecipitation and immunofluorescence. Forimmunofluorescence and immunoelectron microscopy studies, the anti-bodies against the furin subtilisin domain and TGN38 were purifiedfrom the sera by immunoaffinity to furin and TGN38. The subtilisindomain of furin expressed in bacteria and the TGN38-3-galactosidasefusion protein were purified, subjected to SDS-PAGE, electrophoreticallytransferred to nitrocellulose membranes (NC), visualized by PonceauRed staining, and the respective bands excised from the NC. The NCstripes were blocked with 3% low fat milk powder in PBS and incubatedwith a 1:50 dilution of anti-furin serum or anti-TGN38 serum whichhad been deprived of antibodies against P-galactosidase before passingthrough a column containing f-galactosidase (Sigma, Deisenhofen,Germany) immobilized on Affigel 10 (BIORAD, Munich, Germany).After a 3 h incubation at 4°C, the NC stripes were washed extensivelywith PBS and finally with water. The antibodies were eluted from theNC stripes at 4°C with 5 mM glycine-HCl, 500 mM NaCl, at pH 2.8and successively at pH 2.2, and immediately neutralized by addition ofI M Tris-HCl, pH 8.8, to a final concentration of 6 mM. The procedureof incubation and elution was repeated several times. The pooledantibodies were concentrated 1 000-fold in an Amicon ultrafiltrationdevice using a 10 PM30 filter (Amicon Corp., Danvers, MA, USA), andstabilized by addition of bovine serum albumin and NaN3 to finalconcentrations of 4% and 0.02%, respectively.

Radiolabeling, cell surface biotinylation andimmunoprecipitationAt 2.5 h p.i., NRK cells (diameter of the culture dishes 35 mm) werestarved for methionine in modified Eagle's medium (MEM) withoutmethionine for I h, then labeled with 100 lCi [35Sjmethionine (1000 Ci/mmol; Amersham, Braunschweig, Germany) in 0.5 ml methionine-freeMEM. The radioactive label was chased by adding 50 1 MEM containing100 mM methionine for various times as indicated. Cycloheximide(100 ,ug/ml) was added to the chase medium to interrupt protein synthesisin some experiments. For the following steps, the cells, all buffers andreagents were kept on ice until the cells were lysed. After removal ofthe medium, the cell cultures were washed three times with PBScontaining 0.1 mM CaC-2and 1 mM MgCI, (PBS-Ca/Mg). Sulfa-succinimidobiotin (sulfo-NHS-biotin) (Pierce, Ba oud Beijerland,Holland) was freshly dissolved in PBS-Ca/Mg (0.5 mg/ml) and added

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W.Schafer et aL

twice to the cells at 10 min intervals. Residual sulfo-NHS-biotin wasquenched with DMEM supplemented by 5% FCS for 5 min. Cells werewashed three times with PBS-Ca/Mg, lysed with 500 1g of RIPA buffer,pH 7, containing 1% Triton X- 100, 1% deoxycholate, 0.1% SDS, 20 mMTris, 0.15 M NaCl, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride(PMSF), 0.5 ,ug/ml aprotinin and 10 mM iodoacetamide. Cell nucleiwere removed by centrifugation for 20 min at 15 000 g, before thesupernatant was immunoprecipitated by adding 2 ,ul anti-furin serumfrom rabbit (final dilution of 1:500) and 30 gl of a suspension ofprotein A-Sepharose CL-4B (Sigma, Deisenhofen, Germany). Mediumcontaining secreted furin was pre-cleared by centrifugation at 1500 gfor 10 min, diluted 1:2 by RIPA-buffer and immunoprecipitated by anti-furin serum. Immunocomplexes were washed three times with RIPAbuffer and suspended in sample buffer for SDS-PAGE or subjectedto a second precipitation using streptavidin-agarose (Pierce, Ba oudBeijerland, Holland) before SDS-PAGE and fluorography was performed.Quantification of radioactive bands was done by using the Phosphor-Imager (Molecular Dynamics, Krefeld, Germany).

Immunofluorescence microscopyNRK cells grown on coverslips were infected and transfected as describedabove. At 5.5 h p.i., cycloheximide was added to the medium to afinal concentration of 100 gg/ml. In some experiments, BFA (Sigma,Deisenhofen, Germany) was added to the medium 6.5 h p.i. at aconcentration of 5 ,ug/ml. At 7.5 h p.i., cells were washed on ice withPBS supplemented with 0.1 mM CaCl, and 1 mM MgCI2, fixed with4% paraformaldehyde (PFA) in PBS for 20 min, rinsed with PBS andquenched with 50 mM NH4CI in PBS. Antibodies were diluted in PBScontaining 0.1% bovine serum albumin. For surface immunolabeling,the cells were incubated directly with the diluted antibodies. Otherwise,they were permeabilized with 0.2% saponin (Sigma, Deisenhofen,Germany) which was dissolved in PBS. After two rinses in PBS, thecells were incubated with purified antibodies from rabbit specific forfurin (diluted 1:100) and from guinea pig specific for TGN38 (diluted1:50) either with the monoclonal antibody 2A ll-H6 raised against HAIof FPV (diluted 1:100) or antibodies from rabbit specific for FPV(diluted 1:200). The primary antibodies were detected by incubationwith anti-rabbit immunoglobulin (Ig) from donkey conjugated with TexasRed (diluted 1:100) (Amersham, Braunschweig, Germany), rhodamine-conjugated F(ab')2 fragments of anti-mouse IgG from goat (diluted1 :100) (Dianova, Hamburg, Germany), FITC-conjugated anti-mouse IgGfrom goat (diluted 1:100) (Sigma, Deisenhofen, Germany) and FITC-conjugated monoclonal anti-guinea pig antibody (diluted 1:50) (Sigma,Deisenhofen, Germany). Finally, the cells were mounted in Mowiol(Hoechst, Frankfurt, Germany) and 10% 1,4-diazabicyclo[2.2.2]octane(Sigma, Deisenhofen, Germany), and the fluorescence label wasvisualized by either a Zeiss Axiophot microscope equipped with UVoptics or a Compact Confocal Microscope at the EMBL, Heidelberg,Germany. The latter was used at excitation wavelengths of 476 nm forfluorescein and 529 nm for Texas Red fluorescence. Pictures wereindividually recorded, pseudocolored, and superimposed. Images werephotographed on Fujichrome 100 film by a Polaroid Freeze FrameRecorder.

For visualization of the endocytotic pathway. cells were treated with5 mg/ml lysine-fixable FITC-dextran (10 000 mol. wt) (MolecularProbes, Eugene OR, USA) at 37°C in culture medium I h prior to fixation.

Immunoelectron microscopyNRK cells infected with furin recombinant vaccinia virus were rinsedonce with PBS containing CaC12 and MgCI2, at 37°C and fixed for30 min with 4% PFA containing 5% sucrose in PBS at room temperature.Then the cells were saturated with 5% sucrose in PBS containing 50 mMNH4CI for 2X10 min, and permeabilized with 0.2% saponin and 5%sucrose in PBS. After two rinses with PBS, the cells were incubatedovernight at 4°C with either affinity-purified anti-furin antibodies fromrabbits (diluted 1:100) or anti-TGN38 antibodies from guinea pigs(diluted 1:100). Subsequently, the cells were incubated for 2 h at 4°Cwith secondary antibodies labeled with peroxidase. After three washes,the cells were fixed for 30 min with 0.5% glutaraldehyde in PBS andwashed again with PBS. The peroxidase reaction and the furtherprocedures were performed according to Tooze and Hollinshead (1992).

AcknowledgementsWe thank Esther Hedderich and Meike Teuchert for help with theconstruction of pSG5:bfur vectors, Brigitte Agricola for electron micro-

scopy and Volkwin Kramer for photographic assistence. We are alsograteful to Angus Lamond and Karsten Weis at the EMBL, Heidelberg,Germany for help with the confocal fluorescence microscopy. This workwas supported by the Deutsche Forschungsgemeinschaft (SFB 286) andthe Fonds der Chemischen Industrie. Essential parts of this work weredone by W.Schafer in partial fulfilment of the requirements for a Ph.D.degree from the Philipps-Universitat, Marburg, Germany. The data onintracellular localization of furin reported here were presented at theIXth International Congress of Virology, Glasgow, Scotland, 8-13August 1993.

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