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Home , Desmoplakin, Intermediate filament, Plakophilin

Journal of Science 113, 2471-2483 (2000) 2471 Printed in Great Britain © The Company of Biologists Limited 2000 JCS4693

Interaction of with and intermediate filament : an in vitro analysis

Ilse Hofmann1,*, Claudia Mertens1, Monika Brettel1, Volker Nimmrich1,‡, Martina Schnölzer2 and Harald Herrmann1 1Division of /A0100 and 2Protein Analysis Facility/R0800, German Cancer Research Center, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany *Author for correspondence (e-mail: [email protected]) ‡Present address: Suny, Downstate Medical Center, New York, USA

Accepted 19 April; published on WWW 14 June 2000

SUMMARY

Plakophilin 1 and 2 (PKP1, PKP2) are members of the arm- proteins is saturable at an approximately equimolar ratio. repeat family. They are both constitutively In extracts from HaCaT cells, distinct soluble complexes expressed in most cells, in two splice forms containing PKP1a and desmoplakin I (DPI) have been named a and b, and display a remarkable dual location: identified by co-immunoprecipitation and sucrose density they occur in the nuclei of cells and, in epithelial cells, at fractionation. The significance of these interactions of the plasma membrane within the desmosomal plaques. We PKP1a with IF proteins on the one hand and desmoplakin have shown by solid phase-binding assays that both PKP1a on the other is discussed in relation to the fact that PKP1a and PKP2a bind to intermediate filament (IF) proteins, in is not bound – and does not bind – to extended IFs in vivo. particular to (CKs) from epidermal as well as We postulate that (1) effective cellular regulatory simple epithelial cells and, to some extent, to . In mechanisms exist that prevent plakophilins from line with this we show that recombinant PKP1a binds unscheduled IF-binding, and (2) specific desmoplakin strongly to IFs assembled in vitro from CKs 8/18, 5/14, interactions with either PKP1, PKP2 or PKP3, or vimentin or and integrates them into thick (up to combinations thereof, are involved in the selective 120 nm in diameter) IF bundles extending for several µm. recruitment of plakophilins to the desmosomal plaques. The basic amino-terminal, non-arm-repeat domain of PKP1a is necessary and sufficient for this specific interaction as shown by blot overlay and centrifugation Key words: , Desmoplakin, , Intermediate experiments. In particular, the binding of PKP1a to IF filament, , Junction

INTRODUCTION 1994; Schmidt et al., 1997; PKP2: Mertens et al., 1996, 1999; PKP3: Bonné et al., 1999; Schmidt et al., 1999; for a review Among the diverse forms of the plaque-bearing adhering see Hatzfeld, 1999). In addition, some other less-well-studied junctions (for a review, see Schmidt et al., 1994) the proteins are found within the desmosomal plaque (e.g. Tsukita are characterized by their molecular composition and Tsukita, 1985; Schwarz et al., 1990; Hatzfeld and and by their specific anchorage of bundles of intermediate Nachtsheim, 1996; Kowalczyk et al., 1999a). filaments (IFs; Schwarz et al., 1990; Kowalczyk et al., 1999a). The basic protein PKP1a (‘band 6 protein’, Kapprell et al., They represent clusters of isoforms of two types of 1988), known to bind cytokeratins (CKs) in vitro (Kapprell et transmembrane glycoproteins, the (Dsg1-3) and al., 1988; Hatzfeld et al., 1994; Smith and Fuchs, 1998), has (Dsc1-3), both members of the larger family of been identified on the basis of its amino acid (aa) sequence, . In their carboxy-terminal, cytoplasmic domains, together with its splice variant PKP1b, as a member of a large these desmosomal cadherins assemble the common plaque family of proteins characterized by variable numbers of so- protein, (Cowin et al., 1986; Franke et al., 1987a,b, called arm-repeats comprising a motif of a mean number of 42 1989; Fouquet et al., 1992), and a distinct set of desmosomal aa (Schäfer et al., 1993; Hatzfeld et al., 1994; Heid et al., 1994; plaque proteins. These include general desmosomal proteins Schmidt et al., 1994). This arm-repeat motif, first identified in such as desmoplakin I (DPI) and cell type-specific proteins the developmentally defined armadillo of Drosophila such as desmoplakin II (DPII; Franke et al., 1982; Mueller and (Peifer and Wieschaus, 1990; Peifer et al., 1994), has been Franke, 1983; Cowin et al., 1985) as well as members of the found in more than a dozen other junctional plaque and nuclear plakophilin (PKP) subfamily of arm-repeat proteins (PKP1: proteins, including plakoglobin (Franke et al., 1989) and β- Kapprell et al., 1988, 1990; Hatzfeld et al., 1994; Heid et al., (McCrea et al., 1991). 2472 I. Hofmann and others

The plakophilins (PKP1-PKP3) are remarkable as they occur restriction sites and appropriate oligonucleotide pairs to introduce stop constitutively in the nucleoplasm of cells normally forming codons or start codons, respectively. desmosomes, cells induced to form desmosomes and cells cDNAs coding for the complete CK5 and CK14, respectively, were devoid of desmosomes (e.g. Mertens et al., 1996; Schmidt isolated from a human cDNA library (Clontech, Palo Alto, ′ 32 et al., 1997; Bonné et al., 1999). In certain states of CA, USA; 5 -Stretch Plus, HL 1112b) using P-labelled partial differentiation, plakophilins are recruited to the plasma clones of human CK5 and 14 (provided by L. Langbein; Langbein et al., 1993) employing standard procedures (Sambrook et al., 1989). membrane in a cell type-specific manner, targeted to very Both clones were mutagenized to include their start codons as part of specific ensembles of plaque proteins where specific IF unique NdeI sites, and were subsequently cloned into the prokaryotic proteins are inserted. PKP1a, originally isolated from bovine expression vector pET-21b (Novagen, Madison, WI, USA). muzzle as ‘band-6-protein’ under harsh extractive conditions, has been found primarily in desmosomes of Protein purification stratified and complex epithelia (Franke et al., 1983; Kapprell Total human vimentin (Herrmann et al., 1993), the human vimentin et al., 1988, 1990; Hatzfeld et al., 1994; Heid et al., 1994; rod domain (Rogers et al., 1995), mouse desmin (Rogers et al., 1995), Schmidt et al., 1997). By contrast, PKP2, also present in total CK8 and CK18 (Hofmann and Franke, 1997), the CK8 rod and two splice forms designated a and b, is characteristic of the CK18 rod (Bader et al., 1991), all subcloned into the pDS5 desmosomes of one-layered (‘simple’) epithelia and certain plasmid, were introduced into E. coli, strain TG1. Human vimentin, mouse desmin (Li et al., 1994) and the mouse desmin rod domain non-epithelial, desmosome-possessing tissues such as were purified according to Hofmann et al. (1991), and the human myocardium, but has also been localized to certain complex vimentin rod domain according to Herrmann et al. (1996). CK5, and stratified epithelia where it colocalizes with PKP1a and/or CK14, CK8, CK18, CK8 rod and CK18 rod were prepared as PKP3 (e.g. Mertens et al., 1996, 1999). PKP3 has been found described (Coulombe and Fuchs, 1990; Hofmann and Franke, 1997). in desmosomes of both simple and stratified epithelia but not E. coli strain BL21 was transformed with plasmids containing the in hepatocytes and in myocardium (Bonné et al., 1999; cDNA of human PKP1a, various subdomains derived from PKP1a, Schmidt et al., 1999). neurofilament protein NF-L (Heins et al., 1993) and LIII (Stick, DPI and DPII, the latter being a splice 1988), subcloned into the pET-vector system. For the generation of variant of DPI (Green et al., 1990; Virata et al., 1992), together recombinant proteins, transformed bacteria were grown overnight in with , , and bullous pemphigoid 400 ml TB medium at 37°C under rigorous shaking. PKP1a and truncated versions of PKP1a were enriched in body fractions antigen 1 (BPAG1), have been grouped into a distinct protein and purified as described (Hofmann et al., 1991). The inclusion body family, the (Ruhrberg and Watt, 1997). All these fraction was dissolved in 40 ml of column buffer I (8 M urea, 5 mM proteins have been localized, in one or the other cell type, to Tris-HCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, pH 7.5) and certain IFs and their plasma membrane anchorage sites and centrifuged for 90 minutes in a Beckman Ti 50 rotor at 35,000 rpm share a common domain structure, i.e. a central rod domain (Beckman Instruments, München, Germany). The supernatant was flanked by globular amino- and carboxy-terminal domains. directly applied to a 30 ml DEAE-sepharose column in column buffer Given their expression pattern, abundance, and the absence of I. The proteins PKP1a, PKP1a-N268, PKP1a-N353 and PKP1a- a homologous protein in -anchoring cell junctions, DPI N331his were recovered in the flow-through fraction and were directly and DPII have always been prime candidates for a role in the applied to a 15 ml CM-sepharose column equilibrated in column specific connections between desmosomes and IFs. buffer I. Bound protein was eluted in a 50 ml gradient of NaCl (0-0.3 M) with column buffer I. Peak fractions were monitored by SDS- Correspondingly, transient transfection studies using cDNA PAGE and purified protein was pooled and stored at −80°C until use. constructs encoding truncated DPI have suggested that the NF-L bound to the DEAE-sepharose column was eluted in a 50 ml carboxy-terminal desmoplakin domain interacts with IFs gradient of NaCl (0-0.3 M) in column buffer I. It did not, however, (Stappenbeck and Green, 1992; Stappenbeck et al., 1993; bind to CM-sepharose in column buffer I. Therefore fractions eluted Bornslaeger et al., 1996). This concept has been confirmed by from the DEAE-sepharose column were dialzyed into column buffer in vitro studies using CKs and the recombinant carboxy- II (8 M urea, 30 mM sodium formate, 1 mM EDTA, 1 mM EGTA, 1 terminal desmoplakin domain (Kouklis et al., 1994), as well mM DTT, pH 4.0) and subsequently applied to a CM-sepharose as by observations made in yeast two-hybrid experiments column equilibrated in column buffer II. PKP1a-324C and the (Meng et al., 1997). rod were not deposited in bacterial inclusion bodies and were Using blot overlay studies and reconstitution experiments in therefore enriched and purified according to Herrmann et al. (1996) with minor modifications. The S-100 gel filtration column followed vitro, we have systematically examined the direct interaction by concentration on a DEAE sepharose column was omitted and the between PKP1a and IF proteins and have also identified, by protein was directly applied to a CM-sepharose column equilibrated immunoprecipitation of solubilized cellular proteins, in column buffer II in the case of PKP1a-324C or in column buffer I desmoplakin as a PKP1a complex partner. for the myosin rod. Assembly experiments and electron microscopy MATERIALS AND METHODS IF proteins were dialyzed by stepwise lowering of the urea concentration into low salt buffers (for type III IF proteins: 5 mM Tris- Cloning and mutagenesis HCl, pH 8.4; see Hofmann et al., 1991; for CKs: 2 mM Tris-HCl, pH For expression of complete human PKP1a (accession number 9.0; cf. Hofmann and Franke, 1997; Hofmann, 1998). IF assembly Z34974) in Escherichia coli the BsiWI/BamHI 2400 bp fragment was was started by adding the corresponding tenfold concentrated buffer subcloned into the corresponding sites of a derivative of pET-21d, (‘filament buffer’; final concentrations, for type III IF proteins: 50 mM containing a 33-mer oligonucleotide pair coding for the first eleven NaCl, 25 mM Tris-HCl, pH 7.5; for CKs: 10 mM Tris-HCl, pH 7.5). authentic PKP1a amino acids and introducing a BsiWI site, to yield For the assembly of NF-L, LIII and myosin rods, the conditions pET-21d(PKP1a). The constructs of various subdomains, as shown in employed with type III IF proteins were used. PKP1a or mutated Fig. 1, were generated from pET-21d(PKP1a) using endogenous forms of PKP1a were dialyzed in parallel by stepwise lowering of the Plakophilin binding to desmoplakin and intermediate filaments 2473 urea concentration into 10 mM Tris-HCl, pH 7.5, 1 mM DTT, and [35S]methionine, using the reticulocyte lysate system and T3 RNA were then added to soluble forms of IF proteins, either simultaneously polymerase (Promega, Mannheim, Germany). Filters were incubated with filament buffer or 30 minutes after IF assembly start. After either with 35S-labelled CKs or with 35S-labelled full-length PKP1a, addition of PKP1a the mixtures were further incubated for 60 minutes full-length PKP2a or the amino-terminal head domain (aa 1-367) of at room temperature or, in the case of human vimentin and mouse PKP2a. Binding assays with [35S]methionine-labelled PKP2 were desmin, at 37°C. Molar ratios (PKP1a:IF protein) ranging from 1:130 performed according to Merdes et al. (1991). Blots were incubated to 8:1 were examined, at total protein concentrations ranging from 0.1 three times for 10 minutes with washing buffer (20 mM Tris-HCl, pH to 0.2 mg/ml. CK5/14 was dialyzed by stepwise lowering of the urea 7.3, 154 mM NaCl, 0.1% Tween-20). Incubation was performed at concentration into assembly buffer (10 mM Tris-HCl, 1 mM MgCl2, 4°C overnight in ‘gelatin buffer’ (20 mM Tris-HCl, pH 7.3, 154 mM 1 mM DTT, pH 7.25). PKP1a was added either in urea or after dialysis NaCl, 1 mM MgCl2, 1 mM DTT, 0.1% Tween-20, 0.2% (w/v) gelatin into assembly buffer to preformed CK5/14 IFs and incubated for 60 (Merck, Darmstadt, Germany)). After blotting filters were washed minutes at room temperature. four times for 30 minutes each in gelatin buffer, air-dried and Procedures for negative staining as well as for pelleting assembled processed for autoradiography. material and further processing for electron microscopy have been described (Hofmann and Franke, 1997). Specimens were examined Affinity determinations using a Zeiss electron microscope model 10 or 900 (Carl Zeiss, Affinities of IF proteins to immobilized PKP1a were determined by Oberkochen, Germany). surface plasmon resonance detection using a Bialite apparatus (Biacore AB, Freiburg i. Br., Germany; cf. Hofmann and Franke, Centrifugation assays 1997; Hofmann, 1998). Samples of vimentin or desmin rod domains For centrifuation studies, assembly of IF proteins (0.2 mg/ml) was and equimolar mixtures of CK8 rod and CK18 rod were dialyzed into started in Airfuge polyallomer centrifugation tubes (Beckman 2 mM Tris-HCl, pH 9.0, diluted to a concentration of 0.1 mg/ml in Instruments). The molar ratio of PKP1a or mutants thereof to IF ‘running buffer’ (10 mM Hepes, 150 mM NaCl, 1 mM DTT, 0.005% protein ranged from 1:80 to 8:1, as specified in Results. PKP1a or its surfactant P20, pH 7.4) and subsequently injected into the apparatus. truncated forms were added to soluble complexes of IF proteins, either Protein association was monitored at a flow rate of 5 µl/minutes at simultaneously with filament buffer or 30 minutes after initiation of 25°C over a 7 minute period. The surface of the sensor chip was IF assembly, and incubated for 60 minutes. In the case of CK5/14, regenerated between each sample injection by washing with 50 mM PKP1a was either added to urea-solubilized CKs and carried with NaOH. In parallel, a ‘naked’ surface without immobilized PKP1a was them through the various dialysis steps, or dialyzed into low salt used to measure nonspecific binding to the dextran matrix. The PKP1a buffers (see above) and mixed with CK5/14 samples containing binding curves obtained were corrected for nonspecific binding. preformed IFs. Assembled material was pelleted by centrifugation for 15 minutes at 10 psi in the Airfuge, the supernatant was withdrawn, Sucrose gradient centrifugation and the pellet dissolved in an equal volume of SDS sample buffer Cultured human keratinocytes of line HaCaT (Boukamp et al., 1988) containing 6 M urea followed by heating at 95°C for 3 minutes. were grown to confluency in a 10 cm dish. They were rinsed in PBS Protein samples of pellet material and supernatant fractions were buffer and incubated with 1 ml extraction buffer (80 mM KCl, 20 mM analyzed by SDS-PAGE. NaCl, 5 mM EDTA, 1 mM DTT, 250 mM sucrose, 15 mM Hepes, pH 7.5, 0.2% Nonidet-P40) for 5 minutes at 4°C. Cells were then gently Blot binding assay scraped off with a rubber policeman, disrupted by pipetting up and Protein samples separated by SDS-PAGE were transferred to down in a narrow bore pipette and centrifuged at 13,000 g for 10 nitrocellulose. Coupled in vitro transcription-translation of cDNA minutes at 4°C. The supernatant fraction was directly loaded on top clones was performed at 30°C for 90 minutes in the presence of of a 5%-30% (w/v) or 10%-60% (w/v) linear sucrose gradient

arm-Repeats 377 1 837 12 3434 565 6 7 8 9 PKP2a

1 270 727 12 34 56 7 8 9 PKP1a

1 268 PKP1a-N268 1 353 1 2 PKP1a-N353

1 331 1 2 H PKP1a-N331his

324 727 2 3 4 5 6 7 8 9 PKP1a-324C

Fig. 1. Schematic depictions of the structural organization of human plakophilin 2a (PKP2a), plakophilin 1a (PKP1a) and recombinant fragments of PKP1a used in this study: PKP1a-N268 (aa 1-268); PKP1a-N353 (aa 1-353); PKP1a-N331his (aa 1-331 including a C-terminal his-tag); and PKP1a-324C, a carboxy-terminal fragment covering most of the arm-repeat domain (aa 324-727). H, his-tag. The individual arm- repeats are indicated by numbered boxes. 2474 I. Hofmann and others

Immunoblotting for PKP1a was performed with monoclonal antibodies (mAbs) 2D6, 5C2 or 9E7 (Heid et al., 1994). Desmoplakin was detected with desmoplakin- specific rabbit antibodies (Natutec GmbH, Frankfurt, Germany; Arnemann et al., 1993) or a mixture of mAbs specific for DPI and DPII (2.15, 2.17, 2.20; Cowin et al., 1985). Detection of bound horseradish peroxidase-coupled secondary antibodies was performed using the ECL-system (Amersham Pharmacia Biotech).

RESULTS

Solid-phase binding assays The full-length human PKP1a polypeptide and various subdomains (Fig. 1) were synthesized in E. coli, isolated and purified to homogeneity. In order to test if the recombinant PKP1a was able to bind to CKs, as previously shown for native PKP1a (Kapprell et al., 1988; Hatzfeld et al., 1994), blot binding assays were performed (Fig. 2). The immobilized proteins (immunoblots are shown in Fig. 2A) were overlaid with the 35S-labelled epidermal cytokeratins CK5 or CK15. Both cytokeratins bound to full-length PKP1a. Binding was also detected for the amino-terminal half Fig. 2. Reciprocal blot binding assays employing 35S-labelled CKs and (PKP1a-N331his) whereas the carboxy-terminal half plakophilins. (A) Samples of purified PKP1a (lanes 1), PKP1a-N331his (lanes (PKP1a-324C) remained undecorated (Fig. 2Ab,c). 2) and PKP1a-324C (lanes 3) were separated by SDS-PAGE, transferred to 35 The same type of binding was demonstrated for the nitrocellulose and overlaid with mAb 5C2 (a, immunoblot: IB); with S- 35 α labelled CK5 (b, CK5); or with 35S-labelled CK15 (c, CK15). Bound antibodies S-labelled -helical rod domains of CK8 and CK18 were visualized using secondary antibodies in conjunction with the alkaline (data not shown). In the reverse experiment, when phosphatase system. 35S-labelled proteins were detected by autoradiography. cytokeratins were bound to nitrocellulose, both PKP1a (B) Purified recombinant CK5 (lanes 1), CK14 (lanes 2), CK8 (lanes 3), CK18 and PKP2a bound strongly to CK5, CK14, CK8 and (lanes 4) and vimentin (lanes 5) blotted onto nitrocellulose were either stained CK18 and to some extent to vimentin (Fig. 2Bb,c). In with Coomassie Brilliant Blue (a, Coomassie), or overlaid with 35S-labelled addition, the amino-terminal, non-arm-repeat domain PKP1a (b, PKP1a) and with 35S-labelled PKP2a (c, PKP2a), respectively. Bound of PKP2 (aa 1-367), bound to CK8, CK18 and proteins were detected by autoradiography. Relevant molecular mass markers vimentin in a very similar way to that seen with the are indicated at the left side in kDa. complete molecule (data not shown). Hence, both PKP1a and PKP2a interact strongly with various IF proteins, the major binding activity probably residing buffered with 10 mM Tris-HCl, pH 7.5. Centrifugation was performed in the amino-terminal non-arm-repeat domain. in a SW40 rotor (Beckman Instruments) at 35,000 rpm for 19 hours at 4°C. 15 fractions of 0.8 ml each were collected from top to the Electron microscopic studies bottom of the gradient. Marker proteins (bovine serum albumin To study interactions of PKP1a and IFs at the ultrastructural (BSA), catalase, thyroglobulin; all from Sigma, München, Germany) level we performed in vitro reconstitution experiments, were separated on parallel gradients. followed by negative staining of the assembled structures. For Immunoprecipitations IF proteins, especially cytokeratins, pathways for productive refolding from the fully denatured state, as obtained in 9.5 M Protein was extracted from confluent HaCaT cultures in ice-cold immunoprecipiation (IP)-buffer (140 mM NaCl, 5 mM EDTA, 20 mM urea, to assembly-competent soluble complexes, mainly Hepes, pH 7.5, 1% Nonidet-P40) followed by centrifugation at tetramers, have been established and used extensively (see 13,000 g for 10 minutes at 4°C. Samples were cleared by addition of Hofmann and Franke, 1997, and references therein; for a protein A sepharose beads (Amersham Pharmacia Biotech, Freiburg review see Herrmann and Aebi, 1999). We therefore employed i. Br., Germany) for 2 hours on a rotating wheel at 4°C. The beads the same type of dialysis regimen as previously used for were then pelleted, and the supernatants transferred to a tube cytokeratins with PKP1a. When dialyzed from 8 M urea into containing protein A sepharose beads preloaded with guinea pig 10 mM Tris-HCl, pH 7.5, 1 mM DTT, PKP1a was still present antibodies specific for PKP1a (B6-4; Schmidt et al., 1997). After as a monomer, as revealed by sedimentation equilibrium overnight incubation at 4°C on a rotating wheel the protein A experiments with the analytical ultracentrifuge (I. Hofmann, N. sepharose beads were washed four times in ice-cold IP-buffer, then Mücke, J. Reed, H. Herrmann and J. Langowski, unpublished). boiled in sample buffer, processed by SDS-PAGE and stained either with Coomassie Brilliant Blue or blotted to PVDF membranes. As a Moreover, according to circular dichroism data as well as control unrelated guinea pig antibodies were processed in parallel. results obtained by endoproteinase AspN digestion, the Protein bands were excised and digested in the gel strip for peptide recombinant PKP1a evidently folded into a distinct mass fingerprinting by matrix-assisted laser desorption/ionization conformation (data not shown). (MALDI) mass spectrometry as described (cf. Schmelz et al., 1998). We started our analysis with CKs 8 and 18, since for this Plakophilin binding to desmoplakin and intermediate filaments 2475

Fig. 3. Electron microscopic analysis of negatively stained CK8/18 (A-E) and CK5/14 IFs (F) interacting with PKP1a (A-C, F), PKP1a-N268 (D) and PKP1a-324C (E). The recombinant plakophilin derivatives were either incubated with assembled, mature IFs (A,C,D,F) or with soluble CK8/18 complexes (B,E). Molar ratios: 1:1.25 (A-C), 1:1.4 (D), 1:2 (E, F). Note that PKP1a induces the formation of either large, tightly packed or smaller, loosely associated IF bundles (some are indicated by arrows in A and B). Non-bundled IFs are also present protruding out of IF bundles (arrowhead in A). Occasionally, the thick IF bundles form loops (C,F). Bars, 200 nm. 2476 I. Hofmann and others cytokeratin pair the in vitro assembly is well characterized fibrils of laterally aggregated IFs, up to 120 nm in diameter, (Hofmann and Franke, 1997); furthermore, PKP1a and CK8/18 were observed, side by side with normal-looking 8-14 nm IFs. are bona fide interaction partners in vivo, being colocalized in The thick fibrils often folded back on themselves, thereby desmosomes in HaCaT cells and cells of other cultured lines forming loops (Fig. 3C). Even at rather low molar ratios of (Boukamp et al., 1988; Schmidt et al., 1997). PKP1a was added PKP1a to CK8/18, such as 1:30, thick fibrils were observed either to preformed CK8/18 IFs (Fig. 3A) or to precursors of and by increasing the proportion of PKP1a added to CK8/18 CK8/18 IFs simultaneously with the assembly initiation by IFs the frequency of individual IFs was drastically reduced. In rapid mixing with filament buffer (Fig. 3B). In both cases, thick the absence of PKP1a thick fibrils of this kind were not found

Fig. 4. Electron microscopic analysis of structures obtained by interaction of PKP1a-N331his (A-D) or PKP1a (E-G) with IFs assembled from human recombinant vimentin. The N- terminal, non-arm-repeat fragment of PKP1a was reacted in fourfold molar excess (A-D) with mature IFs, resulting in the formation of thick bundles of tightly associated IFs eventually bending back on themselves and forming loops (thick arrow in A). Within individual bundles, single filaments are clearly seen eventually extending as free-running IFs. (B) A ‘blow-up’ of a bundle as it converts into individual IFs. (C) An area of a bundle where individual IFs appear to be surrounded by loosely associated, fibrous material. (D) A fully bundled group of IFs, some of which are still visible through the masses of ‘crosslinking’ protein. (E) When PKP1a was used at lower molar excess (1.3-fold), areas with parallel oriented individual IFs connected by knob-like amorphous material are frequently observed on the grid (arrowheads). (F,G) Ultrathin sections through pelleted vimentin IFs bundled by addition of PKP1a before centrifugation (molar ratio: 1:1.5; the thick bundles, which are sometimes connected by individual IFs (arrows), are indeed formed by and represent laterally associated, regularly oriented groups of individual IFs held together by amorphous material. The number of individual IFs integrated into a single bundle may vary as demonstrated in the cross-section in G. Bars, 100 nm (B-E,G); 200 nm (A,F). Plakophilin binding to desmoplakin and intermediate filaments 2477 in CK8/18 IF assembly experiments (data not shown). In an Since simple epithelial CKs such as CK8 and CK18 attempt to narrow down the IF binding domain of PKP1a, we differ quite significantly from CKs found in epidermis, such employed the amino-terminal, non-arm-repeat domain of as CK5, CK14 and CK15, and since PKP1a colocalizes PKP1a (aa 1-268) as well as slightly longer fragments (see Fig. with CK5/14 in desmosomes of the basal cell layer of 1) in analogous assembly experiments, demonstrating that the epidermis (Heid et al., 1994; Moll et al., 1997), we also first third of PKP1a was as effective in bundling CK8/18 IFs investigated the in vitro interaction of PKP1a with as the whole molecule (Fig. 3D and data not shown). In sharp reconstituted CK5/14 IFs (for assembly of CK5/14, see contrast, a truncated version containing most of the arm-repeat Coulombe and Fuchs, 1990). In both principal types of co- domain, PKP1a-324C (aa 324-727), had only little effect on assembly experiments, addition of PKP1a to assembled IFs the higher order interaction of CK8/18 IFs (Fig. 3E). or presence of PKP1a during dialysis into assembly buffer,

Fig. 5. Sedimentation analysis of PKP1a and deletion mutations thereof with wild-type CK8/18 (CK8wt and CK18wt), α-helical rods of CK8/18 or single CKs. After 60 minutes of incubation, samples were centrifuged at 10 psi for 15 minutes in an Airfuge. Portions of the total sample prior to centrifugation (T), the supernatant (S) and the pelleted fraction (P) were separated by SDS-PAGE and visualized by Coomassie Brilliant Blue staining. (A) Purified CK8/18 (lane 1) was mixed with PKP1a (lane 2) at a molar ratio of 1:1.3 (lane 3). Addition of PKP1a was either to soluble CKs (CKsol, lanes 4, 5) or to preassembled IFs (CK(IF), lanes 8, 9). For controls, PKP1a (−CK, lanes 6, 7) and CK8/18 (−PKP1a, lanes 10, 11) were processed individually (supernatant fractions: lanes 4, 6, 8, 10; pellet fractions: lanes 5, 7, 9, 11). Note that PKP1a on its own remains in the supernatant (lane 6) but is pelleted in the presence of CK8/18 IFs (lanes 5 and 9). (B) The recombinant PKP1a fragments PKP1a-N268 (N268, lanes 1-3) and PKP1a-324C (324C, lanes 4-6) were added to soluble CK8/18 (molar ratio of 1:2) together with IF assembly buffer (total samples: lanes 1, 4; supernatant fractions: lanes 2, 5; pelleted fractions: lanes 3, 6). Note that in contrast to PKP1a- N268, PKP1a-324C does not efficiently cosediment with CK8/18 IFs (compare lanes 5 and 6). (C) Mixing experiments employing PKP1a with complexes made from various combinations of CK8 rod, CK18 rod, CK8 wt and CK18 wt, or with CK8 wt and CK18 wt alone (molar ratios 1:1). IF assembly was started concomitantly by addition of ‘filament buffer’. Note that the rod domain complexes but also both the individual wild-type CKs, CK8 and CK18, are sufficient to mediate the sedimentation of PKP1a. Lanes 1-3, CK8 rod and CK18 rod; lanes 4-6, CK8 wt and CK18 rod; lanes 7-9, CK8 rod and CK18 wt; lanes 10-12, CK8 wt and CK18 wt; lanes 13-15, CK8; lanes 16-18, CK18 (total samples, lanes 1, 4, 7, 10, 13, 16; supernatant fractions, lanes 2, 5, 8, 11, 14, 17; pelleted fractions, lanes 3, 6, 9, 12, 15, 18). Molecular mass standards are indicated at the right in kDa. 2478 I. Hofmann and others

Fig. 6. Stoichiometry and specificity of the PKP1a interaction with various IF proteins. PKP1a was mixed with the various proteins as indicated above the corresponding groups of gel lanes: BSA, bovine serum albumin (molar ratio 1:1); myo-R, a recombinant part of the α-helical myosin rod (molar ratio 3:1); for CK5/14, CK8/18 and Vim (vimentin), respectively, molar ratios of 1:1 and 4:1 were employed as indicated. Portions of the total sample prior to centrifugation (T), the supernatant (S) and pelleted fraction (P) were analyzed by SDS-PAGE (visualised by Coomassie Brilliant Blue staining) following centrifugation in the Airfuge (10 psi, 15 minutes). Although most of the 38 kDa myosin rod fragment is recovered in the pellet fraction only traces of PKP1a cosediment. In contrast, IF proteins pulled down approximately equal molar amounts of PKP1a (fractions labelled P). Unbound PKP1a evidently remained in the supernatant (fractions labelled S). Molecular mass standards are indicated at the right in kDa.

PKP1a induced the formation of thick fibers containing density (Fig. 4G). Thus, a bundle of 100 nm diameter may, numerous individual IFs (Fig. 3F). theoretically, contain up to 100 IFs. The small cross-sectioned The apparently similar type of interaction of two different bundle in the upper left apparently consists of eleven IFs, CK pairs with PKP1a prompted us to investigate if making this estimate indeed realistic. Fibers formed by CK8/18 representatives of the other assembly groups (see Herrmann IFs and PKP1a were processed in parallel and gave principally and Aebi, 2000) would also interact with PKP1a. The solid- the same results (data not shown). phase binding experiments had shown that vimentin bound both PKP1a as well as PKP2a to some extent (see Fig. 2B). In Centrifugation experiments conventional assembly experiments, as introduced above (Fig. The nature of the PKP1a-IF interaction was further examined 3), both PKP1a (data not shown) and the amino-terminal by centrifugation experiments, when PKP1a was added either domain PKP1a-N331his induced the formation of highly to preformed CK8/18 IFs or together with the buffer that ordered fibrils, bundling numerous individual vimentin IFs into initiates IF assembly of CK8/18. The experiments were structures persisting for more than 4 µm in length (Fig. 4A), terminated by centrifugation and the resulting soluble and eventually fusing with another fiber that partially folded back pelletable material was analyzed by SDS-PAGE (Fig. 5). When on itself, thereby forming a loop (thick arrow). Enlargements PKP1a was present in near stochiometric amounts, both the of different types of structures formed by PKP1a-N331his CK8/18 IFs and PKP1a were almost quantitatively pelleted, and vimentin are shown in Fig. 4B-E. In Fig. 4B a bundle irrespective of whether PKP1a was added to preformed IFs or terminates in few individual IFs; Fig. 4C depicts the transition added to the IF assembly mixture. In contrast, PKP1a on its from a tightly packed bundle into a zone of loose association own remained entirely in the supernatant fraction (Fig. 5A). back into a more condensed bundle; Fig. 4D shows the Also, the smallest N-terminal PKP1a fragment (PKP1a-N268) appearance of a ‘normal’ bundle, with the contours of bound strongly to CK8/18 and cosedimented whereas most of individual IFs being visible in the upper middle part among the C-terminal PKP1a fragment (PKP1a-324C) remained in the the ‘cross-bridging’ PKP1-derived material. The type of supernatant (Fig. 5B). Furthermore, in order to identify the IF organization of PKP1a molecules on individual IFs is shown protein domains involved in PKP1a binding, we formed all in Fig. 4E (arrowheads). Amorphous material in the form of possible pair combinations between wild type (wt) CK8, wt knobs with a diameter of 30 nm is frequently seen lying on and CK18, the CK8 rod and CK18 rod domain, and studied their connecting several filaments to loose, parallel running fiber interaction with PKP1a in solution (Fig. 5C). Notably, the arrays. For desmin the addition of PKP1 mainly gave the same heterodimer consisting of both rod domains was sufficient to result (data not shown). Further insight into the thick, densely generate structures that sedimented quantitatively. Finally, we packed fibrils was obtained with ultrathin sections of pelleted investigated if one CK partner alone could bind and mediate material (Fig. 4F). Often, normal-looking IFs protruded out of the cosedimentation of PKP1a. On their own, CK8 as well as these fibers and, on occasion, longitudinally sectioned bundles CK18, like PKP1a, remained in the supernatant fraction (data revealed individual IFs making a connection to the next bundle not shown), but when mixed with PKP1a, both were recovered (arrows). Moreover, the distinct minimal inter-IF spacing, in the pellet together with PKP1a (Fig. 5C). Similarly, two IF typical for vimentin filaments (e.g. Hofmann et al., 1991), was proteins from sequence class IV and V, i.e. the low molecular lost and these bundles appeared to be packed with maximal weight neurofilament triplet protein NF-L and Xenopus lamin Plakophilin binding to desmoplakin and intermediate filaments 2479

rod domains of CK8/18, desmin and vimentin, since we had shown that the CK8/18 rod by themself already interacted strongly with PKP1 (see Fig. 5C). Various amounts of these IF protein fragments were allowed to react and increases in resonance (RU, resonance units) were monitored and compared (Fig. 7). All three proteins bound to the immobilized PKP1a as indicated by the increase of resonance, although with somewhat differing affinities. For the vimentin rod, final RU values of more than 800 were attained. The rod domain of desmin and CK8/18 complexes showed RU values of around 400 and 100, respectively, indicative of a lower relative affinity under these experimental conditions. Binding of either CK8 rod or CK18 rod alone resulted in final RU values comparable to those seen with the mixture of type I and type II CKs (data not shown). In all combinations examined, practially no dissocation was observed (less than 50 RU), and therefore the determination of affinity constants was not possible. Wild-type IF proteins could not be analyzed as they rapidly assembled in the physiological buffer conditions used. Evidently filaments cannot be analyzed in the flow-through system of the biacore Fig. 7. Binding kinetics of recombinant α-helical rod fragments from instrument. various IF proteins to PKP1a as measured by surface plasmon resonance. PKP1a was immobilized to the carboxymethylated layer Immunoisolation of soluble PKP1a-desmoplakin of a CM5 chip and exposed to the rod domains of vimentin (Vim), complexes from cell extracts desmin (Des) and CK8/18 complexes dissolved in running buffer Having shown the direct interaction of PKP1a with IF proteins (100 µg/ml). Measurements were performed at a flow rate of 5 µ  and IFs in vitro, we searched for protein complexes containing l/minute using a Bialite Biosensor. Resonance, measured during PKP1a in vivo. When HaCaT keratinocyte cultures were lysed 500 seconds of exposure, is plotted on the ordinate in arbitrary units (RU). with Nonidet-P40-containing buffers, and the soluble proteins obtained used for immunoprecipitation with PKP1a antibodies, substantial amounts of PKP1a were recovered by LIII, bound quantitatively to PKP1a yielding structures that immunoprecipitation (Fig. 8A, open arrowhead). A Coomassie sedimented in the Airfuge (data not shown). Blue-stained band with less electrophoretic mobility than the Since PKP1a is a basic protein with an isoelectric point of heavy chain myosin of the gel standard (212 kDa) was also 9.25, we examined the possibility that the observed binding of found specifically enriched in these immunoprecipitates (Fig. IF proteins was due merely to an electrostatic interaction. In 8A, filled arrowhead) and was identified by peptide mass centrifugation assays, the soluble, acidic protein bovine serum fingerprinting and immunoblotting as desmoplakin I (DPI). albumin (BSA) did not cosediment with PKP1a but both The amount of DPI clearly exceeded that of DPII as verified proteins remained in the supernatant (Fig. 6). Further support in immunoblots using a serum specific for both proteins (Fig. for the specificity of the interaction between IF proteins and 8B). The Coomassie Blue-stained band comigrating with PKP1a came from experiments employing another coiled-coil myosin was not further characterized as it also showed up in forming protein, the myosin rod domain, known to exhibit the control to some extent. clusters of negative charge on its surface. Most of the myosin Other known desmosomal proteins such as and rod sedimented on its own but leaving, however, PKP1a added plakoglobin did not co-immunoprecipitate with PKP1a. in a several-fold molar excess nearly entirely in the supernatant Neither were CKs found to be enriched in soluble PKP1a (Fig. 6). complexes (data not shown). This, however, is in accordance After establishing these qualitative data, we attempted to with the fact that most of the IF proteins are in an assembled analyze the PKP1a-IF protein interaction quantitatively. In our state within cells and therefore are not present in cell extracts sedimentation experiments the molar ratio of PKP1a to IF that have been cleared by centrifugation. In the reverse proteins was varied from 1:130 to 8:1. However, the amount of experiment, when desmoplakin-specific antibodies were used pelleted PKP1a increased only up to a molar ratio of for immunoprecipitation, PKP1a was co-isolated with approximately 1:1. When present in excess over IFs, surplus desmoplakin, whereas desmoglein (see e.g. Pasdar et al., 1991) PKP1a remained in the supernatant fraction. This saturation and plakoglobin were not (data not shown). effect was obtained for the epidermal CK pair, CK5/14, and To characterize the soluble PKP1a complexes in more detail, the simple epithelial CK pair, CK8/18, as well as for vimentin sucrose density gradient centrifugations of cell extracts was (Fig. 6). performed. PKP1a was found in two peaks. The main portion in 5%-30% sucrose gradients was found in the last fraction Surface plasmon resonance detection (Fig. 9A), a minor one fractionated together with soluble In a first attempt to evaluate the relative affinities of PKP1a to desmoplakin (approx. 8 S). In addition, a band of lower different IF proteins, we applied surface plasmon resonance molecular mass of approximately 50 kDa was visible, located technology with PKP1a immobilized on the sensor surface. We in fractions 4-7. This band was recognized by the PKP1a- decided to investigate the binding properties of the α-helical specific serum and probably corresponds to a degradation 2480 I. Hofmann and others

bundles is somewhat similiar to that of CK bundles (‘macrofibrils’) induced by filaggrin, an upper strata-specific protein of squamous stratified epithelia, notably epidermis (Dale et al., 1978, 1988, 1993, 1997; Steinert et al., 1981; Mack et al., 1993; Parry and Steinert, 1995; see also, however, Weidenthaler et al., 1993; Ishida-Yamamoto et al., 1994). Both PKP1a and filaggrin are basic proteins and bind to the IF rod domain in a protein-specific manner. For filaggrin it has been shown that basic 20-mer peptides consisting of a repeated amino acid motif (SGSR/X, X being a charged residue) interact as efficiently with IFs as does full-length filaggrin. A similar type of repeat motif does not, however, occur in the N-terminal Fig. 8. Immunoprecipitation of PKP1a-containing complexes present in detergent extracts from cultured human keratinocytes of line non-arm-repeat domain of PKP1a. Currently we are examining HaCaT. (A) Co-immunoprecipitation of PKP1a with desmoplakin. whether a basic 21-aa stretch in the plakophilin N terminus, Proteins of different fractions were separated by SDS-PAGE and found in PKP1a, PKP2 and PKP3 (cf. Schmidt et al., 1999), is stained with Coomassie Blue. Lane 1, reaction with an unrelated involved in IF interaction. The question of how PKP1a induces guinea pig serum, used as a negative control; lane 2, precipitates IFs to bundle cannot be unequivocally answered with our obtained with guinea pig antibodies specific for PKP1a (serum B6- current knowledge. It is clear from sedimentation equilibrium 4), without addition of Nonidet-P40-soluble HaCaT proteins as experiments that PKP1a on its own is monomeric in solution serum control; lane 3, immunoprecipitates obtained with serum B6-4 (I. Hofmann, N. Mücke, J. Reed, H. Herrmann and J. from Nonidet-P40-soluble HaCaT cell fractions. The open arrowhead Langowski, unpublished). This does not necessarily imply that indicates PKP1a, the filled arrowhead, DPI, identified by mass within the PKP1a head domain two binding sites are located. spectroscopy fingerprinting. Lane R, reference proteins (from top to bottom, in kDa): 212, 158, 116, 97, 66, 55, 42, 36. (B) Immunoblot As has been shown for a filaggrin-derived peptide, short basic analysis of the different protein fractions employing PKP1a-specific peptides derived from PKP1a may function as an ionic zipper mAb 2D6. Lane 1, HaCaT cell lysate prior to immunoprecipitation; and may be able to bundle IFs. On the other hand, binding of lane 2, immunoprecipitate obtained with antibodies B6-4 specific for PKP1a to IFs might induce intramolecular, conformational PKP1a; lane 3, precipitate obtained with unrelated serum. The changes such that PKP1a oligomerizes, forming a glue intense reactive bands in lanes 1 and 2 contain PKP1a. (B′) embedded in IFs. Immunoblot analysis corresponding to B using antibodies specific Our finding that the rod domain of type III IFs or CK8/18 is for desmoplakin. The reactive bands in lines 1 and 2 represent capable for PKP1a interaction and sufficient to mediate bundle desmoplakins DPI and DPII (the latter is only a minor component in formation, seems to be at variance with results reported for the lane 2). The slight differences in molecular mass (compare lanes 1 epidermal CK5 by Smith and Fuchs (1998). Using blot and 2) are most probably due to differences in sample composition α (total cell lysate versus immunoprecipitate). overlays they identified a sequence motif in the non- -helical head domain of CK5 as responsible for PKP1a binding. Clarification of this apparent difference will have to await product of PKP1a. In parallel experiments with recombinant future experiments. However, we do not exclude the existence PKP1a, the purified protein sedimented at 4.3 S (data not of other interaction sites outside the α-helical rod domain. shown). In order to investigate more closely the composition In addition to PKP1, desmoplakin is so far the only other of the fast-sedimenting PKP1a-containing material, we desmosomal protein reported to bind to IF proteins. In transient analyzed corresponding cell extracts on 10%-60% sucrose transfection studies using simple epithelial cells or fibroblasts, gradients (Fig. 9B,B′). The main portion of PKP1-containing an N-terminally truncated DPI-construct colocalized with IF complexes was found in a distinct peak of around 50 S. This fibrils and resulted in a gradual disruption of the endogenous fraction did not contain any of the other known desmosomal IF network (Stappenbeck and Green, 1992). In further proteins. In addition, PKP1a complexes with S-values of analyses, Stappenbeck et al. (1993) pointed to the requirement approximately 8 cosedimenting with desmoplakin were of the last 68 aa of desmoplakin for its binding to IF proteins, detected (cf. Duden and Franke, 1988; Pasdar and Nelson, a region known to harbour a repeated amino acid motif (GSRS; 1988). Further characterization of the high molecular mass cf. Franke et al., 1989; Green et al., 1990) resembling the complex of PKP1a is in progress. filaggrin motif mentioned above. For desmosome assembly not only IF-binding plaque proteins are needed but also the transmembrane cadherins, DISCUSSION desmoglein and . It has been repeatedly demonstrated that the cytoplasmic portions of both In the present study we have investigated the interaction of desmosomal cadherins can bind to plakoglobin (Troyanovsky PKP1a with various IF proteins by a series of different in vitro et al., 1993, 1994a,b; Mathur et al., 1994; Chitaev et al., 1996; assays. We show that this interaction is specific for IF proteins. Wahl et al., 1996; Witcher et al., 1996). Observations made Moreover, the PKP1a head domain alone, i.e. the N-terminal with DP null mouse embryos suggest that DP is important not non-arm-repeat domain (aa 1-268), is sufficient to mediate only in establishing IF cytoskeletal architecture but also for bundling of IFs. With respect to the IF proteins, their α-helical assembly and stabilization of the desmosome (Gallicano et al., rod domain enables the formation of macromolecular 1998). Moreover, Smith and Fuchs (1998) have recently structures with PKP1a. reported, mostly in blot overlay experiments, in vitro The electron microscopic appearance of PKP1a-induced IF interactions of PKP1a with major desmosomal components, Plakophilin binding to desmoplakin and intermediate filaments 2481 i.e. desmoglein, desmocollin and desmoplakin. Given their On the other hand it should not be overlooked that in vivo a close amino acid , PKP2 and PKP3 would decoration of IFs with PKP1a, or any other plakophilin, is not be expected to display similar characteristics of protein observed (Kapprell et al., 1988; Schmidt et al., 1994, 1997, interactions but this needs to be further studied. 1999; Mertens et al., 1996). Only recently the decoration of IFs In search of PKP1a complexes in vivo we have identified, in with fragments of PKP1a has been reported to occur in some immunoprecipitation experiments, soluble or solubilized transfected cells (Klymkowsky, 1999). This suggests that, in cytoplasmic DPI as a specifically interacting partner. We tend normal living cells, the association of PKP1a with IFs and with to conclude that this is a genuine cytoplasmic complex, DPI is tightly regulated to prevent plakophilins from binding although so far we cannot rule out that part of this complex has to IFs and PKP1a-mediated IF bundling. Of course, PKP1a – been extracted from desmosomes. Both proteins cofractionate or other plakophilins – might bind to IF protein configurations during sucrose density centrifugation at approximately 8 S, (‘roots’) deep in the desmosomal plaque where plakophilins whereas the purified proteins reveal lower S-values. have been localized, at least in certain cell types, relatively Recombinant PKP1a sediments at 4.3 S, and purified DPI has close to the membrane (e.g. Kapprell et al., 1990; Mertens et been reported at 6.7 S (O’Keefe et al., 1989). Desmoplakin al., 1996; North et al., 1999; Schmidt et al., 1999). Obviously, from cultured epithelial cells grown in low calcium medium the regulatory mechanisms governing plakophilin-IF protein has been shown to occur in soluble complexes of 7.3-9 S interactions in vivo will have to be elucidated in future (Duden and Franke, 1988; Pasdar and Nelson, 1988), in fair experiments. agreement with our observation. A direct PKP1a-desmoplakin Finally as most, if not all, cells contain plakophilins, usually interaction is also suggested from results obtained with the in the nucleus, we still have to find the regulatory principles yeast two-hybrid system and from transient transfection for recruiting the specific plakophilins to, or excluding them experiments (Kowalczyk et al., 1999b). Obviously, the PKP1a- from, the desmosomes of a given cell type. Site-specific desmoplakin complex shown here is different from the of desmoplakin, for example, has been shown large vesicle-associated structures containing desmoplakin, to regulate its interaction with IFs (Stappenbeck et al., 1994). desmoglein and plakoglobin isolated with vesicle fractionation However, other regulatory principles, including additional techniques (Demlehner et al., 1995). The concept that the interaction of PKP1a with IFs as well post-translational modifications, should not be excluded a as with desmoplakin is important in vivo receives further priori as determinants in the observed topological sorting. support from reports that mutations in both alleles of the We thank Reimer Stick and Lutz Langbein for helpful discussions, PKP1a gene result in a form of congenital Jutta Osterholt for skillful photographic work and Eva Ouis for (McGrath et al., 1997, 1999). In the epidermis of these patients, arranging the manuscript. Moreover, we gratefully acknowledge the the association between IFs and desmosomes, specifically technical assistance of Sonja Reidenbach. Alfred Wittinghofer desmoplakin, appears disturbed although plakoglobin, provided the prokaryotic expression plasmid encoding the myosin-rod desmogleins and desmocollins are still localized to fragment and Ansgar Schmidt helped with the design of the PKP1a desmosome-like sites at cell borders. constructs. W. W. Franke is thanked for his generous support and

Fig. 9. Extracts of Nonidet-P40-soluble proteins from HaCaT cells were analyzed by sucrose gradient centrifugation (A,A′: 5%-30% sucrose; B,B′: 10%-60% sucrose), fractions were divided into portions from top to bottom and the portions analyzed by immunoblotting either with PKP1a antibodies B6-4 (A,B) or with desmoplakin-specific antibodies (A′,B′). The reference proteins were BSA (4.3 S; found in fractions 4 and 5 in A,A′) and catalase (11.3 S; found in fractions 8 and 9 in A,A′). Lane S, sample of total Nonidet-P40-soluble proteins from HaCaT cells. Note that PKP1a and desmoplakin cofractionate at approx. 8 S (fractions 6 and 7 in A,A′; fractions 4 and 5 in B,B′). The main portion of PKP1a was recovered in fractions corresponding to complexes with S-values of roughly 50. 2482 I. Hofmann and others critical comments. The work has been supported by the Deutsche Schiller, D. L. and Cowin P. (1989). 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