The 70-kDa Heat Shock Proteins Associate with Glandular Intermediate Filaments in an ATP-dependent Manner

Abstract

Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins expressed preferentially in glandular epithelia. We describe the identification, by co-immunoprecipitation from normal human colonic tissues and cultured cell lines, of the 70-kDa heat shock protein (hsp) and its related heat shock cognate protein as K8/18-associated proteins (hsp/c). The association is significant but sub-stoichiometric and occurs preferentially with the soluble rather than the cytoskeletal K8/18 fractions. Heat stress increases the level of soluble K8/18 in association with an increase in hsp70 levels and an increase in the stoichiometry of K8/18-hsp70 association. Identity of the associated proteins was confirmed by microsequencing of a tryptic digest of the purified associated protein and by using anti-hsp/c70-specific antibodies. The K8/18-hsp/c70 complex can be dissociated in a Mg-ATP-dependent manner that requires ATP hydrolysis. Binding of hsp to K8/18 can be reconstituted using purified bovine hsp70 and human K8/18 immunoprecipitates that have been depleted of bound hsp/c70 and increases slightly in the presence of ATP. The reconstituted K8/18-hsp70 complex can be again released in the presence of Mg-ATP. In addition, hsp70 binds to K8/18 without having a significant effect on in vitro filament assembly when added during or after assembly. Using an overlay assay, hsp70 binds exclusively to K8 in the presence of ATP. Our results show direct association of the hsp/c70 proteins with K8/18. This interaction may serve, at least in part, to regulate the function of these two abundant protein groups. Keratin polypeptides 8 and 18 (K8/18) are intermediate filament proteins expressed preferentially in glandular epithelia. We describe the identification, by co-immunoprecipitation from normal human colonic tissues and cultured cell lines, of the 70-kDa heat shock protein (hsp) and its related heat shock cognate protein as K8/18-associated proteins (hsp/c). The association is significant but sub-stoichiometric and occurs preferentially with the soluble rather than the cytoskeletal K8/18 fractions. Heat stress increases the level of soluble K8/18 in association with an increase in hsp70 levels and an increase in the stoichiometry of K8/18-hsp70 association. Identity of the associated proteins was confirmed by microsequencing of a tryptic digest of the purified associated protein and by using anti-hsp/c70-specific antibodies. The K8/18-hsp/c70 complex can be dissociated in a Mg-ATP-dependent manner that requires ATP hydrolysis. Binding of hsp to K8/18 can be reconstituted using purified bovine hsp70 and human K8/18 immunoprecipitates that have been depleted of bound hsp/c70 and increases slightly in the presence of ATP. The reconstituted K8/18-hsp70 complex can be again released in the presence of Mg-ATP. In addition, hsp70 binds to K8/18 without having a significant effect on in vitro filament assembly when added during or after assembly. Using an overlay assay, hsp70 binds exclusively to K8 in the presence of ATP. Our results show direct association of the hsp/c70 proteins with K8/18. This interaction may serve, at least in part, to regulate the function of these two abundant protein groups. INTRODUCTIONIntermediate filaments (IF) 1The abbreviations used are: IFintermediate filamentshscheat shock cognate proteinhspheat shock proteinHSPheat shock protein familyMAbmonoclonal antibodyPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisS and P fractionssoluble and pellet fractionsIFAPintermediate filament-associated proteinsBSAbovine serum albuminATP γ Sadenosine 5′-O-(thiotriphosphate). comprise one of the three major cytoskeletal protein groups that include microfilaments and microtubules (for reviews, see Steinert and Roop, 1988; Klymkowsky et al., 1989; Skalli and Goldman, 1991; Fuchs and Weber, 1994). IF expression is generally tissue specific with the keratin IF subfamily being preferentially expressed in epithelial cells. Tissue-specific expression within epithelial subtypes also extends to keratins, which are comprised of at least 20 members (catalogued as K1-K20) that are found in cells as heteropolymeric pairs (Moll et al., 1982, 1990). Although each epithelial cell type may express several keratins, they usually express one pair as their dominant IF network. For example, glandular “simple” type epithelia, as found in the gastrointestinal tract, liver and pancreas express K8 and K18 (K8/18), keratinocytes express K5/14, and esophageal epithelia express K4/13 as their major keratin pair. IF proteins are highly abundant in cells that express them, as noted for K8/18, which make up ∼ 5% of the total protein in cultured colonic cell lines (Chou et al., 1993), and for keratinocyte keratins, which account for ∼ 30% of the cellular protein (Sun et al., 1979).A number of intermediate filament-associated proteins (IFAP) have been described that may be involved in regulating the function of IF or that may utilize binding to IF to regulate their function (Foisner and Wiche, 1991). These IFAP may interact directly or indirectly with IF and include kinases, membrane-associated proteins, and nuclear proteins. Examples of IF-kinase interactions include the association of protein kinase C-related kinases with K8/18 (Omary et al., 1992a) and vimentin (Spudich et al., 1992; Murti et al., 1992) and the association of cGMP-dependent protein kinase with vimentin (Wyatt et al., 1991; MacMillan-Crow and Lincoln, 1994). Other IFAP include ankyrin and lamin B (Georgatos et al., 1987; Djabali et al., 1991), desmoplakin (Stappenbeck et al., 1993), a 140-kDa desmosomal antigen (Cartaud et al., 1990), IFAP 300 (Skalli et al., 1994), plectin (Foisner et al., 1991), gyronemin (also called filamin) (Brown and Binder, 1992), and KAP85 (Chou et al., 1994).Another highly abundant protein family is the heat shock protein family (HSP) with major classes including HSP25, −60, −70, −80, −90, and −110 (numbers correspond to approximate Mr of class members) (Gething and Sambrook, 1992; Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993; Becker and Craig, 1994). An important function of HSP proteins is to act as molecular chaperones, and in doing so they form complexes with a variety of cellular proteins (e.g. see Schlesinger, 1990). In mammalian cells, two cytosolic members of the HSP70 family are the heat-inducible hsp70 (Mr 72,000) and the constitutively expressed hsc70 (Mr 73,000) (Becker and Craig, 1994). Several indirect reports and reviews have suggested an interaction of HSP70-like proteins with IF, although most describe an association of HSP70 with microtubules (e.g. Schlesinger, 1990; Gupta, 1990). For example, 1) a ubiquitous 70-kDa protein termed β-internexin was initially identified as a microtubule-associated protein and shown indirectly to associate with IF (Napolitano et al., 1985) and then was subsequently shown to be identical to hsp70 (Green and Liem, 1989); 2) hsp70 was shown to co-purify with microtubules in murine mastocytoma cells (Ohtsuka et al., 1986) and with retinal microtubule and IF fractions (Clark and Brown, 1986); 3) in vitro interaction of purified yeast HSP70 with vimentin and nuclear lamins in an ATP-dependent manner has been reported (Georgatos et al., 1989); of cultured cells with the in vimentin and hsp70 levels in the although direct association was shown et al., which are related to the shown to with vimentin in bovine and to in vitro filament assembly of vimentin and protein and of the of K8/18, noted a 70-kDa by that with K8/18 (Chou et al., In show that 70-kDa to hsp/c70 and that its association with K8/18 increases with heat K8/18-hsp/c70 association occurs preferentially with the soluble K8/18 with of hsp in the presence of Mg-ATP. K8/18 and hsp70 binding and can be reconstituted using purified hsp70 and K8/18 immunoprecipitates that have been depleted of bound In vitro binding of hsp70 to using an overlay occurs exclusively with of purified hsp70 to K8/18 during or after filament assembly assembly. The of the K8/18-hsp70 association is bovine and ATP γ from to K8/18 that K8/18 and and (Chou and 1991), which was used as or to protein Other used which a cell (Omary et al., and (Omary et al., which an epithelial cell termed or to as et al., 1994). The two to as by the to and hsp/c70 from and and from and and cell lines from the and at in with and For heat cells at ∼ at for in from for a by the for the of on at of cells with was by cells in with serum by of the for or then by of the and with In cells for by in normal after the in in and using a in and then to with for was and the was used for cells by in by to the soluble The was in by to the P then using the or after to a of cells or or the P For of cells or with 20 of or or of The complexes then using protein to using gel (Chou et al., 1992) and et al., as described that an was used as by the levels of K8/18 and hsp/c70 using of of bovine serum as a protein on the gel the proteins of and of K8/18 to filament assembly using a two The involved as described et al., et al., This a highly K8/18, which is then in was by of the and then a in a protein K8/18 then using a with the K8/18 being and of purified keratin was ∼ of cells. The B and then at assembly was by K8/18 for with two was also in the presence of bovine hsp70 or filaments for for and then with or hsp70 for at was by by as described (Chou et al., of the soluble K8/18 after assembly in the presence or of hsp70 or was by and then of fractions of the soluble and pellet of hsp/c70 from K8/18 and of K8/18-hsp70 immunoprecipitates from cells at or in the or presence of ATP γ ATP γ or immunoprecipitates and then with for by with to released K8/18 immunoprecipitates depleted of hsp/c70 then with of of bovine hsp70 in the presence or of or by to hsp70 and then by Binding Using an highly keratin was by (Chou et al., by using and using and and then using of and was then by to two was with and the was with in for by with of bovine hsp70 for in the presence of in 20 and with 20 in the was with in for and then with hsp was using of K8/18 with of K8/18 from the colonic epithelial cell noted co-immunoprecipitation of a 70-kDa with K8/18. This is in a 70-kDa is noted to with K8/18 using a of using described to and and K8/18 or the 70-kDa The (Mr ∼ is abundant in cells and can be by the antigen can be after (Omary et al., of the 70-kDa shown in by of the and microsequencing of one of the the This with of human hsp70 and hsc70 proteins and The association of K8/18 with hsp/c70 by co-immunoprecipitation was noted also in normal human colonic and in colonic tissue cell lines including and The of a that with K8/18 in normal colonic tissue was that K8 and K18 and is usually with and to K8 or a related keratin on tryptic the of association of hsp/c70 with K8/18, used three that K8/18 three in co-immunoprecipitation of hsp/c70 with K8/18 that hsp/c70 co-immunoprecipitation is to of In addition, the of the 70-kDa as hsp70 hsc70 was confirmed by of K8/18 immunoprecipitates using two to HSP70 proteins and We to K8/18 the two used hsp/c70 of Heat on K8/18 and with the effect of heat stress on K8/18 association with hsp/c70 and the association the soluble and cytoskeletal K8/18 shown in the of HSP70 associated with K8/18 ∼ after heat stress and and with the expression of hsp/c70 and This was confirmed by of K8/18 immunoprecipitates and from normal and cells using of K8/18 immunoprecipitates from the and P fractions of cells to of association of hsp/c70 with K8/18 heat stress in fractions a by the in and 8 is with K8/18 from cells. This which may a form of K8 that was in cells (Chou and 1994), with of heat stress preferentially with the soluble K8/18 with and with K8/18 by heat cells or cells in by using of the soluble after and 2) or K8/18 immunoprecipitates from the and cells and by by The pellet and soluble used for with as described and of cells and directly in or by and of the and then fractions on cell of the also used for of K8/18 and by or by using cells at that HSP70 association with K8/18 occurs preferentially with the soluble K8/18 with association noted after heat heat stress results in an increase in the soluble K8/18 shown in the soluble K8/18 increases after heat stress when by and or by of the total soluble and or after and For the shown in of cells at and which total keratin and stoichiometry of hsp/c70 associated with soluble K8/18 was ∼ and for cells at and as after using This binding of hsp/c70 to K8 or K18 and an Mr of for and for This stoichiometry when the or of hsc70 and hsp70 to the and K8/18 70-kDa K8/18-associated in gel two that hsp70 and hsc70 associated with K8/18 used and that hsp70 or hsp/c70 to K8/18 association with hsp/c70 after of the complex from the soluble and cytoskeletal pellet fractions. shown in the a hsp and the two to hsp70 and hsc70 and The for hsp70 and hsc70 and which are to reported for bovine hsp/c70 The hsc70 (Mr 73,000) slightly than the hsp70 (Mr 72,000) as noted by and hsp70 and hsc70 associate with soluble and cytoskeletal gel and of the associated 70-kDa heat shock proteins from soluble and keratin cells by by to the soluble and pellet fractions. K8/18-hsp/c70 from the and P fractions by in the and then in the then by using and used in and also to the of and which are by and are shown in and levels of the and P the K8/18-hsp/c70 association in of K8/18 and shown in the of K8/18 a is in the soluble and is also associated with for or in of K8/18 from the soluble to the cytoskeletal with association with hsp/c70 noted in the level for the as by of the immunoprecipitates from the and P association with the soluble K8/18 for by results that to the in This that the K8/18-hsp/c70 association occurs at the of but also to with K8/18 during cells in for and then with for or cells and then with cells using by of the and P fractions and then of by and by of Binding on that the protein and vimentin in vitro filament assembly in an manner and 1994), the effect of purified hsp70 on K8/18 in vitro filament assembly. This was by bovine hsp70 to K8/18 proteins during assembly of the which for filament or after filaments have shown in of bovine hsp70 to filaments for by in binding of a of hsp70 to the cytoskeletal pellet in a the of protein during binding of to the was noted of the filaments by effect of hsp70 on filament assembly of hsp70 during filament assembly in binding of of the hsp70 to the K8/18 in a and with filament assembly binding of to the was noted a of hsp70 in by filament assembly and then the hsp70 in the soluble hsp70 the of K8/18 the soluble when added after or during filament K8/18 filaments to K8/18 was purified from cells using of cells to for keratin proteins by using as described and in of to the and filament assembly. filaments with the of bovine hsp70 or as a protein by to the soluble and fractions fractions then by or in vitro assembly of keratin intermediate K8/18 by and then used for in vitro assembly in or in the presence of the of or bovine hsp70 in assembly was by for with by or by of of the and P fractions assembly was for the and is by the two shown in hsp70 and hsp70 noted by hsp70 in the in the of K8/18, was assembly of ATP on K8/18-hsp/c70 Binding and of Binding and in that the association of heat shock proteins with a number of proteins is ATP (e.g. and et al., 1991), the interaction of hsp/c70 with K8/18 is also ATP This was by ATP or to 1) by or 2) immunoprecipitates by to released shown in of to by of K8/18 in the of hsp/c70 from the K8/18-hsp/c70 of γ or This that ATP is important for the of hsp/c70 from the results when immunoprecipitates of K8/18 of cellular as by the of hsp/c70 from K8/18 and of K8/18 immunoprecipitates in the presence of or of results to in In addition, and in of the K8/18-hsp70 results to association with K8/18 is ATP and can be reconstituted in a soluble of cells was by cell by from the soluble was using normal to or with to in the presence of of γ and ATP γ of K8/18 from cells at and then with K8/18 immunoprecipitates depleted of hsp/c70 identical to in then with of bovine hsp70 a total of in the presence or of and as to hsp70 and then using by also the of hsp70 binding to K8/18 immunoprecipitates that depleted of bound hsp/c70 for example, the shown in in of hsp70 to immunoprecipitates in binding of hsp70 to K8/18, which in the presence of ATP but or This that ATP the binding of hsp70 to K8/18. binding of hsp70 to in the presence of hsp70 and may to the of hsp70 from K8/18 immunoprecipitates binding is related to the hsp70 in the reconstituted binding which the binding rather than the of K8/18-hsp/c70 complex and the reconstituted K8/18-hsp70 can be dissociated in a manner to the K8/18-hsp/c70 complex from or cells. shown in the of K8/18-hsp/c70 association by co-immunoprecipitation by in the presence of by can be by hsp70 again in the presence of cells at a of the heat hsp70 (Mr of in cells at a of the hsc70 (Mr of in K8/18 immunoprecipitates from cells of bound bovine hsp70 within from K8/18 immunoprecipitates from cells at in a This in the of bovine hsp70 is highly and may be related to the effect of of K8/18 in cells on its or to the of bovine hsp70 with human of reconstituted of K8/18 from cells at or after heat stress The hsp/c70 was released from K8/18 by with by and of the binding by with bovine hsp70 to the reconstituted was with for the then to released hsp70 and then by in Binding of hsp70 to K8 and K18 form and Roop, and immunoprecipitates of K8/18 also to which of the hsp70 For used an overlay binding bovine hsp70 was with purified and as as as a shown in hsp70 binds to K8 without significant binding to or of the proteins and to the as shown in a in binding of hsp70 to and purified and and then as described and to a and then using an identical to that shown in was with and then with bovine the was with by of bound hsp70 as described and results reported in the 1) hsp70 and hsc70 associate with K8/18 as by co-immunoprecipitation from normal colonic cells and cell lines, of binding in and binding to filaments in vitro during or after filament assembly. 2) In cell hsp/c70 preferentially to the soluble of K8/18 with binding to soluble and cytoskeletal K8/18 after heat The binding of hsp70 to K8/18 after heat stress is also associated with an increase in the soluble of K8/18. 3) Binding of purified hsp70 to K8/18 is by and of the complex in cell or in immunoprecipitates requires and ATP hydrolysis. Using an overlay assay, K8 specific and binding to hsp70 in the presence of ATP as with K18 and Binding of hsp70 to K8/18 filament assembly in of hsp/c70 Binding to in a specific hsp/c70 and K8/18 interaction that is to and an in vitro binding is by which for binding is noted by co-immunoprecipitation after as in 1) and is noted after cell and then of the soluble in in the of binding of hsp70 to in an overlay specific binding to K8 without binding to or overlay binding keratin for binding to and of hsp70 binding to K8 these may an in to be the binding is of hsp/c70 to and intermediate filaments and binding of hsp70 to K8/18 is on a of K8 or is on a of K8/18 or although a direct interaction of hsp/c70 with K8/18, the that interaction may be by one or proteins that form a that hsp/c70 is the major K8/18 as by proteins are involved in a they have to be to in the K8/18-hsp/c70 interaction of hsp/c70 with a number of cellular proteins may in an ATP-dependent or For example, hsp/c70 with et al., proteins et al., 1993), and et al., in an ATP-dependent manner in with the association with et al., 1994). In the of hsp70 interaction with K8/18, ATP to the in vitro binding with of the complex being by ATP association of hsp70 with K8/18 is but is We that the of stoichiometry of association is of hsp/c70 to of K8 that binding occurs preferentially with K8 as suggested by the overlay in This is for the in cells at and is a in cells to a of ∼ In cells, that 5% of the total cellular protein is K8/18, and ∼ 5% of K8/18 is soluble (Chou et al., the co-immunoprecipitation K8/18-hsp/c70 association in cells, and that hsp/c70 make up of the total cellular protein et al., then that of hsp/c70 is bound to K8/18. The cytosolic of hsp/c70 and the of of hsp70 with cytoskeletal 1985) that a significant of hsp/c70 is bound to K8/18. This that the of total hsp/c70 that is associated with K8/18 is to be to than to of to bound hsp/c70 from K8/18 and then the binding and in the presence of that the interaction of hsp/c70 with K8/18 is highly to in the interaction of hsp/c70 with K8/18 include as and proteins as For example, and important for of the but γ was Although was reported to be important for the of hsp70 from its et al., 1993), in K8/18-hsp/c70 This may the rather than the K8/18 in of a protein or a as of K8/18-hsp/c70 interaction to be to a that K8/18 in cells are which may account for the hsp/c70 association with K8/18 in cells. the heat increase in K8/18-hsp/c70 association may be related to hsp70 Other may be involved in K8/18-hsp/c70 For example, yeast interaction with protein was found to be by a of termed et al., that several proteins that are by (e.g. (Chou et al., also with K8/18. The that these proteins in K8/18-hsp/c70 interaction is to for the of hsp/c70 with from a number of has for a number of for These include of and interaction with proteins and protein et al., 1990; and Sambrook, 1992), to 1990), et al., 1992; and 1992), and et al., 1994), and of binding of et al., is to that the interaction of two highly abundant proteins as hsp/c70 and may be a that cells can to have the two protein groups regulate each This co-immunoprecipitation and with the noted significant stoichiometry of association. For example, of hsp/c70 that is bound to K8/18 then the to interact with one or of its The identified K8/18-hsp/c70 association a in of the heat shock protein Other to for K8/18-hsp/c70 association are to the of molecular in of et al., 1992) and and et al., and the association of with vimentin with of cytoskeletal vimentin a soluble and 1994). a increase in the association of hsp/c70 with soluble cytoskeletal K8/18 and that the of K8/18 in association with heat stress is with levels of a significant effect of hsp70 on K8/18 or assembly a for hsp/c70 in the or of keratin is is important to that hsp/c70 with of proteins in a or 1990; et al., 1990). the interaction of hsp/c70 with K8/18 has from a the of keratins, the stoichiometry of and the ATP are INTRODUCTIONIntermediate filaments (IF) 1The abbreviations used are: IFintermediate filamentshscheat shock cognate proteinhspheat shock proteinHSPheat shock protein familyMAbmonoclonal antibodyPBSphosphate-buffered salinePAGEpolyacrylamide gel electrophoresisS and P fractionssoluble and pellet fractionsIFAPintermediate filament-associated proteinsBSAbovine serum albuminATP γ Sadenosine 5′-O-(thiotriphosphate). comprise one of the three major cytoskeletal protein groups that include microfilaments and microtubules (for reviews, see Steinert and Roop, 1988; Klymkowsky et al., 1989; Skalli and Goldman, 1991; Fuchs and Weber, 1994). IF expression is generally tissue specific with the keratin IF subfamily being preferentially expressed in epithelial cells. Tissue-specific expression within epithelial subtypes also extends to keratins, which are comprised of at least 20 members (catalogued as K1-K20) that are found in cells as heteropolymeric pairs (Moll et al., 1982, 1990). Although each epithelial cell type may express several keratins, they usually express one pair as their dominant IF network. For example, glandular “simple” type epithelia, as found in the gastrointestinal tract, liver and pancreas express K8 and K18 (K8/18), keratinocytes express K5/14, and esophageal epithelia express K4/13 as their major keratin pair. IF proteins are highly abundant in cells that express them, as noted for K8/18, which make up ∼ 5% of the total protein in cultured colonic cell lines (Chou et al., 1993), and for keratinocyte keratins, which account for ∼ 30% of the cellular protein (Sun et al., 1979).A number of intermediate filament-associated proteins (IFAP) have been described that may be involved in regulating the function of IF or that may utilize binding to IF to regulate their function (Foisner and Wiche, 1991). These IFAP may interact directly or indirectly with IF and include kinases, membrane-associated proteins, and nuclear proteins. Examples of IF-kinase interactions include the association of protein kinase C-related kinases with K8/18 (Omary et al., 1992a) and vimentin (Spudich et al., 1992; Murti et al., 1992) and the association of cGMP-dependent protein kinase with vimentin (Wyatt et al., 1991; MacMillan-Crow and Lincoln, 1994). Other IFAP include ankyrin and lamin B (Georgatos et al., 1987; Djabali et al., 1991), desmoplakin (Stappenbeck et al., 1993), a 140-kDa desmosomal antigen (Cartaud et al., 1990), IFAP 300 (Skalli et al., 1994), plectin (Foisner et al., 1991), gyronemin (also called filamin) (Brown and Binder, 1992), and KAP85 (Chou et al., 1994).Another highly abundant protein family is the heat shock protein family (HSP) with major classes including HSP25, −60, −70, −80, −90, and −110 (numbers correspond to approximate Mr of class members) (Gething and Sambrook, 1992; Georgopoulos and Welch, 1993; Hendrick and Hartl, 1993; Becker and Craig, 1994). An important function of HSP proteins is to act as molecular chaperones, and in doing so they form complexes with a variety of cellular proteins (e.g. see Schlesinger, 1990). In mammalian cells, two cytosolic members of the HSP70 family are the heat-inducible hsp70 (Mr 72,000) and the constitutively expressed hsc70 (Mr 73,000) (Becker and Craig, 1994). Several indirect reports and reviews have suggested an interaction of HSP70-like proteins with IF, although most describe an association of HSP70 with microtubules (e.g. Schlesinger, 1990; Gupta, 1990). For example, 1) a ubiquitous 70-kDa protein termed β-internexin was initially identified as a microtubule-associated protein and shown indirectly to associate with IF (Napolitano et al., 1985) and then was subsequently shown to be identical to hsp70 (Green and Liem, 1989); 2) hsp70 was shown to co-purify with microtubules in murine mastocytoma cells (Ohtsuka et al., 1986) and with retinal microtubule and IF fractions (Clark and Brown, 1986); 3) in vitro interaction of purified yeast HSP70 with vimentin and nuclear lamins in an ATP-dependent manner has been reported (Georgatos et al., 1989); of cultured cells with the in vimentin and hsp70 levels in the although direct association was shown et al., which are related to the shown to with vimentin in bovine and to in vitro filament assembly of vimentin and protein and of the of K8/18, noted a 70-kDa by that with K8/18 (Chou et al., In show that 70-kDa to hsp/c70 and that its association with K8/18 increases with heat K8/18-hsp/c70 association occurs preferentially with the soluble K8/18 with of hsp in the presence of Mg-ATP. K8/18 and hsp70 binding and can be reconstituted using purified hsp70 and K8/18 immunoprecipitates that have been depleted of bound In vitro binding of hsp70 to using an overlay occurs exclusively with of purified hsp70 to K8/18 during or after filament assembly assembly. The of the K8/18-hsp70 association is