Báo cáo khoa học: The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum ) a quantitative approach potx

The heat shock protein 70 molecular chaperone network in the pancreatic endoplasmic reticulum ) a quantitative approach Andreas Weitzmann, Christiane Baldes, Johanna Dudek and Richard Zimmermann Medizinische Biochemie und Molekularbiologie, Universitat des Saarlandes, Homburg, Germany ă Keywords BiP; endoplasmic reticulum; J-domains; nucleotide exchange factors; molecular chaperones Correspondence R Zimmermann, Medizinische Biochemie und Molekularbiologie, Universita des ăt Saarlandes, D-66421 Homburg, Germany Fax: +49 6841 1626288 Tel: +49 6841 1626510 E-mail: bcrzim@uks.eu (Received 15 June 2007, revised August 2007, accepted 10 August 2007) doi:10.1111/j.1742-4658.2007.06039.x Traditionally, the canine pancreatic endoplasmic reticulum (ER) has been the workhorse for cell-free studies on protein transport into the mammalian ER These studies have revealed multiple roles for the major ER-luminal heat shock protein (Hsp) 70, IgG heavy chain-binding protein (BiP), at least one of which also involves the second ER-luminal Hsp70, glucose-regulated protein (Grp) 170 In addition, at least one of these BiP activities depends on Hsp40 Up to now, five Hsp40s and two nucleotide exchange factors, Sil1 and Grp170, have been identified in the ER of different mammalian cell types Here we quantified the various proteins of this chaperone network in canine pancreatic rough microsomes We also characterized the various purified proteins with respect to their affinities for BiP and their effect on the ATPase activity of BiP The results identify Grp170 as the major nucleotide exchange factor for BiP, and the resident ER-membrane proteins ER-resident J-domain protein plus ER-resident J-domain protein ⁄ Sec63 as prime candidates for cochaperones of BiP in protein transport in the pancreatic ER Thus, these data represent a comprehensive analysis of the BiP chaperone network that was recently linked to two human inherited diseases, polycystic liver disease and MarinescoSjogren ă syndrome The initial step in the biogenesis of approximately 30% of eukaryotic proteins is their integration into the membrane or their transport into the lumen of the endoplasmic reticulum (ER) Protein integration or transport into the ER can occur cotranslationally or post-translationally, and typically requires signal peptides at the N-terminus of the precursor proteins and the transport machinery Post-translational protein transport into the yeast ER involves the Sec complex in the membrane, comprising the Sec61p subcomplex [1,2], the putative signal peptide receptor subcomplex [3,4], the heat shock protein (Hsp) 40, termed Sec63p [5], and the luminal Hsp70 proteins Kar2p [6] and Lhs1p [7] Sec63p and Kar2p are essential proteins in yeast and, together with Lhs1p, are also involved in cotranslational protein transport into the ER [8,9] Cotranslational protein transport into dog pancreas microsomes involves a similar Sec61 complex [10–12] Furthermore, protein transport into the mammalian ER involves Hsp70-type molecular chaperones and their Hsp40-type cochaperones: Hsp70-type molecular chaperones of the ER lumen, IgG heavy chain-binding protein [BiP (also termed glucose-regulated protein (Grp) 78, HspA5) and Grp170 (Orp150) are involved in cotranslational and post-translational insertion of precursor polypeptides into the Sec61 complex [13] Abbreviations BiP, IgG heavy chain-binding protein; ER, endoplasmic reticulum; ERj, endoplasmic reticulum-resident J-domain protein; Grp, glucoseregulated protein; GSH, glutathione; GST, glutathione S-transferase; Hsp, heat shock protein; RM, rough microsome; SPR, surface plasmon resonance FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5175 Pancreatic endoplasmic reticulum chaperone network A Weitzmann et al ERj1 yeast ortholog Kar2p in Sec61 gating [18] A mammalian ortholog of yeast protein Sec63p was shown to be an abundant protein in canine pancreatic microsomes and was found in association with the Sec61 complex [19–21] The Sec63-related protein ER-resident J-domain protein (ERj) was observed to be associated with translating ribosomes on the ER surface [22–24] and to be able to complement a yeast mutant that is deficient in Sec63p [25] In the ER of Saccharomyces cerevisiae, four Hsp40 proteins with a luminal J-domain have been identified: the two membrane proteins Sec63p [5] and Scj2p [26], and the two luminal proteins Scj1p [27] and Jem1p [28] In the ER of various mammalian cells, five Hsp40 proteins have been identified: the three membrane proteins Sec63 (alternative names: ERj2, DnaJC2) [19–21], ERj1 (Mtj1p, DnaJC1) [22,29,30], and ERj4 (MDG1, DnaJB9) [31,32], and the two luminal proteins ERj3 (HEDJ, Dj9, DnaJB11) [33–35] and ERj5 (JPDI, DnaJC10) [36,37] (Fig 1, Table 1) Furthermore, nucleotide exchange factors ) Sil1p in yeast [38,39] and Sil1 (also termed BAP) in mammalian cells [40] ) have been identified, and Lhs1p and Grp170 were shown to be alternative nucleotide exchange factors for Kar2p and BiP, respectively [41,42] Both LHS1 and SIL1 are nonessential genes in yeast However, simultaneous deletion of both genes results in synthetic lethality Here we quantitatively characterized the Hsp70 chaperone network in the rough ER of a single ERj4 ERj2/Sec63 N C cytosol C ER membrane ER lumen N C GF J-domain J-domain J domain C N C N Cys GF N ATPase TRX PB BiP domain domain N C J domain J domain ERj3 N Sil1 C N ATPase PB Grp170 domain domain C ERj5 Fig The established network of Hsp70-type molecular chaperones in the lumen of the mammalian ER The cartoon summarizes data from different cell types The putative domain organization of the various proteins is indicated (PB, peptide-binding domain; GF, glycine ⁄ phenylalanine-rich region; Cys, cysteine-rich region; TRX, thioredoxin-like domains) The quantitative aspects are summarized in Table BiP was also identified as a luminal protein that is involved in the completion of protein translocation [14,15] Furthermore, BiP was shown to seal the luminal end of the mammalian Sec61 complex in the absence of protein translocation and at several stages during cotranslational translocation of preproteins [16–18] BiP was shown to involve an unidentified resident ER Hsp40 and could not be substituted by its Table Hsp70 chaperones and their cochaperones of the ER in the canine pancreas The concentrations refer to a suspension of RMs, with a concentration of heterotrimeric Sec61 complexes of 2.12 lM [54] The affinities of Hsp40s for BiP are based on the SPR experiments shown in Fig or were determined previously [21,22] The ATPase experiments shown in Fig are the basis for the stimulatory effects of Hsp40, Sil1, and Grp170 The experimental details are given in Experimental procedures ND, not determined Protein (alternative name) BiP (Grp78, HspA5) Grp170 (Orp150) ERj1 (Mtj1p, DnaJC1) ERj1J ERj2 (Sec63p, DnaJC2) ERj2J ERj3 (HEDJ, Dj9, DnaJB11) ERj3J ERj4 (MDG1, DnaJB9) ERj5 (JPDI, DnaJC10) ERj5J Sil1 (BAP) 5176 5.00 0.60 0.36 1.98 GST–J-domain (91–189) 0.29 2.00 0.005 Recombinant protein (amino acid residues) Affinity for BiP in the presence of ATP (KD in lM) Stimulation of ATPase activity of BiP (fold) BiP)6His (20–655) – – ND GST–J-domain (44–140) Concentration in suspension of RMs (lM) 5.00 GST–ERj3 (18–336) GST–J-domain (18–119) GST–ERj4 (23–222) GST–ERj5 (26–793) GST–J-domain (26–113) GST–Sil1 (39–461) Further stimulation of BiP ATPase by Grp170 (fold) Sil1 (fold) – 1 – 1.5 – 0.12 5.2 5.6 2.4 3.5 3.60 3.50 6.07 0.45 0.59 ND 5.7 1.8 ND 1.6 3.9 1.8 1.5 2.2 1.9 ND 1.5 ND 4.3 – 1.3 ND 1.1 ND – FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS Pancreatic endoplasmic reticulum chaperone network mammalian tissue, canine pancreas, which predominantly comprises exocrine cells Except for ERj4, all mammalian Hsp40s of the ER were detected As Sil1 was found at very low concentrations, Grp170 (ortholog of yeast Lhs1p) appears to act as the predominant nucleotide exchange factor for BiP–ADP in the pancreatic ER The interactions of the various J-domains with BiP were characterized by pull-down experiments, surface plasmon resonance (SPR) spectroscopy, and ATPase experiments These data provide the first comprehensive and quantitative analysis of a chaperone network that was recently linked to two human hereditary diseases, autosomal dominant polycystic liver disease (OMIM 174050) and the neurodegenerative MarinescoSjogren syndrome (OMIM 248800) ă [4346] colour intensity (arbitrary units) A Weitzmann et al Results B A 400 300 200 100 0 0,5 1,5 protein (µg) 2,5 Previously, we had determined the concentrations of BiP, Grp170, ERj1, ERj2 and ERj3 in suspensions of dog pancreas microsomes [21,22,33,34] In order to determine how abundant ERj4, ERj5 and Sil1 are in these pancreatic microsomes, purified glutathione S-transferase (GST) hybrid proteins and specific antibodies were employed in western blotting, according to the established procedure (Fig 2; Table 1) In all cases, two different antibodies were used that recognized the respective recombinant protein In the case of ERj5 and Sil1, these antibodies recognized an identical band in the pancreatic microsomes We determined concentrations of lm for ERj5 and nm for Sil1 in the microsomal suspensions In the case of ERj4, the antibodies failed to identify a common antigen in the pancreatic microsomes Therefore, we conclude that ERj4 is not an abundant protein in dog pancreas microsomes under physiological conditions This view is also supported by the fact that we failed to characterize ERj4 in proteomic analysis of these microsomes It follows from the concentrations of Hsp70 and Hsp40 proteins in the ER lumen (Table 1) that all Hsp40s can be associated with BiP at any given time The J-domains of all mammalian ER-resident Hsp40s productively interact with BiP but differ in their affinities for BiP To determine whether the mammalian ER-resident Hsp40s contain a functional J-domain, hybrid proteins colour intensity (arbitrary units) 1500 Two Hsp70s, four Hsp40s and Sil1 form a chaperone network in the pancreatic ER 1000 500 0 microsomes (µL) Fig Quantitation of proteins in dog pancreas microsomes Serial dilutions of BSA (filled squares) were run on SDS polyacrylamide gels in parallel with two samples of purified recombinant protein (arrowheads, A) The proteins were stained with Coomassie Brilliant Blue, and the staining intensity was quantified by densitometry (Personal Densitometer; Applied Biosystems, Krefeld, Germany) The same purified protein (arrowhead) was run on SDS polyacrylamide gels in parallel with serial dilutions of dog pancreas microsomes (filled circles, B) Subsequently, the proteins were transferred to poly(vinylidene difluoride) membranes and incubated with rabbit antibodies that were directed against the protein of interest and with a peroxidase conjugate of goat anti-(rabbit IgG) serum The bound antibodies were made visible by incubation with enhanced chemiluminescence (ECL) and exposure to X-ray film The intensity of silver precipitation was quantified by densitometry The calculation of the molar concentration of the respective protein in microsomal suspensions was based on the predicted molecular mass of the protein, as calculated by the protean option of the LASERGENE DNASTAR sequence analysis software (GATC, Konstanz, Germany) FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5177 Pancreatic endoplasmic reticulum chaperone network A Weitzmann et al A B C D E F G 5178 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS A Weitzmann et al comprising GST and the respective protein or J-domain were constructed, purified, and subjected to three activity assays In the case of the membrane proteins ERj1, ERj 2, and ERj4, the transmembrane domains were absent form the GST hybrids (Table 1) Thus, only the ER-luminal domains were analyzed in these cases In the case of the two luminal Hsp40s ERj3 and ERj5, GST hybrids were analyzed that contained either the J-domains or the full-length proteins When compared to each other, the two types of GST hybrids behaved quite similarly in the functional assays that were employed here (Table 1) In the first series of experiments, ‘pull-down assays’ were carried out with detergent extracts of dog pancreas microsomes as described previously [21] GST served as a negative control GST or GST hybrids were immobilized on glutathione (GSH)–Sepharose and incubated with detergent extracts of dog pancreas microsomes in the absence or presence of ATP The bound proteins were eluted and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue (Fig 3) All GST hybrids selectively pulled down BiP from the detergent-solubilized microsomal proteins in the presence of ATP and less efficiently in its absence (Fig 3A–F, lane versus lane 4) From our results, we conclude that BiP interacts with all ERjs in a productive manner, as: (a) GST did not pull down BiP (Fig 3G, lanes and 6); and (b) the other major molecular chaperones, present in the detergent extract of dog pancreas microsomes (such as Grp94 and calreticulin), did not bind to the GST hybrids (Fig 3A–F, lane versus lane 5) We note, however, that the different GST hybrids were different in their BiP pull-down efficiencies (see below) We next characterized the interaction of BiP with the GST hybrids by SPR spectroscopy as described previously [21] (Fig 4) We determined the apparent affinities in the presence of ATP (KD), which are given in Table In summary, BiP has an approximately 10fold higher affinity for ERj1 and ERj5 as compared to ERj2, ERj3 and ERj4 However, we note that these apparent affinities have to be treated with caution, as the kinetics could not be fitted perfectly to a : binding model The generally accepted explanation for this fact is that after J-domain-mediated ATP hydrolysis and in the absence of a real substrate, BiP binds Hsp40 as a substrate [47] Accordingly, this interaction Pancreatic endoplasmic reticulum chaperone network is not seen when only the ATPase domain of BiP is employed instead of full-length BiP (data not shown) We note that it was also observed for yeast Kar2p plus Sec63p that a stable interaction between this Hsp70– Hsp40 pair is possible in the absence of any substrate polypeptide [47], and stable interactions were also seen previously between BiP and mammalian ERj2 ⁄ Sec63 [21] and ERj1 [22], in both pull-down and SPR experiments Next, we investigated whether the GST hybrids stimulate BiP’s ATPase activity under steady-state conditions, i.e in the presence of 500 lm ATP BiP was incubated with [32P]ATP[cP] in the absence or presence of GST hybrid After various times of incubation, the samples were analyzed by TLC and phosphorimaging (Fig 5A–E; Table 1) According to the time-dependent hydrolysis of ATP under the different conditions, all J-domains stimulated the ATPase activity of BiP GST had no such stimulatory effect, even at much higher concentrations [21] Therefore, it seems to be unlikely that the observed stimulation of BiP’s ATPase activity by the GST–J-domain hybrid was due to a BiP–substrate rather than a BiP–cochaperone interaction In the case of ERj1, ERj3, and ERj4, the stimulatory effects of the ERjs correlated with their affinities for BiP However, for unknown reasons, this was not the case for ERj2 ⁄ Sec63 and ERj5 Grp170 serves as an efficient and general nucleotide exchange factor for BiP Our previous attempts to purify Grp170 with ATPaffinity chromatography resulted in a mixture of Grp170 and BiP [13] Here, we employed gel filtration chromatography in the absence or presence of ATP as a subsequent and final purification step (Fig 6A,B) In the absence of ATP, a proportion of BiP cofractionated with Grp170, with an elution maximum of both proteins at a position that corresponded to a molecular mass of 240 kDa (Fig 6A) In addition, the vast majority of BiP was observed at a position that corresponded to its monomeric molecular mass (70 kDa) In the presence of ATP, however, there was hardly any overlap of the two proteins, and they more or less eluted according to their theoretical molecular masses (70 and 140 kDa; Fig 6B) Apparently, the two Hsp70type molecular chaperones can form a heterodimeric Fig Functional characterization of Hsp40-type cochaperones of the ER: selective binding of BiP GST or GST hybrid was immobilized and incubated with detergent extract of microsomes in the absence or in the presence of ATP as described in Experimental procedures The unbound and bound proteins were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue We note that: (a) the band that is labeled BiP was identified as such by western blotting; and (b) we failed to detect Grp170 in the bound fractions under these conditions FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5179 Pancreatic endoplasmic reticulum chaperone network A Weitzmann et al Fig Functional characterization of Hsp40-type cochaperones of the ER: affinity for BiP SPR analysis was carried out with immobilized ERj3 (A), ERj3J (B), ERj5 (C), ERj5J (D), and ERj4 (E) and recombinant mouse BiP as described in Experimental procedures We note that for all Hsp40s there was no interaction observed in the absence of ATP or when ATPcS was used instead of ATP (not shown) The calculated affinities are given in Table We note that prior to application of BiP, dissociation of previously applied BiP was allowed to reach completion (not shown) 5180 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS A Weitzmann et al Pancreatic endoplasmic reticulum chaperone network A B C D E F G H I J K L Fig Functional characterization of Hsp40-type cochaperones and nucleotide exchange factors of the ER: effect on the ATPase activity of BiP ATP hydrolysis assays were carried out under steady-state conditions as described in Experimental procedures The concentrations were: ATP, 500 lM; Sil1, lM; BiP and Kar2p, lM; Hsp40, lM; and Grp170, 0.25 lM complex in the absence of free ATP ) i.e when at least BiP can be expected to be in the ADP form – and this complex is dissociated in the presence of an excess of free ATP In order to confirm this interpretation, an immobilized antibody that recognizes native Grp170 was employed A fraction from the gel filtration chromatography in the absence of ATP that contained approximately stoichiometric amounts of both chaperones was incubated with immobilized antibodies to Grp170 either in the absence or in the presence of ATP Subsequently, the antibody-bound and unbound proteins were analyzed by SDS ⁄ PAGE and protein staining (Fig 6C, lanes and versus lanes and 5) The antibody to Grp170 coimmunoprecipitated BiP more efficiently in the absence than in the presence of ATP; that is, a significant amount of BiP remained in the unbound fraction in the presence of ATP (Fig 6C, lane 3) Thus, the two chaperones are indeed able to form a stable complex in the absence of ATP In the next experiment, the ability of Grp170 to interact with Hsp40 was examined BiP served as an internal control for this experiment In order to keep the two Hsp70 chaperones from forming a complex, ATP was present A mixture of both Hsp70-type chaperones was incubated with an immobilized J-domain (ERj1J) Subsequently, the J-domain-bound and unbound proteins were analyzed by SDS ⁄ PAGE and protein staining (Fig 6D, lane versus lane 2) As expected, BiP was efficiently bound by the immobilized J-domain In contrast, Grp170 was not bound by the immobilized J-domain, i.e remained in the unbound fraction (Fig 6D, lane 2) Thus, in contrast to BiP, Grp170 appears to be unable to form a stable complex with Hsp40-type proteins Grp170 has a low basal ATPase activity that was hardly stimulated by ERj1J and serves as a nucleotide exchange factor for BiP in the presence of ERj1 [42] In order to analyze the nuleotide exchange activity of Grp170 in the presence of the other ERjs, steady-state FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5181 Pancreatic endoplasmic reticulum chaperone network 150 B - ATP 100 Grp170 + BiP 50 450 + ATP Protein (arbitrary units) Protein (arbitrary units) A A Weitzmann et al BiP 300 Grp170 150 BiP 0 10 15 fraction number 20 C 10 15 fraction number 20 D Fig Characterization of Grp170: functional interactions Superose gel filtration was carried out as described in Experimental procedures in the absence (A) and presence (B) of ATP Fractions were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue Staining intensity was quantified by densitometry Grp170 (open squares) and BiP (filled circles) were identified as such by western blotting An aliquot of fraction of the gel filtration in the absence of ATP (termed input and shown in lane 1) was incubated with immobilized antibodies to Grp170 in the absence or presence of ATP as indicated (C) The unbound (lanes and 3) and bound (lanes and 5) proteins were collected and subjected to SDS ⁄ PAGE and subsequent staining with Coomassie Brilliant Blue An aliquot of an ATP eluate of ATP–agarose chromatography was incubated with GSH–Sepharose (– ERj1J) or immobilized ERj1J (+ ERj1J) in the presence of ATP (D) Subsequently, unbound (lanes and 2) and bound (lanes and 4) material were separated by centrifugation, and analyzed by SDS ⁄ PAGE and protein staining with Coomassie Brilliant Blue The protein ladder was run on the same gel (lane 5) ATPase assays were carried out that involved BiP and Grp170 ) in a physiological ratio ) plus Hsp40 (Fig 5A–E; Table 1) The established nucleotide exchange factor Sil1 ) at about two-fold molar excess ) served as a positive control in these experiments (Fig 5F–J) Under conditions of stimulation of BiP’s ATPase activity by any ER-resident Hsp40, Grp170 led to further acceleration of ATP hydrolysis Thus Grp170 can serve as a nucleotide exchange factor for BiP after stimulation of BiP by any ER-resident Hsp40 Grp170 was more efficient than Sil1 in this respect We note that Sil1 had previously been shown to stimulate the ATPase activity of BiP in the presence of ERj4 under slightly different conditions [40] There5182 fore, the apparent lack of nucleotide exchange factor activity of Sil1 in the presence of ERj3, Erj4 and ERj5 in our experiments should not be taken as an indication of a specialized function of Sil1 (Fig 5H–J; Table 1) However, the data point to the fact that the two nucleotide exchange factors have different efficiencies Both ERj1 and ERj2 ⁄ Sec63 as well as Grp170 functionally interact with the yeast BiP ortholog Kar2p The yeast ortholog of BiP that is termed Kar2p was observed to be unable to substitute for BiP in facilitating Sec61 channel gating in canine pancreatic FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS A Weitzmann et al Pancreatic endoplasmic reticulum chaperone network ERj1 0.18mM 0.12µM ERj2 ERj3 0.99mM ERj4 0.29mM* 5µM 3.6 µM 6µM UPR BiP5mM* sensors: PERK IRE1 ATF6 Grp170 Sil1 0mM* 0.6mM* ERj5 2mM* 0.45µM 0.005mM Fig The BiP network in the pancreatic ER The estimated ER-luminal concentration and the measured affinities are indicated The observed concentrations for RMs (Table 1) were corrected for the facts that about 50% of the luminal proteins leak out of the organelle during tissue homogenization and that the inner volume of the RM is small as compared to the total volume of the RM suspension (approximately : 500), in order to estimate the concentration of the luminal proteins in the ER of pancreatic cells UPR, unfolded protein response; *UPR-inducible microsomes [18] In fact, it even had a dominant negative effect on BiP in these experiments Furthermore, this gating activity of BiP was shown to involve an unidentified resident ER Hsp40 [18] In analogy to the situation in yeast, the respective Hsp40 is expected to be a membrane protein in pancreatic microsomes As lack of interchangeability of various Hsp70s has been observed previously [48,49], it seemed reasonable that the failure of Kar2p to support a complete ATPase cycle in concert with one of the two most likely candidate Hsp40s in channel gating, ERj1 and ERj2, or the major pancreatic nucleotide exchange factor, Grp170, was responsible for the effects of Kar2p in channel gating in mammalian microsomes At first, we addressed the question of whether Kar2p functionally interacts with the two relevant J-domains Kar2p was stimulated in its ATPase activity by the two J-domains to an extent that is comparable to their stimulation of BiP (Fig 5K,L) The stimulation by ERj1J was 3-fold and that by ERj2J was 2.5-fold, as compared to 5.2-fold and 3.5-fold (Table 1) Next, we deterrmined whether Grp170 functionally interacts with Kar2p after stimulation of its ATPase activity by ERj1J or ERj2J Grp170 stimulated the ATPase activity of Kar2p to an extent that is comparable to the stimulation of BiP (Fig 5K,L) The stimulation by Grp170 in the presence of ERj1J was 2.4-fold and that of ERj2J was 2.2-fold, as compared to 5.6-fold and 5.7-fold (Table 1) The observed differences appear to be too small to provide an explanation for the inability of Kar2p to substitute for BiP in Sec61 gating in mammalian microsomes Thus, lack of interchangeability between BiP and Kar2p at the level of the Hsp40s ERj1 and ERj2 and at the level of the nucleotide exchange factor Grp170 does not appear to be responsible for the observed effect of Kar2p in channel gating experiments [18] Discussion The pancreatic network of ER-luminal chaperones under steady-state conditions From the concentrations of the various chaperones and cochaperones in the lumen of canine pancreatic rough microsomes (RMs), one can extrapolate to the situation in the corresponding rough ER (Table 1, Fig 7) It has to be taken into account that during preparation of microsomes, about 50% of the luminal content is lost into the postribosomal supernatant and that the luminal volume of the microsomes is only a minor fraction of the total volume of the microsomal suspension (we estimate : 500) Taken together, we estimate that in the rough ER, the concentrations of the most abundant chaperones such as BiP and ERj5 or their J-domains, such as in the case of ERj2 ⁄ Sec63, are in the low millimolar range Furthermore, the total amounts of Hsp70 and Hsp40 proteins in the ER lumen (Table 1) and the observed affinities (Table 1) allow one to conclude that, in principle, all J-domains can be associated with BiP at any given time In reality, however, a large proportion of BiP will be engaged with polypeptide substrates Therefore, one can assume that under these steady-state conditions, the determined affinities become relevant On the basis of the analogies with yeast, and the facts that human ERj1 can complement deletion of the SEC63 gene in yeast, and that ERj4 appears to be absent from pancreatic rough ER under nonstress conditions, the two Hsp40s FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5183 Pancreatic endoplasmic reticulum chaperone network A Weitzmann et al ERj1 and ERj2 ⁄ Sec63 appear to be the prime candidates for alternatively cooperating with BiP in protein transport This may explain why disruption of SEC63 ⁄ ERJ2 in autosomal dominant polycystic liver disease is not lethal, in contrast to the situation in yeast [44] Grp170 was characterized as an alternative nucleotide exchange factor for BiP [42] This is in perfect agreement with the fact that disruption of SIL1 ⁄ BAP in humans and mice does not cause lethality [45,46,50] Thus, as in yeast, the presence of the additional nucleotide exchange factor Grp170 may compensate for the loss of Sil1 The network of ER-luminal chaperones under stress conditions When misfolded proteins accumulate in the ER, various signal transduction pathways are activated that increase the biosynthetic capacity and decrease the biosynthetic burden of the ER This phenomenon is termed the unfolded protein response Most of the members of the chaperone network discussed here are under control of the unfolded protein response (indicated by an asterisk in Fig 7) Therefore, one would expect ERj4 to be present in pancreatic microsomes under stress conditions Furthermore, the GRP170 gene would be expected to be overexpressed after disruption of SIL1 ⁄ BAP in humans and mice Thus, overexpression of GRP170 may compensate for the loss of Sil1 in most tissues However, for unknown reasons, this does not seem to work for certain areas of the cerebellum in patients suffering from MarinescoSjogren syndrome ă [45,46] and in the so-called woozy mice [50] Experimental procedures Materials The protein ladder (10–200 kDa) was obtained from Life Technologies (Grand Island, NY, USA) ATP-C8-agarose, thrombin and peroxidase conjugate of goat anti-(rabbit IgG) serum were obtained from Sigma Chemical Company, Taufkirchen, Germany) [32P]ATP, ATP, GSH–Sepharose Fast Flow, protein A–Sepharose and protein G–Sepharose Fast Flow, Superose 6B, X-ray films and the enhanced chemiluminescence (ECL) were obtained from GE Healthcare (Freiburg, Germany) Poly(vinylidene difluoride) membranes and Centricon devices were obtained from Millipore (Schwalbach, Germany) Chaps was obtained from Calbiochem (Schwalbach, Germany) Hepes and Coomassie Brilliant Blue were purchased from Serva (Heidelberg, Germany) Purification of proteins from dog pancreas The ATPase cycle of BiP In principle, BiP’s ATPase cycle follows the well-established, paradigmatic functional cycle of DnaK [51] Briefly, BiP–ATP has a low affinity for polypeptide substrates Proteins with BiP-reactive J-domains have a high affinity for BiP–ATP and can therefore bind to the underside of the ATP-binding cleft Owing to their additional domains, the Hsp40s ERj3, ERj4 and ERj5 may be able to bind polypeptide substrates and deliver them to peptide-binding domains of BiP in the course of their interaction with BiP In contrast, in the case of ERj1 and ERj2, BiP appears to be recruited to the substrate polypeptides by spatial proximity to the Sec61 complex and ribosomes, respectively In any case, interaction of the J-domain with the ATP-binding cleft triggers ATP hydrolysis and a subsequent conformational change in the peptide-binding domain Apparently, in the in vitro analysis in the absence of 5184 polypeptide substrates, this leads to reversible trapping of the J-domain or neighboring parts of Hsp40 in the peptide-binding domain In the presence of BiP substrates, Hsp40s dissociate from BiP, and the polypeptide substrates are trapped by BiP Next, BiP-bound ADP is exchanged for ATP, and the above-mentioned conformational change in the peptide-binding domain is reversed Substrate is released, and BiP is ready for the next round of the cycle Typically, ADP–ATP exchange is catalyzed by a nucleotide exchange factor, such as Grp170 in the pancreatic ER, that has a high affinity for BiP–ADP We note that our observation of a stable complex of Grp170 and BiP is perfectly in line with previous observations of chaperone complexes in the ER lumen [52,53] Dog pancreas microsomes were prepared as previously described [13] The microsomes were stripped with respect to ribosomes according to published procedures [11] After reisolation of microsomes by centrifugation, the pellets were resuspended in extraction buffer (20 mm Hepes ⁄ KOH, pH 7.5, 400 mm KCl, mm EDTA, 1.5 mm MgCl2, mm dithiothreitol, 15% w ⁄ v glycerol, 0.65% w ⁄ v Chaps), resulting in a crude extract Typically, the ribosomes were pelleted by centrifugation for 30 at °C and 240 000 g in a Beckman TLA 100.3 rotor Purification of ATP-binding proteins was carried out on ATP-C8-agarose as described previously [13] Where indicated, the ATP eluate was concentrated in Centricon devices and subjected ă to Akta chromatography in a Superose 6B column (16 · 500 mm) (GE Healthcare) The running buffer was identical to the extraction buffer, except that glycerol was omitted, KCl was reduced to 200 mm, and mm Mg-ATP was added where indicated FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS A Weitzmann et al Purification of recombinant proteins Purification of recombinant GST and GST hybrid proteins was carried out on GSH-Sepharose Fast Flow in a manner similar to that described for purification of a GST– Sec63J hybrid [21] Sil1 was purified after cleavage of GST-Sil1 hybrid with thrombin Purification of His-tagged murine BiP is described elsewhere [33] Antibodies Antibodies against Sil1 were raised after cleavage of GSTSil1 hybrid with thrombin Antibodies against Grp170 (LAVMSVDLGSEC), ERj4 (TVQTENRFHGSSKHC) and ERj5 (CLRNQGKRNKDEL) were raised against the indicated peptides plus an additional N-terminal or C-terminal cysteine as previously described [21] The antipeptide antibodies were affinity purified and immobilized on a mixture of protein A–Sepharose and protein G–Sepharose as previously described [21] Immunoaffinity purification was carried out in the above-defined gel filtration buffer as previously described [21] Quantitation of proteins in dog pancreas microsomes The amount of protein present in the band of a gel of a GST hybrid preparation was determined by comparison with protein standards that were separated on the same gel and were stained with Coomassie Brilliant Blue simultaneously Subsequently, an aliquot of the same sample of purified GST hybrid protein was separated on the same gel together with increasing amounts of microsomes [21] The known amount of purified protein served as a standard for the western blot signals, as determined by luminescence and densitometry of the X-ray films Pull-down assay GST or a GST hybrid was immobilized on GSH–Sepharose The supernatant, derived from the detergent extract of microsomes, was diluted by the addition of one volume of salt-free extraction buffer and applied to the immobilized proteins in the absence or in the presence of ATP (2 mm) The columns were washed with application buffer [21] The bound proteins were eluted and subjected to SDS ⁄ PAGE, and subsequent staining with Coomassie Brilliant Blue SPR spectroscopy SPR spectroscopy was carried out in a BIAlite upgrade system as previously described [21] Briefly, monoclonal goat anti-GST serum (BIACORE, Freiburg, Germany) was immobilized on a CM5 research grade sensor chip Pancreatic endoplasmic reticulum chaperone network (BIACORE) by amine coupling according to the manufacturer’s protocol The chip was equilibrated with application buffer supplemented with ATP (final concentration: mm) and Tween-20 (final concentration: 0.1%), termed running buffer (flow rate: 15 lLỈmin)1) GST–J-domain hybrid was bound to the immobilized antibodies in the measuring cell Similarly immobilized GST in the reference cell served as a negative control Subsequently, solutions containing increasing concentrations of purified BiP (0.05–12 lm) were passed over the chip in the presence of ATP Each BiP application was followed by application of running buffer The analysis was carried out employing the BIA evaluation software version 2.2.4 (BIACORE) Steady-state ATPase assay Steady-state assays were incubated for 60 at 37 °C in 40 mm Hepes ⁄ KOH (pH 7.4), 25 mm KCl, and 2.5 mm MgCl2 [21] The standard protein concentrations were: Sil1, lm; BiP, lm; Hsp40, lm; and Grp170, 0.25 lm Reactions were started by adding 5% (v ⁄ v) of an ATP cocktail that contained 0.1 lCi of [32P]ATP[cP] (4500 CiỈmmol)1) per 100 lL plus 10 mm unlabeled ATP Aliquots were removed from the assay at the indicated times and quenched with an equal volume of 60 mm EDTA The samples were analyzed by TLC on polyethyleneimide–cellulose in m formiate 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cochaperone of BiP Nat Genet 37, 974–979 51 Mayer M, Reinstein J & Buchner J (2003) Modulation of the ATPase cycle of BiP by peptides and proteins J Mol Biol 330, 137–144 52 Tatu U & Helenius A (1997) Interactions between newly synthesized glycoproteins, calnexin and a network of resident chaperones in the endoplasmic reticulum J Cell Biol 136, 555–565 53 Meunier L, Usherwood Y-K, Chung KT & Hendershot LM (2002) A subset of chaperones and folding enzymes form multiprotein complexes in endoplasmic reticulum to bind nascent proteins Mol Biol Cell 13, 4456–4469 54 Guth S, Volzing C, Muller A, Lerner M, Jung M & ă ă Zimmermann R (2004) Protein transport into canine pancreatic microsomes: a quantitative approach Eur J Biochem 271, 3200–3207 FEBS Journal 274 (2007) 5175–5187 ª 2007 The Authors Journal compilation ª 2007 FEBS 5187 ... material were separated by centrifugation, and analyzed by SDS ⁄ PAGE and protein staining with Coomassie Brilliant Blue The protein ladder was run on the same gel (lane 5) ATPase assays were carried... there was hardly any overlap of the two proteins, and they more or less eluted according to their theoretical molecular masses (70 and 140 kDa; Fig 6B) Apparently, the two Hsp70type molecular chaperones.. .Pancreatic endoplasmic reticulum chaperone network A Weitzmann et al ERj1 yeast ortholog Kar2p in Sec61 gating [18] A mammalian ortholog of yeast protein Sec63p was shown to be an abundant protein