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• W2130619361 abstract "Article15 March 1999free access The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis Geoffrey C. Meacham Geoffrey C. Meacham Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Zhen Lu Zhen Lu Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Scott King Scott King Departments of Physiology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Eric Sorscher Eric Sorscher Departments of Physiology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Albert Tousson Albert Tousson Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Douglas M. Cyr Corresponding Author Douglas M. Cyr Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Geoffrey C. Meacham Geoffrey C. Meacham Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Zhen Lu Zhen Lu Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Scott King Scott King Departments of Physiology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Eric Sorscher Eric Sorscher Departments of Physiology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Albert Tousson Albert Tousson Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Douglas M. Cyr Corresponding Author Douglas M. Cyr Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA Search for more papers by this author Author Information Geoffrey C. Meacham1, Zhen Lu1, Scott King2,3, Eric Sorscher2,3, Albert Tousson1,3 and Douglas M. Cyr 1,3 1Departments of Cell Biology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA 2Departments of Physiology, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA 3Gregory Fleming James Cystic Fibrosis Center, School of Medicine and Dentistry, University of Alabama Medical Center, Birmingham, AL, 35209 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:1492-1505https://doi.org/10.1093/emboj/18.6.1492 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The cystic fibrosis transmembrane conductance regulator (CFTR) is a chloride ion channel constructed from two membrane-spanning domains (MSDs), two nucleotide-binding domains (NBD) and a regulatory (R) domain. The NBDs and R-domain are cytosolic and how they are assembled with the MSDs to achieve the native CFTR structure is not clear. Human DnaJ 2 (Hdj-2) is a co-chaperone of heat shock cognate 70 (Hsc70) which is localized to the cytosolic face of the ER. Whether Hdj-2 directs Hsc70 to facilitate the assembly of cytosolic regions on CFTR was investigated. We report that immature ER forms of CFTR and ΔF508 CFTR can be isolated in complexes with Hdj-2 and Hsc70. The ΔF508 mutation is localized in NBD1 and causes the CFTR to misfold. Levels of complex formation between ΔF508 CFTR and Hdj-2/Hsp70 were ∼2-fold higher than those with CFTR. The earliest stage at which Hdj-2/Hsc70 could bind CFTR translation intermediates coincided with the expression of NBD1 in the cytosol. Interestingly, complex formation between Hdj-2 and nascent CFTR was greatly reduced after expression of the R-domain. In experiments with purified components, Hdj-2 and Hsc70 acted synergistically to suppress NBD1 aggregation. Collectively, these data suggest that Hdj-2 and Hsc70 facilitate early steps in CFTR assembly. A putative step in the CFTR folding pathway catalyzed by Hdj-2/Hsc70 is the formation of an intramolecular NBD1–R-domain complex. Whether this step is defective in the biogenesis of ΔF508 CFTR will be discussed. Introduction How cytosolic regions of polytopic membrane proteins fold and assemble into native conformations is a fundamental question in biology. Biogenesis of multiple domain proteins is proposed to proceed via a sequential pathway with sub-domain folding initiating co-translationally and final assembly occurring after completion of synthesis (Fedorov and Baldwin, 1997; Netzer and Hartl, 1997). The folding of extracellular domains on membrane proteins in the ER has been the subject of intense study in recent years and numerous lumenal protein-folding catalysts which facilitate this process have been identified (Hurtley and Helenius, 1989; Gaut and Hendershot, 1993). However, many membrane proteins possess large sub-domains which are exposed to the cytosol and how the cell facilitates the folding/assembly of this class of polypeptide is not clear. For instance, little information is available to describe steps in the assembly pathway of ATP binding cassette proteins (Higgins, 1992) such as the cystic fibrosis transmembrane conductance regulator (CFTR) (Riordan et al., 1989). CFTR is a 140 kDa glycoprotein which contains two membrane-spanning domains (MSDs) and two large cytosolic regions (Riordan et al., 1989). The first cytosolic region of CFTR is the size of an average protein (∼500 amino acid residues) and contains two sub-domains, a nucleotide-binding domain (NBD1) and a regulatory (R) domain. The second cytoplasmic region is separated from the first by MSD2 and contains an additional nucleotide-binding fold termed NBD2. The sub-domains of CFTR assemble to form a channel which is proposed to conduct small anions, water and ATP (Anderson et al., 1991). The phosphorylation state of the R-domain controls the channel activity of CFTR via a mechanism which involves modulation of NBD1's affinity for ATP (Winter and Welsh, 1997). Assembly of the CFTR channel initiates with its synthesis and folding in the ER and it can attain its native conformation in this cellular location (Pasyk and Foskett, 1995). Numerous reports indicate that early steps in CFTR biogenesis are inefficient. The majority of CFTR and almost all of ΔF508 CFTR, the mutant identified in most cystic fibrosis cases, never reach the plasma membrane and are degraded in a pre-Golgi compartment via a pathway which involves the proteasome (Yang et al., 1993; Lukacs et al., 1994; Ward and Kopito, 1994; Jensen et al., 1995; Ward et al., 1995). Inefficiencies in ΔF508 CFTR processing can be ameliorated by the growth of cells at low temperature (Denning et al., 1992) or by addition of chemical chaperones to cell culture media (Brown et al., 1996; Sato et al., 1996). Thus, folding/assembly defects appear to cause nascent ΔF508 CFTR to be diverted from the biosynthetic pathway to degradative pathways (Denning et al., 1992). Defective biogenesis of ΔF508 CFTR contributes to the clinical pathology of cystic fibrosis (Welsh and Ostedgaard, 1998). The pathway and components that facilitate the folding and assembly of CFTR are currently being identified. A fragment of CFTR that contains MSD1, NBD1 and the R-domain is functionally active, which suggests that these domains can fold and assemble independently of MSD2 and NBD2 (Sheppard et al., 1994; Xiong et al., 1997; Schwiebert et al., 1998). However, studies on the processing of the many CFTR mutants identified indicate that regions within MSD2 and NBD2 are also critical for proper folding and assembly (Welsh and Smith, 1993; Welsh and Ostedgaard, 1998). Thus, regions within the N- and C-terminus must interact in order for CFTR to form a stable ion channel. Molecular chaperones localized in the ER lumen (calnexin) and cytosol (Hsp70) have been observed to transiently interact with immature CFTR (Yang et al., 1993; Pind et al., 1994). ΔF508 CFTR–Hsp70 and ΔF508 CFTR–calnexin complexes have a longer half-life than similar complexes with CFTR. Therefore Hsp70 and calnexin may function in the ‘quality control’ system which recognizes misfolded ER proteins (Yang et al., 1993; Pind et al., 1994). CFTR was also detected in complexes with Hsp70 and calnexin, therefore these chaperones may also function to facilitate normal folding of this membrane protein. Indeed, Hsp70 was recently shown to promote the folding of a CFTR fragment that corresponds to NBD1 (Strickland et al., 1997). To facilitate protein folding, Hsp70 must interact with co-chaperone proteins that regulate its ability to bind and release non-native regions of proteins (Cyr, 1997; Frydman and Hohfeld, 1997; Hohfeld and Jentsch, 1997; Takayama et al., 1997). A major class of Hsp70 co-chaperone proteins are members of the Hsp40 (DnaJ-related) family (Cyr et al., 1994) whose progenitor is Escherichia coli DnaJ (Georgopoulos et al., 1980; Zylicz et al., 1985). The Hsp40 family is structurally diverse and members function to stimulate the Hsp70 ATP hydrolytic cycle (Liberek et al., 1991; Cyr et al., 1992) and some have the capacity to function as molecular chaperones (Wickner et al., 1991; Langer et al., 1992; Lu and Cyr, 1998a). Hsp40 proteins also act as specificity factors which direct Hsp70 to catalyze specific reactions in cellular protein metabolism (Silver and Way, 1993). Hsp40 proteins with the potential to help Hsp70 facilitate CFTR biogenesis are Human DnaJ-1 (Hdj-1) and Hdj-2. Hdj-1 interacts with Hsp70 to fold nascent polypeptides as they emerge from cytosolic ribosomes and helps protect cells from thermal stress (Ohtsuka, 1993; Frydman et al., 1994; Terada et al., 1997). Hdj-2 contains a CaaX box (C, cysteine; a, any aliphatic amino acid; X, a polar amino acid) on its C-terminus (Chellaiah et al., 1993) and is predicted to be modified with isoprenoids (Zhang and Casey, 1996). A portion of farnesylated Hsp40 proteins are typically localized to the cytosolic surface of the ER (Caplan et al., 1993). If Hdj-2 is localized to the ER, this would position it to direct cytosolic Hsp70 to facilitate the folding of CFTRs cytosolic sub-domains just as they emerge from membrane tethered ribosomes. We report that Hdj-2 is indeed localized to the cytoplasmic face of the ER and that it interacts with Heat shock cognate 70 (Hsc70) to suppress the aggregation of NBD1 on CFTR. Co-translational binding of Hdj-2/Hsc70 to NBD1 appears to stabilize it in an assembly-competent conformation and thereby promote protein assembly events which enable CFTR to reach its native state. One such assembly event appears to be the formation of intramolecular contacts between surfaces of NBD1 and the R-domain. These data suggest that Hdj-2/Hsc70 is an Hsp40/Hsp70 chaperone pair which can function to facilitate the biogenesis of membrane proteins that have large cytoplasmic domains. Results Hdj-2 co-localizes with ER markers The localization of Hsp40 co-chaperone proteins plays a critical role in determining their ability to specify Hsp70 action in cellular protein metabolism (Brodsky et al., 1993; Ungermann et al., 1994; Cyr and Neupert, 1996). To determine whether Hdj-1 or Hdj-2 was more likely to function with Hsc70 to facilitate CFTR biogenesis, the localization of these Hsp40 proteins in cultured cells was compared by indirect immunofluorescence and digital confocal microscopy (Figure 1A and B). In 0.5 μm confocal sections of cells, Hdj-2 antibodies stained the cytosol and a perinuclear compartment. Perinuclear Hdj-2 staining overlapped extensively with the pattern of the ER marker calnexin (Figure 1B). On the other hand, Hdj-1 exhibited a diffuse cytoplasmic staining pattern that exhibited little overlap with calnexin. A portion of Hdj-2 is therefore localized to the ER, while another pool appears to be soluble. Hdj-1, on the other hand, does not appear to exhibit a specific staining pattern. In related studies we determined that Hdj-2, but not Hdj-1, is modified post-translationally with the isoprenoid farnesyl (data not shown). The partial localization of Hdj-2 to the ER surface is consistent with the localization observed for other farnesylated Hsp40 proteins (Caplan et al., 1992). Hdj-2 is positioned within the cell to promote reactions in protein metabolism that occur on the ER membrane surface. Figure 1.Hdj-2 co-localizes with ER markers. (A) Decoration of CFPAC cells with rabbit polyclonal Hdj-1 and mouse monoclonal calnexin antibodies. (B) Decoration of CFPAC cells with mouse monoclonal Hdj-2 and rabbit polyclonal calnexin antibodies. To visualize the staining patterns of indicated primary antibodies, respective coverslips were decorated with goat anti-rabbit IgG–Oregon Green and goat anti-mouse IgG–Texas Red secondary antibodies. The localization of respective chaperone proteins was then determined in 0.5 μM optical sections of cells by digital confocal microscopy (see Materials and methods for details). Download figure Download PowerPoint Cytosolic Hsp70 and Hsp40 proteins can be co-immunoprecipitated with newly synthesized proteins To examine the role of Hsc70 and its co-chaperones in CFTR biogenesis, we established experimental conditions to immunoprecipitate these and other cytosolic chaperones from cell extracts (Figure 2). Hsc70, Hdj-1, Hdj-2 and Tcp-1, a subunit of the cytosolic chaperonin Tric (Frydman et al., 1994), have all been shown to participate in cellular protein folding (Hartl, 1996). Antibodies to each of these proteins were acquired from commercial sources (see Materials and methods). When the antibodies against Hsc70, Hdj-1, Hdj-2 and Tcp-1 were utilized to probe Western blots of HeLa cell extracts they cross-reacted with only a single band which migrates on SDS–PAGE gels with the proper apparent molecular weight (Figure 2A, lanes 1, 4, 7 and 10). When HeLa cell extracts were incubated with the appropriate quantity of respective chaperone antibody (see Materials and methods for details) and protein G–agarose beads (PG), <80, 100 and 85% of Hsc70, Hdj-1 and Hdj-2 could be immunodepleted. In the case of Tcp-1, we could only deplete ∼50% of it from cell lysates. When lysates were treated with PG alone little chaperone was removed and this suggested that the immunodepletion observed resulted from specific recognition of chaperone proteins by the respective antibodies against them. Figure 2.Hsc70, Hdj-1, Hdj-2 and Tcp-1 form complexes with newly synthesized proteins. (A) Western blot analysis of HeLa cell extracts before and after immunodepletion of Hsc70, Hdj-1, Hdj-2 and Tcp-1. Immunodepletions were carried out as described below for the immunoprecipitation of chaperone proteins from metabolically labeled cell extracts. The symbols (+) and (−) indicate whether or not cell lysates were incubated with the indicated antibody and PG to immunodeplete the chaperone of interest. PG denotes lysates that were treated with only protein G–agarose beads. Numbers at the bottom of the gel in (A) denote quantitation of the signal generated from the respective chaperone proteins on each Western blot. Numbers are normalized to the total signal from lysates to which no antibody or protein G–agarose was added. The * band in lane 3 represents rat IgG that remains in cell extracts after their treatment with PG and is detected by sheep anti-rat IgG-HRP. The * band in lane 9 represents mouse IgG that remains in cell extracts after their treatment with PG and is recognized by goat anti-mouse IgG–HRP. The * band in lane 12 represents rabbit IgG that remains in cell extracts after their treatment with PG and is recognized by goat anti-rabbit IgG–HRP. (B) Immuno- precipitation of cytosolic chaperones from HeLa cell extracts. HeLa cells were metabolically labeled for 20 min with Tran35S -label (100 μCi/1×106 cells). Cells were then lysed at 4°C under non-denaturing conditions in a buffer containing Mg-ATP and an ATP regenerating system as detailed in the Materials and methods section. Lysates were precleared with Pansorbin (2.0%) and antisera specific for Hsc70 (12.5 μg), Hdj-1 (0.5 μg), Hdj-2 (5.0 μg), Tcp-1 (2.5 μg) or mouse IgG (2.5 μg) was added to cell extracts (12.5 μg of total protein). Products of co-immunoprecipitation reactions were analyzed by SDS–PAGE and fluorography. IgG refers to non-immune mouse IgG that was utilized as a control to measure the level of labeled proteins that precipitate non-specifically. In the lanes labeled ‘Total Extract’ 10% of the labeled material used for co-immunoprecipitation reactions was loaded onto the gel. The mobility of molecular mass markers (in kDa) is indicated on the left of the gels. Download figure Download PowerPoint In experiments where HeLa cells were metabolically labeled and then lysed with the non-ionic detergent Triton X-100, radiolabeled Hsc70, Hdj-1, Hdj-2 and Tcp-1 could be immunoprecipitated from cell extracts (Figure 2B). The relative intensity of the immunoprecipitated bands suggested that Hdj-2 was ∼5-fold more abundant than Hdj-1, but present at concentrations that were similar to Hsc70. In Western blots, in which purified proteins were utilized as standards, Hsc70, Hdj-1, and Hdj-2 were found to represent 1.3, 0.05 and 0.23%, respectively, of total cell protein (Terada et al., 1997; data not shown). Purified Tcp-1 was not available to us, but literature results indicate that Tcp-1 is present in the cell at approximately one-third the level of Hsc70 (Frydman et al., 1994). Thus, the relative quantities of radiolabeled Hsc70, Hdj-1, Hdj-2 and Tcp-1 which we immunoprecipitated appear to accurately reflect the levels of these proteins in the cell. A broad smear of newly synthesized protein could be co-immunoprecipitated with Hsc70, Hdj-1, Hdj-2 and Tcp-1 (Figure 2B). The quantity of newly synthesized protein that co-precipitated was proportional to the level of individual chaperones and significantly above the level of non-specific precipitation observed with non-immune IgG (Figure 2B, compare lane 2 with lanes 3–6). These data demonstrate our ability to immunoprecipitate Hsc70, Hdj-1, Hdj-2 and Tcp-1 from cell lysates. In addition, these data also demonstrate that, under native buffer conditions, we are able to isolate by co-immunoprecipitation, the complexes formed between molecular chaperones and newly synthesized polypeptides. Hsc70 and Hdj-2 form complexes with CFTR and ΔF508 CFTR To examine interactions between cytosolic chaperones and biogenic intermediates of CFTR and ΔF508 CFTR these proteins were transiently expressed in HeLa cells (see Materials and methods). Cells were then metabolically labeled and the processing of CFTR was monitored as an indicator of its progression through the secretory pathway (Denning et al., 1992; Ward and Kopito, 1994). On SDS–PAGE gels, the immature ER localized biogenic intermediate of CFTR, termed the B-form, migrates with a greater mobility than the maturely glycosylated C-form which has been transported out of the ER (Ward and Kopito, 1994). Immediately after the initial labeling period, we could detect CFTR and ΔF508 CFTR in the B-form (Figure 3A and B, lane 1) and during the 180 min chase period a significant portion of CFTR was converted to the C-form (Figure 3A, lane 7). ΔF508 CFTR is unable to exit the ER (Denning et al., 1992) and therefore we did not observe conversion of its B-form to C-form (Figure 3B, compare lane 1 with 7). These data establish that the CFTR and ΔF508 CFTR we express in HeLa cells represent biogenic intermediates which behave in a manner similar to forms of these proteins that are naturally expressed in epithelial cells. Figure 3.Hsc70, Hdj-1 and Hdj-2 form complexes with immaturely glycosylated CFTR. HeLa cells were infected with vaccinia virus expressing T7 RNA polymerase (vTF7-3) and transiently transfected with (A) pCDNA-CFTR or (B) pCDNA-ΔF508 CFTR. After an 8 h incubation, cells were metabolically labeled for 45 min with Tran35S-label (100 μCi/1×106 cells). Cells were then analyzed for chaperone–CFTR complexes immediately (lanes 1–6) or incubated for an additional 180 min in DMEM + 10% FBS (lanes 8–12). The sample in lane 1 was prepared by immunoprecipitating CFTR with α-CFTR under denaturing conditions and 10% of the total protein isolated by this method was run on the gel. To isolate chaperone CFTR complexes, cell lysates were made under non-denaturing conditions, precleared with Pansorbin and antisera to the indicated chaperone proteins was added. Immune complexes were then precipitated with PG. To definitively identify CFTR in co-precipitates products of the co-immunoprecipitation reactions were split. One portion was immediately run on SDS–PAGE gels and the results from this analysis are shown in the panel denoted as Co-IP. The other portion was subjected to a second round of immunoprecipitation under denaturing conditions with α-CFTR under denaturing conditions and the results of this analysis are exhibited in the panels denoted as Re-IP. In an attempt to normalize the signals for these different experimental protocols, gels from the Re-IP experiments were exposed to X-ray film for twice as long as the Co-IPs. IgG indicates non-immune mouse IgG used as a control for co-immunoprecipitations. ‘B’ marks the mobility of the ER-localized, core-glycosylated, immature form of CFTR. ‘C’ denotes the position on gels of the complex-glycosylated, mature form of CFTR. Numbers on the left of each panel indicate the migration of molecular mass markers (in kDa). Download figure Download PowerPoint When CFTR–chaperone complexes were analyzed, a band that migrated with a mobility that was identical to that of immature CFTR, but not mature CFTR, could be co-immunoprecipitated with Hsc70, Hdj-1, Hdj-2 and Tcp-1 (Figure 3A). At T = 0, on average (n = 4), the quantity of CFTR which could be co-precipitated with Hsc70, Hdj-1, Hdj-2, and Tcp-1 represented 19, 6, 9 and 2% of total CFTR, respectively. The amount of CFTR that could be co-immunoprecipitated with Hsc70, Hdj-1 and Hdj-2 was several fold above that isolated by non-specific immunoprecipitation with non-immune IgG (Figure 3A, compare lane 2 with lanes 3, 4 and 5). However, levels of CFTR that co-precipitated with Tcp-1 were very to close to the level of background precipitation (Figure 3A, compare lanes 2 and 6). A complication to the interpretation of results from the co-immunoprecipitation experiments presented in Figure 3, was the observation that a number of radiolabeled bands in addition to the putative B-form of CFTR were also co-precipitated with chaperone proteins. These data raised the possibility that the band on gels which migrated with the same mobility as CFTR might be of some other origin. To rule out this possibility we demonstrated that the band designated as the B-form of CFTR in co-immunoprecipitates could be re-immunoprecipitated in a second reaction, under denaturing conditions, with α-CFTR sera (Figure 3A, Re-IP). A number of observations suggest that the CFTR–chaperone complexes isolated represent specific intermediates of the CFTR biogenic pathway. Firstly, the quantity of CFTR that was co-precipitated with Hsc70, Hdj-1 and Hdj-2 was routinely several times greater than the amount that was non-specifically precipitated with non-immune IgG (compare Figure 3A, lane 2 with lanes 3, 4 and 5). The total level of the CFTR–chaperone complexes formed in the cell may be underestimated in these experiments because chaperone–substrate complexes tend to dissociate during isolation (Ungermann et al., 1994, 1996). Secondly, the binding of CFTR appeared to be specific for the chaperone protein in question. CFTR formed much higher levels of complex with Hsp70 and Hsp40 proteins than with Tcp-1 (Figure 3). In contrast, Tcp-1 could co-precipitate quantities of newly synthesized protein that were similar to Hsc70 and Hdj-2 (Figure 2). Finally, the ability of Hsp70 and Hsp40 proteins to bind CFTR appeared to be dependent upon its conformation. Hsc70, Hdj-1 and Hdj-2 were observed to bind immature, but not mature forms, of CFTR (Figure 3A, compare T = 0 with T = 180). ΔF508 CFTR exhibits subtle defects in its folding pathway that cause assembly intermediates to accumulate and limit its ability to reach the native state (Qu et al., 1997; Zhang et al., 1998). To determine whether Hsp70 and Hsp40 proteins bind CFTR and ΔF508 CFTR differentially, we examined their interactions with chaperones. When interactions between ΔF508 CFTR and Hsc70, Hdj-1, Hdj-2 and Tcp-1 were examined at T = 0 and then compared with results obtained with CFTR, the levels of ΔF508 CFTR–Hsc70 and ΔF508 CFTR–Hdj-2 complexes were 1.9±0.2-fold and 1.6±0.1-fold (n = 5) more abundant than similar complexes with CFTR. However, levels of complex formed between ΔF508 CFTR and Hdj-1 and Tcp-1 appeared unchanged. To obtain these data we quantitated the amount of CFTR and ΔF508 CFTR that could be co-immunoprecipitated with chaperone proteins and then normalized these values to the total amount of CFTR and ΔF508 CFTR which was immunoprecipitated directly with α-CFTR (Figure 3A and B). Next, we compared the half-life of CFTR–chaperone and ΔF508 CFTR–chaperone complexes (Figure 4). Since complexes between Hsc70 and Hdj-2 appeared most sensitive to the ΔF508 mutation (Figure 3B), in this set of experiments, only complexes between these chaperones and CFTR were examined. CFTR and ΔF508 CFTR were expressed at similar levels and over the time course of the 90 min chase period, the signal for the immature form of each decayed in a time-dependent manner (Figure 4A). At the beginning of the chase period complexes between ΔF508 CFTR and Hsc70 and Hdj-2 were ∼2-fold more abundant than those with CFTR (Figure 4B and C, compare lanes 1 and 5). Throughout the time course of the chase period, ΔF508 CFTR–chaperone complexes became progressively more abundant than similar complexes with CFTR. Collectively these data suggest that Hsc70 and Hdj-2 exhibit differences in their ability to interact with wild-type and mutant forms of CFTR. Figure 4.Complex formation between ΔF508 CFTR and Hsc70 and Hdj-2 is greater than that with CFTR. HeLa cells were infected with vaccinia virus (vTF7-3) and transiently transfected with pCDNA-CFTR or pCDNA-ΔF508 CFTR. After an 8 h incubation, cells were metabolically labeled for 15 min with Tran35S-label (100 μCi/ 1×106 cells). Complex formation between the respective proteins and molecular chaperones were then analyzed by co-immunoprecipitation as described in the legend to Figure 3. Cell lysates were produced at the indicated time points and were precleared with Pansorbin. In (A), α-CFTR was added to cell lysates and immunoprecipitations were carried out under denaturing conditions. Ten percent of the material isolated in the immunoprecipitation reaction carried out at the respective time points was then loaded on SDS–PAGE gels and analyzed. Quantitation of gels was then performed by densitometric analysis and values shown in lanes 2–4 are normalized to the signal for immature CFTR at T = 0 in lane 1. In (B) and (C), antisera against Hsc70 and Hdj-2, respectively, was added to cell lysates and co-immunoprecipitations were carried out under non-denaturing conditions (see Materials and methods for details). Quantitation of immature CFTR and ΔF508 CFTR which could be co-immuno- precipitated with Hsc70 or Hdj-2 was performed by densitometric analysis and the values shown are expressed as % of the total CFTR and ΔF508 CFTR that could be isolated by immunoprecipitation at T = 0 and shown in (A). ‘B’ denotes the ER localized, core-glycosylated, immature form of CFTR. ‘C’ denotes the complex-glycosylated, mature form of CFTR. Numbers on the left of each panel denote the mobility of molecular mass markers in kDa. Download figure Download PowerPoint Hdj-2 and Hsc70 bind truncated fragments of CFTR in a domain-specific manner What aspects of CFTR biogenesis do Hsc70 and Hdj-2 facilitate and to what regions of the" @default.
• W2130619361 created "2016-06-24" @default.
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• W2130619361 creator A5050704941 @default.
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• W2130619361 date "1999-03-15" @default.
• W2130619361 modified "2023-10-18" @default.
• W2130619361 title "The Hdj-2/Hsc70 chaperone pair facilitates early steps in CFTR biogenesis" @default.
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