Kinetics of interactions of sendai virus envelope glycoproteins, F and HN, with endoplasmic reticulum-resident molecular chaperones, BiP, calnexin, and calreticulin

Sendai virus envelope glycoproteins, F and HN, mature during their transport through the endoplasmic reticulum (ER) and Golgi complex. To better understand their maturation processes in the ER, we investigated the time course of their interactions with three ER- resident molecular chaperones, BiP, calnexin (CNX), and calreticulin (CRT), in Sendai virus-infected HeLa cells. Pulse-chase and immunoprecipitation analyses using antibodies against each virus glycoprotein or ER chaperone revealed that F precursor interacted with CNX transiently (t(1/2)=8 min), while HN protein displayed longer and sequential interactions with BiP (t(1/2)=8 min), CNX (t(1/2)=15 min), and CRT (t(1/2)=20 min). HN interacted with the three ER chaperones not only as a monomer but also as a tetramer for several hours, suggesting mechanism(s) to undergo chaperone-mediated quality control of an assembled HN oligomer in the ER. The kinetics of dissociation of the HN-chaperone complexes exhibited a marked delay in the presence of proteasome inhibitors, suggesting that a part of HN associated with BiP, CNX, and CRT is destined to be degraded in the proteasome-dependent pathway. Further, the associations between virus glycoproteins and CNX or CRT were impaired by castanospermine, an inhibitor of ER glucosidase I and II, confirming that these interactions require monoglucosylated oligosaccharide on F(0) and HN peptides. These findings together suggest that newly synthesized F protein undergoes rapid maturation in the ER through a transient interaction with CNX, whereas HN protein requires more complex processes involving prolonged association with BiP, CNX, and CRT for its quality control in the ER.     

Loss of Specific Chaperones Involved in Membrane Glycoprotein Biosynthesis during the Maturation of Human Erythroid Progenitor Cells

The production of erythrocytes requires the massive synthesis of red cell-specific proteins including hemoglobin, cytoskeletal proteins, as well as membrane glycoproteins glycophorin A (GPA) and anion exchanger 1 (AE1). We found that during the terminal differentiation of human CD34⁺ erythroid progenitor cells in culture, key components of the endoplasmic reticulum (ER) protein translocation (Sec61α), glycosylation (OST48), and protein folding machinery, chaperones BiP, calreticulin (CRT), and Hsp90 were maintained to allow efficient red cell glycoprotein biosynthesis. Unexpected was the loss of calnexin (CNX), an ER glycoprotein chaperone, and ERp57, a protein-disulfide isomerase, as well as a major decrease of the cytosolic chaperones, Hsc70 and Hsp70, components normally involved in membrane glycoprotein folding and quality control. AE1 can traffic to the cell surface in mouse embryonic fibroblasts completely deficient in CNX or CRT, whereas disruption of the CNX/CRT-glycoprotein interactions in human K562 cells using castanospermine did not affect the cell-surface levels of endogenous GPA or expressed AE1. These results demonstrate that CNX and ERp57 are not required for major glycoprotein biosynthesis during red cell development, in contrast to their role in glycoprotein folding and quality control in other cells.The production of red blood cells involves the terminal differentiation of hematopoietic stem cells in the bone marrow followed by release into the peripheral blood (1, 2). Red blood cells remain in circulation for ∼120 days and require the prior production of abundant red cell-specific proteins including hemoglobin, cytoskeletal proteins, and membrane glycoproteins such as anion exchanger 1 (AE1)³ and glycophorin A (GPA). During differentiation, erythroid progenitor cells undergo extensive remodeling of their cytoskeleton and loss of nuclei and other organelles like the endoplasmic reticulum (ER). AE1 and GPA are known to be synthesized late in differentiation when these key cellular components are lost (3). The efficient biosynthesis of these red cell membrane glycoproteins, however, is expected to require robust ER assembly machinery involving protein translocation, N-glycosylation, and protein folding chaperones.The proper folding of membrane glycoproteins engages the quality control function of cytosolic and ER chaperone proteins (4, 5). Newly synthesized proteins undergo cycles of binding and release with chaperones, minimizing aggregation and facilitating folding. Chaperones also play a role in the retention and degradation of misfolded proteins and in apoptosis (6-8). The membrane-bound ER chaperone calnexin (CNX) and its luminal paralog calreticulin (CRT) interact with folding intermediates via their lectin and protein binding domains, thereby preventing aggregation (9). A wide variety of glycoprotein substrates have been identified, with some binding to one or both chaperones, and both have been shown to be vital in the prevention of aggregation and proper maturation of membrane glycoproteins (9, 10). Disruption of interactions with CNX and CRT can allow misfolded membrane glycoproteins to escape the ER and traffic to the plasma membrane (9).In the present study, we examined the integrity of the ER protein translocation, N-glycosylation, and quality control machinery during the differentiation of human CD34⁺ erythroid cells in culture. We found that specific components of the protein quality control system were completely lost (CNX and ERp57) or diminished (Hsc70 and Hsp70) before the production of the major glycoproteins, AE1 and GPA, was completed. Components of the protein translocation (Sec61α) and N-glycosylation machinery (OST48) were, however, maintained. Chaperones that play other roles in erythrocyte maturation and survival (CRT, BiP, and Hsp90) were also retained (11). AE1 was found to traffic efficiently to the plasma membrane in mouse embryonic fibroblasts completely lacking the ER chaperone CNX or CRT. Furthermore, disruption of CNX/CRT-glycoprotein interactions in human K562 cells did not affect the cell-surface expression of GPA or AE1. These results demonstrate that CNX and ERp57 are not required for the efficient synthesis and folding of red cell membrane glycoproteins during terminal erythropoiesis. The lack of engagement with the quality control and disulfide folding machinery may allow the more rapid production of red cell glycoproteins late in differentiation, sacrificing quality for quantity.     

Hepatic lipase maturation: a partial proteome of interacting factors

Tandem affinity purification (TAP) has been used to isolate proteins that interact with human hepatic lipase (HL) during its maturation in Chinese hamster ovary cells. Using mass spectrometry and Western blotting, we identified 28 proteins in HL-TAP isolated complexes, 16 of which localized to the endoplasmic reticulum (ER), the site of HL folding and assembly. Of the 12 remaining proteins located outside the ER, five function in protein translation or ER-associated degradation (ERAD). Components of the two major ER chaperone systems were identified, the BiP/Grp94 and the calnexin (CNX)/calreticulin (CRT) systems. All factors involved in CNX/CRT chaperone cycling were identified, including UDP-glucose:glycoprotein glucosyltransferase 1 (UGGT), glucosidase II, and the 57 kDa oxidoreductase (ERp57). We also show that CNX, and not CRT, is the lectin chaperone of choice during HL maturation. Along with the 78 kDa glucose-regulated protein (Grp78; BiP) and the 94 kDa glucose-regulated protein (Grp94), an associated peptidyl-prolyl cis-trans isomerase and protein disulfide isomerase were also detected. Finally, several factors in ERAD were identified, and we provide evidence that terminally misfolded HL is degraded by the ubiquitin-mediated proteasomal pathway. We propose that newly synthesized HL emerging from the translocon first associates with CNX, ERp57, and glucosidase II, followed by repeated posttranslational cycles of CNX binding that is mediated by UGGT. BiP/Grp94 may stabilize misfolded HL during its transition between cycles of CNX binding and may help direct its eventual degradation.     

Heterologous expression of rice calnexin (OsCNX) confers drought tolerance in Nicotiana tabacum

Calnexin (CNX) is an integral membrane protein of endoplasmic reticulum (ER) and is a critical component of ER quality control machinery. It acts as a chaperone and ensures proper folding of newly synthesised glycoproteins. CNX shares a considerable homology with its luminal counterpart calreticulin (CRT). Together, they constitute CNX/CRT cycle which is imperative for proper folding of nascent proteins. CNX deficient organisms develop severe complications because of improper folding of proteins and consequently ER stress. CNX maintains calcium homeostasis by binding to the Ca²⁺ which is a central node in various signaling pathways. Phosphorylation of cytoplasmic tail of CNX controls the sarco endoplasmic reticulum calcium ATPase and thus the movement of Ca²⁺ in and out of its store-house, i.e. ER. Our studies on Oryza sativa CNX (OsCNX) reveal constitutive expression at various developmental stages and various tissues, thereby proving its requirement throughout the plant development. Further, its expression under various stress conditions gives an insight of the crosstalk existing between ER stress and abiotic stress signaling. This was confirmed by heterologous expression of OsCNX (OsCNX-HE) in tobacco and the OsCNX-HE lines were observed to exhibit better germination under mannitol stress and survival under dehydration stress conditions. The dehydration tolerance conferred by OsCNX appears to be ABA-dependent pathway.     

Calnexin and calreticulin promote folding, delay oligomerization and suppress degradation of influenza hemagglutinin in microsomes.

Calnexin (CNX) and calreticulin (CRT) are molecular chaperones that bind preferentially to monoglucosylated trimming intermediates of glycoproteins in the endoplasmic reticulum. To determine their role in the maturation of newly synthesized glycoproteins, we analyzed the folding and trimerization of in vitro translated influenza hemagglutinin (HA) in canine pancreas microsomes under conditions in which HA's interactions with CNX and CRT could be manipulated. While CNX bound to all folding intermediates (IT1, IT2 and NT), CRT was found to associate preferentially with the earliest oxidative form (IT1). If HA's binding to CNX and CRT was inhibited using a glucosidase inhibitor, castanospermine (CST), the rate of disulfide formation and oligomerization was doubled but the overall efficiency of maturation of HA decreased due to aggregation and degradation. If, on the other hand, HA was arrested in CNX-CRT complexes, folding and trimerization were inhibited. This suggested that the action of CNX and CRT, like that of other chaperones, depended on an 'on-and-off' cycle. Taken together, these results indicated that CNX and CRT promote correct folding by inhibiting aggregation, preventing premature oxidation and oligomerization, and by suppressing degradation of incompletely folded glycopolypeptides.     

Delineation of the lectin site of the molecular chaperone calreticulin

Calreticulin (CRT) is a soluble molecular chaperone of the endoplasmic reticulum that functions to promote protein folding as well as to retain misfolded proteins. Similar to its membrane-bound paralog calnexin (CNX), CRT is a lectin that preferentially interacts with glycoproteins bearing Glc1Man5-9GlcNAc2 oligosaccharides. Although the lectin site of CNX has been delineated through X-ray crystallographic and mutagenic studies, the corresponding site for CRT has not been as well characterized. To address this issue, we attempted to construct lectin-deficient CRT mutants, using the structure of CNX as a guide to identify potential oligosaccharide-binding residues. Mutation of 4 such CRT residues (Y109, K111, Y128, D317) completely abrogated oligosaccharide binding. In contrast, mutation of CRT residues M131 and D160, which correspond to important residues in the lectin site of CNX, had no effect on oligosaccharide binding. These findings suggest that the organization of the lectin site in CRT largely resembles that of CNX but is not identical. The deficiency in oligosaccharide binding by the mutants was not due to misfolding because they exhibited wild-type protease digestion patterns, were capable of binding the thiol oxidoreductase ERp57, and functioned just as efficiently as wild-type CRT in suppressing the aggregation of the nonglycosylated substrate citrate synthase. However, they were impaired in their ability to suppress the aggregation of the glycosylated substrate jack bean alpha-mannosidase. This provides the first direct demonstration of the importance of CRT's lectin site in suppressing the aggregation of nonnative glycoproteins.     

Calreticulin facilitates the cell surface expression of ABCG5/G8

ATP-binding cassette (ABC) G5 (G5) and ABCG8 (G8) heterodimerize and function as sterol transporter that promote biliary excretion of neutral sterols. Both G5 and G8 interact with a lectin-like chaperone, calnexin (CNX), in the endoplasmic reticulum (ER) but the significance of this interaction remains unclear. Here, we show that not only CNX, but also its homologue calreticulin (CRT), is involved in the biosynthesis of G5/G8 sterol transporter. Both CNX and CRT interacted with immature forms of G5 and G8, and stimulated their productive folding by inhibiting their degradation. Interestingly, CRT predominantly enhanced the cell surface expression of mature G5/G8 whereas CNX did not have a similar effect. Inhibitors of N-glycan processing indicated that quality control of G5 and G8 might be differentially regulated in the ER. These findings clarify the role of CNX and CRT in the biosynthesis and quality control of G5/G8 sterol transporter.     

Molecular cloning of calnexin and calreticulin in the Pacific oyster Crassostrea gigas and its expression in response to air exposure

Calnexin (CNX) and calreticulin (CRT) are endoplasmic reticulum (ER) chaperones. CNX is a type I transmembrane protein and CRT is a soluble CNX homologue. In the ER, CNX and CRT are important for Ca(2+) homeostasis and protein maturation. Here, we describe the full-length cDNA of the first mollusk CNX (cgCNX) and a second mollusk CRT (cgCRT) from the oyster Crassostrea gigas. CgCNX, containing 3255bp, was composed of a 1764bp open reading frame (ORF) that encodes a 588-amino acid protein. CgCRT, containing 1727bp, was composed of a 1242bp ORF that encodes a 414-amino acid protein. CgCNX and cgCRT contains an N-terminal 21- and 16-amino acid sequence, respectively, which is characteristic of a signal sequence. At the C-terminus, cgCRT also contains the KDEL (-Lys-Asp-Glu-Leu) peptide motif suggesting that cgCRT localizes in the ER. Northern blot analysis showed that both cgCNX and cgCRT mRNAs are induced by air exposure. The expression patterns of cgCNX mRNA differed from those of cgCRT during air exposure. This suggests that these two molecular chaperones have different roles in the response to air exposure.     

Association of the thyrotropin receptor with calnexin, calreticulin and BiP. Efects on the maturation of the receptor.

The thyrotropin receptor (TSHR) is a member of the G protein-coupled receptor superfamily. It has by now been clearly established that the maturation of the glycoproteins synthesized in the endoplasmic reticulum involves interactions with molecular chaperones, which promote the folding and assembly of the glycoproteins. In this study, we investigated whether calnexin (CNX), calreticulin (CRT) and BiP, three of the main molecular chaperones present in the endoplasmic reticulum, interact with the TSHR and what effects these interactions might have on the folding of the receptor. In the first set of experiments, we observed that in a K562 cell line expressing TSHR, about 50% of the receptor synthesized was degraded by the proteasome after ubiquitination. In order to determine whether TSHR interact with CNX, CRT and BiP, coimmunoprecipitation experiments were performed. TSHR was found to be associated with all three molecular chaperones. To study the role of the interactions between CNX and CRT and the TSHR, we used castanospermine, a glucosidase I and II inhibitor that blocks the interactions between these chaperones and glycoproteins. In K562 cells expressing the TSHR, these drugs led to a faster degradation of the receptor, which indicates that these interactions contribute to stabilizing the receptor after its synthesis. The overexpression of calnexin and calreticulin in these cells stabilizes the receptor during the first hour after its synthesis, whereas the degradation of TSHR increased in a cell line overexpressing BiP and the quantity of TSHR able to acquire complex type oligosaccharides decreased. These results show that calnexin, calreticulin and BiP all interact with TSHR and that the choice made between these two chaperone systems is crucial because each of them has distinct effects on the folding and stability of this receptor at the endoplasmic reticulum level.     

Relationship between calnexin and BiP in suppressing aggregation and promoting refolding of protein and glycoprotein substrates

Calnexin (CNX) is a membrane protein of the endoplasmic reticulum that has been defined primarily as a lectin, yet is capable of functioning as a molecular chaperone with non-glycosylated proteins in vitro. Here, we assess the relative contributions of the oligosaccharide- and polypeptide-binding sites of CNX to its in vitro chaperone functions by comparing it with the Hsp70 chaperone of the endoplasmic reticulum, BiP. Both proteins were equally effective in preventing the aggregation of non-glycosylated citrate synthase, indicating that the polypeptide-binding site of CNX is capable of functioning at a level similar to that of Hsp70. However, when confronted with glycoprotein substrates, the lectin site of CNX provided a significant advantage over BiP in suppressing aggregation. CNX also cooperated with BiP and the J domain of Sec63p in the ATP-dependent refolding of glycoprotein and non-glycosylated substrates. The lectin site of CNX was essential for refolding of the glycoprotein. These findings reinforce the function of CNX as a bona fide chaperone and illustrate how its lectin site confers advantages relative to other chaperones when confronted with glycoprotein substrates.     

Role of calnexin, calreticulin, and endoplasmic reticulum mannosidase I in apolipoprotein(a) intracellular targeting

Apolipoprotein(a) [apo(a)] is a component of atherogenic lipoprotein(a) [Lp(a)]. Differences in the extent of endoplasmic reticulum (ER) associated degradation (ERAD) of apo(a) allelic variants contribute to the >1000-fold variation in plasma Lp(a) levels. Using human apo(a) transgenic mouse hepatocytes, we analyzed the role of the ER chaperones calnexin (CNX) and calreticulin (CRT), and ER mannosidase I in apo(a) intracellular targeting. Co-immunoprecipitation and pulse-chase analyses revealed similar kinetics of apo(a) interaction with CNX and CRT, peaking 15-30 min after apo(a) synthesis. Trapping of apo(a) N-linked glycans in their monoglucosylated form, by posttranslational inhibition of ER glucosidase activity with castanospermine (CST), enhanced apo(a)-CNX/CRT interaction and prevented both apo(a) secretion and ERAD. Delay of CST addition until 20 or 30 min after apo(a) synthesis [when no apo(a) had yet undergone degradation or Golgi-specific carbohydrate modification] allowed a portion of apo(a) to be secreted or degraded. These results are consistent with a transient apo(a)-CNX/CRT association and suggest that events downstream of CNX/CRT interaction determine apo(a) intracellular targeting. Inhibition of ER mannosidase I with deoxymannojirimycin or kifunensine had no effect on apo(a) secretion, but inhibited proteasome-mediated apo(a) ERAD even under conditions where apo(a)-CNX/CRT interaction was prevented. These results suggest a role for an additional, mannose-specific, ER lectin in targeting secretory proteins to the proteasome for destruction.     

Effect of doxycycline-regulated calnexin and calreticulin expression on specific thrombopoietin productivity of recombinant Chinese hamster ovary cells

In an attempt to increase the specific thrombopoietin (TPO) productivity (q(TPO)) of recombinant Chinese hamster ovary (rCHO) cells (CHO-TPO), the effect of expression level of calnexin (CNX) and calreticulin (CRT) on q(TPO) was investigated. To control both CNX and CRT expression levels simultaneously, the Tet-Off system was first introduced in CHO-TPO cells, and stable Tet-Off cells (TPO-Tet-Off) were screened by luciferase assay. The doxycycline-regulated CNX and CRT expression system in rCHO cells (TPO-CNX/CRT) was established by cotransfection of CNX and CRT expression vector and pTK-Hyg vector into TPO-Tet-Off cells and subsequent screening by Western blot analysis of CNX and CRT. The expression levels of CNX and CRT in TPO-CNX/CRT cells could be tightly controlled by adding different concentrations of doxycycline to a culture medium. Compared with the basal level (2 microg/mL doxycyline), a 2.9-fold increase in CNX expression and a 2.8-fold increase in CRT expression were obtained in the absence of doxycycline. This, in turn, resulted in a 1.9-fold increase in q(TPO), not inhibiting cell growth or changing in vivo biological activity of TPO. Taken together, these results demonstrate that a simultaneous overexpression of CNX and CRT can increase the q(TPO) of rCHO cells.     

Systematic deletion of the ER lectin chaperone genes reveals their roles in vegetative growth and male gametophyte development in Arabidopsis

Calnexin (CNX) and calreticulin (CRT) are homologous lectin chaperones in the endoplasmic reticulum (ER) that facilitate glycoprotein folding and retain folding intermediates to prevent their transit via the secretary pathway. The Arabidopsis genome has two CNX (CNX1 and CNX2) and three CRT (CRT1, CRT2 and CRT3) homologs. Despite growing evidence of the biological roles of CNXs and CRTs, little is understood about their function in Arabidopsis growth and development under normal conditions. Here, we report that the deletion of CNX1, but not of CNX2, in the crt1 crt2 crt3 triple mutation background had an adverse effect on pollen viability and pollen tube growth, leading to a significant reduction in fertility. The cnx1 crt1 crt2 crt3 quadruple mutation also conferred severe defects in growth and development, including a shortened primary root, increased root hair length and density, and reduced plant height. Disruption of all five members of the CNX/CRT family was revealed to be lethal. Finally, the abnormal phenotype of the cnx1 crt1 crt2 crt3 quadruple mutants was completely rescued by either the CNX1 or CNX2 cDNA under the control of the CNX1 promoter, suggesting functional redundancy between CNX1 and CNX2. Taken together, these results provide genetic evidence that CNX and CRT play essential and overlapping roles during vegetative growth and male gametophyte development in Arabidopsis.     

Perturbation of Hsp90 interaction with nascent CFTR prevents its maturation and accelerates its degradation by the proteasome.

Maturation of wild-type CFTR nascent chains at the endoplasmic reticulum (ER) occurs inefficiently; many disease-associated mutant forms do not mature but instead are eliminated by proteolysis involving the cytosolic proteasome. Although calnexin binds nascent CFTR via its oligosaccharide chains in the ER lumen and Hsp70 binds CFTR cytoplasmic domains, perturbation of these interactions alone is without major influence on maturation or degradation. We show that the ansamysin drugs, geldanamycin and herbimycin A, which inhibit the assembly of some signaling molecules by binding to specific sites on Hsp90 in the cytosol or Grp94 in the ER lumen, block the maturation of nascent CFTR and accelerate its degradation. The immature CFTR molecule was detected in association with Hsp90 but not with Grp94, and geldanamycin prevented the Hsp90 association. The drug-enhanced degradation was decreased by lactacystin and other proteasome inhibitors. Therefore, consistent with other examples of countervailing effects of Hsp90 and the proteasome, it would seem that this chaperone may normally contribute to CFTR folding and, when this function is interfered with by an ansamycin, there is a further shift to proteolytic degradation. This is the first direct evidence of a role for Hsp90 in the maturation of a newly synthesized integral membrane protein by interaction with its cytoplasmic domains on the ER surface.     

Glucosidase II and N-glycan mannose content regulate the half-lives of monoglucosylated species in vivo

Glucosidase II (GII) sequentially removes the two innermost glucose residues from the glycan (Glc(3)Man(9)GlcNAc(2)) transferred to proteins. GII also participates in cycles involving the lectin/chaperones calnexin (CNX) and calreticulin (CRT) as it removes the single glucose unit added to folding intermediates and misfolded glycoproteins by the UDP-Glc:glycoprotein glucosyltransferase (UGGT). GII is a heterodimer in which the α subunit (GIIα) bears the active site, and the β subunit (GIIβ) modulates GIIα activity through its C-terminal mannose 6-phosphate receptor homologous (MRH) domain. Here we report that, as already described in cell-free assays, in live Schizosaccharomyces pombe cells a decrease in the number of mannoses in the glycan results in decreased GII activity. Contrary to previously reported cell-free experiments, however, no such effect was observed in vivo for UGGT. We propose that endoplasmic reticulum α-mannosidase-mediated N-glycan demannosylation of misfolded/slow-folding glycoproteins may favor their interaction with the lectin/chaperone CNX present in S. pombe by prolonging the half-lives of the monoglucosylated glycans (S. pombe lacks CRT). Moreover, we show that even N-glycans bearing five mannoses may interact in vivo with the GIIβ MRH domain and that the N-terminal GIIβ G2B domain is involved in the GIIα-GIIβ interaction. Finally, we report that protists that transfer glycans with low mannose content to proteins have nevertheless conserved the possibility of displaying relatively long-lived monoglucosylated glycans by expressing GIIβ MRH domains with a higher specificity for glycans with high mannose content.     

Abundant accumulation of the calcium-binding molecular chaperone calreticulin in specific floral tissues of Arabidopsis thaliana.

Calreticulin (CRT) is a calcium-binding protein in the endoplasmic reticulum (ER) with an established role as a molecular chaper-one. An additional function in signal transduction, specifically in calcium distribution, is suggested but not proven. We have analyzed the expression pattern of Arabidopsis thaliana CRTs for a comparison with these proposed roles. Three CRT genes were expressed, with identities of the encoded proteins ranging from 54 to 86%. Protein motifs with established functions found in CRTs of other species were conserved. CRT was found in all of the cells in low amounts, whereas three distinct floral tissues showed abundant expression: secreting nectaries, ovules early in development, and a set of subepidermal cells near the abaxial surface of the anther. Localization in the developing endosperm, which is characterized by high protein synthesis rates, can be reconciled with a specific chaperone function. Equally, nectar production and secretion, a developmental stage marked by abundant ER, may require abundant CRT to accommodate the traffic of secretory proteins through the ER. Localization of CRT in the anthers, which are degenerating at the time of maximum expression of CRT, cannot easily be reconciled with a chaperone function but may indicate a role for CRT in anther maturation or dehiscence.     

Effects of cellular, chemical, and pharmacological chaperones on the rescue of a trafficking-defective mutant of the ATP-binding cassette transporter proteins ABCB1/ABCB4

The ATP-binding cassette transporter ABCB4 is a phosphatidylcholine translocator specifically expressed at the bile canalicular membrane in hepatocytes, highly homologous to the multidrug transporter ABCB1. Variations in the ABCB4 gene sequence cause progressive familial intrahepatic cholestasis type 3. We have shown previously that the I541F mutation, when reproduced either in ABCB1 or in ABCB4, led to retention in the endoplasmic reticulum (ER)/Golgi. Here, Madin-Darby canine kidney cells expressing ABCB1-GFP were used as a model to investigate this mutant. We show that ABCB1-I541F is not properly folded and is more susceptible to in situ protease degradation. It colocalizes and coprecipitates with the ER chaperone calnexin and coprecipitates with the cytosolic chaperone Hsc/Hsp70. Silencing of calnexin or overexpression of Hsp70 have no effect on maturation of the mutant. We also tested potential rescue by chemical and pharmacological chaperones. Thapsigargin and sodium 4-phenyl butyrate were inefficient. Glycerol improved maturation and exit of the mutant from the ER. Cyclosporin A, a competitive substrate for ABCB1, restored maturation, plasma membrane expression, and activity of ABCB1-I541F. Cyclosporin A also improved maturation of ABCB4-I541F in Madin-Darby canine kidney cells. In HepG(2) cells transfected with ABCB4-I541F cDNA, cyclosporin A allowed a significant amount of the mutant protein to reach the membrane of bile canaliculi. These results show that the best strategy to rescue conformation-defective ABCB4 mutants is provided by pharmacological chaperones that specifically target the protein. They identify cyclosporin A as a potential novel therapeutic tool for progressive familial intrahepatic cholestasis type 3 patients.