XB-ART-7077J Biol Chem. August 9, 2002; 277 (32): 28948-58.
Cysteine string protein interacts with and modulates the maturation of the cystic fibrosis transmembrane conductance regulator.
The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-regulated chloride channel whose phosphorylation regulates both channel gating and its trafficking at the plasma membrane. Cysteine string proteins (Csps) are J-domain-containing, membrane-associated proteins that have been functionally implicated in regulated exocytosis. Therefore, we evaluated the possibility that Csp is involved in regulated CFTR trafficking. We found Csp expressed in mammalian epithelial cell lines, several of which express CFTR. In Calu-3 airway cells, immunofluorescence colocalized Csp with calnexin in the endoplasmic reticulum and with CFTR at the apical membrane domain. CFTR coprecipitated with Csp from Calu-3 cell lysates. Csp associated with both core-glycosylated immature and fully glycosylated mature CFTRs (bands B and C); however, in relation to the endogenous levels of the B and C bands expressed in Calu-3 cells, the Csp interaction with band B predominated. In vitro protein binding assays detected physical interactions of both mammalian Csp isoforms with the CFTR R-domain and the N terminus, having submicromolar affinities. In Xenopus oocytes expressing CFTR, Csp overexpression decreased the chloride current and membrane capacitance increases evoked by cAMP stimulation and decreased the levels of CFTR protein detected by immunoblot. In mammalian cells, the steady-state expression of CFTR band C was eliminated, and pulse-chase studies showed that Csp coexpression blocked the conversion of immature to mature CFTR and stabilized band B. These results demonstrate a primary role for Csp in CFTR protein maturation. The physical interaction of this Hsc70-binding protein with immature CFTR, its localization in the endoplasmic reticulum, and the decrease in production of mature CFTR observed during Csp overexpression reflect a role for Csp in CFTR biogenesis. The documented role of Csp in regulated exocytosis, its interaction with mature CFTR, and its coexpression with CFTR at the apical membrane domain of epithelial cells may reflect also a role for Csp in regulated CFTR trafficking at the plasma membrane.
PubMed ID: 12039948
Article link: J Biol Chem.
Grant support: DK56490 NIDDK NIH HHS
Genes referenced: alb calu canx cftr hspa8 myc rcan1 stx1a
Article Images: [+] show captions
|Figure 1 Csps are expressed in various epithelial cell lines. Panel A, homogenates (15 μg of protein) from the indicated cell lines were probed with Csp antiserum. Panel B, reverse transcription-PCR was performed as described under “Experimental Procedures.” No cDNA was added in the PCR designated Control.|
|Figure 2 Colocalization of Csp with CFTR or with calnexin in Calu-3 cells. Calu-3 cells were labeled with polyclonal Csp antiserum and monoclonal CFTR R-domain or calnexin antibodies. Labeling was detected by Alexa488 (greenindicating Csp) or Alexa568 (red indicating CFTR inpanels A–E or calnexin in panel F). Serial images in the xy plane were collected every 0.5 μm through the specimen depth; xy images (panels A–D), taken from cell apex to base, are separated by 2.5 μm. Panel E shows the xz scan of the region indicated in panel A. Scale bar, 10 μm. Images were collected by confocal microscopy and analyzed using Metamorph (Universal Imaging) software.|
|Figure 3 Coimmunoprecipitation of Csp and CFTR. Calu-3 membrane extract was mixed with Csp or CFTR antibody and precipitated as described under “Experimental Procedures.” Immunoblots were probed with anti-CFTR (1:2,000, panel A) or anti-Csp antibodies (1:2,000, panel B). Calu-3 cell membrane extract was loaded as a positive control (panels A andB, lanes 1). Antibodies used for immunoprecipitation were nonimmune IgG and anti-Csp (panel A, lanes 2 and 3) and anti-GST and anti-CFTR-NBD1 (panel B, lanes 2 and3). The lower band denoted Csp inpanel B likely represents deacylated Csp, produced by the coimmunoprecipitation conditions, which include overnight incubation with detergent. This outcome was noted by others (43, 47).Hc indicates antibody heavy chain.|
|Figure 4 Csp interacts with the R-domain and N terminus of CFTR. Bead-immobilized GST-CFTR fusion proteins were incubated with Calu-3 membrane extracts. After washing, samples were resolved on 12% SDS-PAGE. Coomassie Blue staining was used to determine protein loading (panel A); membranes were blotted with Csp antiserum (panel B). GST-CFTR fusion proteins:lane 1, GST; lane 2, GST-R; lane 3, GST-C; lane 4, GST-N; lane 5, GST-N random;lane 6, GST-syntaxin 1A. BSA, bovine serum albumin.|
|Figure 5 Quantitative Csp interactions with the CFTR R-domain and N terminus. Immobilized GST-CFTR fusion proteins were incubated with 35S-Csp1 or 35S-Csp2, washed, and samples were resolved by 15% SDS-PAGE. Csp binding was detected by autoradiography (panel A) and liquid scintillation counting (panel B). Mean data from three independent experiments are shown. Asterisks indicate a significant difference from the GST control. Lane 1, GST; lane 2, GST-R;lane 3, GST-C; lane 4, GST-N; lane 5, GST-NBD1; S1 or S2, 1 μl of 35S-Csp1 or35S-Csp2 loaded as a positive control. The higher molecular mass bands are thought to represent Csp dimers and have been observed previously (47).|
|Figure 6 Affinity of Csp binding to CFTR-R and CFTR-N. Panels A and B, immobilized GST-CFTR fusion proteins were incubated with 35S-Csp1 in the presence and absence of five concentrations of unlabeled Csp1 and binding detected by autoradiography (upper panels) or densitometric quantification after autoradiography (lower panels).Lane B, 35S-Csp1 omitted as a negative control;lane C, equivalent amount of 35S-Csp1 added as a positive control.|
|Figure 8 Csp coexpression reduces oocyte CFTR protein levels. cRNAs encoding CFTR (1 ng) and Csp isoforms (5 ng) were injected into Xenopus oocytes as indicated. Upper panel, results from CFTR immunoprecipitation. Immunoprecipitation was performed with anti-CFTR monoclonal M3A7, and the immunoblot was probed with anti-CFTR polyclonal R3195 antibodies. Lane 1, uninjected control; lane 2, CFTR alone; lane 3, CFTR plus Csp1; lane 4, CFTR plus Csp2; lane 5, CFTR plus Csp2 double mutant; lane 6, HEK293 cells transiently expressing CFTR used as a positive control. Lower panel, cAMP-stimulated chloride currents recorded from the oocyte populations used for the immunoprecipitations. Data are from five pooled oocytes for immunoprecipitation (50 μg of protein/lane) and four or five oocytes for ΔI Cl under each condition. Recordings/immunoprecipitations were performed 4 days after injection.|
|Figure 9 Effect of Csp on CFTR biogenesis. Panel A, HEK293 cells were transfected with CFTR (lane 2), CFTR plus Myc-Csp1 (lane 3), CFTR plus Myc-Csp2 (lane 4), and CFTR plus Myc-Csp2 double mutant (lane 5).Lane 1, nontransfected control. After 24 h, 50 μg of cell extract was resolved by 7% or by 15% mini-SDS-PAGE, and blots were probed with polyclonal anti-CFTR (R3195, 1:2,000), monoclonal anti-Hsc70 (1:2,000), or monoclonal anti-c-Myc (9E10, 1:2,000, Sigma) antibodies. B and C indicate the positions of immature and mature CFTR, respectively. Panel B, HEK293 cells were transfected with VSV-G (lane 1) or VSV-G plus Csp1 (lane 2); blots were probed with anti-VSV-G polyclonal (1:5,000). Experiments were otherwise performed as in panel A.|
|Figure 10 Pulse-chase analysis of CFTR biogenesis in HEK293 cells, performed as described under “Experimental Procedures.” TTF indicates the transient transfection conditions. 35S-Labeled CFTR was immunoprecipitated using 3 μg of monoclonal CFTR antibody M3A7. Samples were resolved on SDS-PAGE, and CFTR was revealed by autoradiography. The intensities of CFTR C and B forms at the indicated times were quantified by densitometry and are expressed as a percentage of CFTR B form at chase time = 0 (taken as 100%).|