Cystic Fibrosis Transmembrane Conductance Regulator Trafficking Is Mediated by the COPI Coat in Epithelial Cells

Abstract

Cystic fibrosis (CF) is caused by defects in the CF transmembrane conductance regulator (CFTR) that functions as a chloride channel in epithelial cells. The most common cause of CF is the abnormal trafficking of CFTR mutants. Therefore, understanding the cellular machineries that transit CFTR from the endoplasmic reticulum to the plasma membrane (PM) is important. The coat protein complex I (COPI) has been implicated in the anterograde and retrograde transport of proteins and lipids between the endoplasmic reticulum and the Golgi. Here, we investigated the role of COPI in CFTR trafficking. Blocking COPI recruitment to membranes by expressing an inactive form of the GBF1 guanine nucleotide exchange factor for ADP-ribosylation factor inhibits CFTR trafficking to the PM. Similarly, inhibiting COPI dissociation from membranes by expressing a constitutively active ADP-ribosylation factor 1 mutant arrests CFTR within disrupted Golgi elements. To definitively explore the relationship between COPI and CFTR in epithelial cells, we depleted ␤-COP from the human colonic epithelial cell HT-29Cl.19A using small interfering RNA. ␤-COP depletion did not affect CFTR synthesis but impaired its trafficking to the PM. The arrest occurred pre-Golgi as shown by reduced level of glycosylation. Importantly, decreased trafficking of CFTR had a functional consequence as cells depleted of ␤-COP showed decreased cAMP-activated chloride currents. To explore the mechanism of COPI action in CFTR traffic we tested whether CFTR was COPI cargo. We discovered that the ␣-, ␤-, and ␥-subunits of COPI co-immunoprecipitated with CFTR. Our results indicate that the COPI complex plays a critical role in CFTR trafficking to the PM.

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“…Intra-Golgi transport mechanisms rely heavily on COPI vesicles mediating anterograde and retrograde cargo movement. The important role of COPI in trafficking of the CFTR, which is structurally similar to P-gp, has been reported (32,45). Several sorting motifs were identified in membrane proteins, (such as KKXX or RXR motifs) regulating proper protein maturation.…”

“…P-gp K665A and R666A mutations were introduced using the QuikChange site-directed mutagenesis kit (Stratagene). Myc-tagged inactive GBF1/E794K mutant, hemagglutinin (HA)-tagged wild-type ADP-ribosylation factor 1 (ARF1), and HA-tagged constitutively active Arf1/Q71L mutant plasmids used to examine the role of the coat protein complex I (COPI) in P-gp trafficking were provided by Dr. E. Sztul (University of Alabama, Birmingham, AL) (32).…”

“…To further confirm the involvement of COPI complex in P-gp trafficking, we introduced a dominant-negative mutant protein that alters COPI dynamics and investigated its effect on sensitivity of transiently transfected cells to doxorubicin. Wildtype ARF, inactive mutant of the ARF guanine nucleotide exchange factor GBF1 (GBF1/E794K), and GTP-restricted mutant of ARF (ARF1/Q71L) coding plasmids and pcDNA4.0/ TO/myc-his (Invitrogen) as a vector control (32) were cotransfected with P-gp. It was shown by others that introduction of ARF and GBF1 mutants interferes with proper COPI function (32).…”

“…Wildtype ARF, inactive mutant of the ARF guanine nucleotide exchange factor GBF1 (GBF1/E794K), and GTP-restricted mutant of ARF (ARF1/Q71L) coding plasmids and pcDNA4.0/ TO/myc-his (Invitrogen) as a vector control (32) were cotransfected with P-gp. It was shown by others that introduction of ARF and GBF1 mutants interferes with proper COPI function (32). Results of the HEK-293T survival experiments are shown in Fig.…”

“…Participation of all three types of vesicles (clathrin-coated, COPI, and COPII-coated) has been reported for different stages of the transport, a variety of cells and conditions. Recently, significant attention has been devoted to COPI-mediated transport activity due to the discovery of the Arg-based retention signals, ability to mediate retrograde and anterograde traffic, and existence of different populations of COPI-coated vesicles (27,28,32). ABCB1 [P-glycoprotein (P-gp), ABCB subfamily], ABCC [multidrug resistance-associated proteins (MRPs)], and ABCG2 (ABCG subfamily) are examples of ABC proteins that confer drug resistance (20).…”