Cystic fibrosis transmembrane conductance regulator degradation depends on the lectins Htm1p/EDEM and the Cdc48 protein complex in yeast

Abstract

Cystic fibrosis is the most widespread hereditary disease among the white population caused by different mutations of the apical membrane ATP-binding cassette transporter cystic fibrosis transmembrane conductance regulator (CFTR). Its most common mutation, DeltaF508, leads to nearly complete degradation via endoplasmic reticulum-associated degradation (ERAD). Elucidation of the quality control and degradation mechanisms might give rise to new therapeutic approaches to cure this disease. In the yeast Saccharomyces cerevisiae, a variety of components of the protein quality control and degradation system have been identified. Nearly all of these components share homology with mammalian counterparts. We therefore used yeast mutants defective in the ERAD system to identify new components that are involved in human CFTR quality control and degradation. We show the role of the lectin Htm1p in the degradation process of CFTR. Complementation of the HTM1 deficiency in yeast cells by the mammalian orthologue EDEM underlines the necessity of this lectin for CFTR degradation and highlights the similarity of quality control and ERAD in yeast and mammals. Furthermore, degradation of CFTR requires the ubiquitin protein ligases Der3p/Hrd1p and Doa10p as well as the cytosolic trimeric Cdc48p-Ufd1p-Npl4p complex. These proteins also were found to be necessary for ERAD of a mutated yeast "relative" of CFTR, Pdr5(*)p.

Figure 1.

Expression of human CFTR in yeast. A total of 3 A₆₀₀ per time point were labeled with 20 μCi/A₆₀₀ TRANS-Label. After cell lysis, protein was immunoprecipitated and detected as described under Materials and Methods. (A) Detectable expression levels of CFTR are dependent on the PDR1-3 mutation. Wild-type CFTR (WT-CFTR) and mutated protein (ΔF508-CFTR) were detected. The strain W303-1B was taken as a control without CFTR. (B) Yeast cells grown at different temperatures show decreasing CFTR expression with increasing temperatures. The highest amount of labeled CFTR was set to 100% for WT and ΔF508 CFTR independently.

Figure 2.

Influence of the yeast lectin Htm1p and its mammalian orthologue mEDEM on the degradation of CFTR. The wild-type strain HTM1 and the Δhtm1 deletion strain carried the empty yeast expression vector p413TEF as a control. mEDEM was expressed in Δhtm1 strains either from the low-copy expression plasmid p413TEF-mEDEM or the high-copy expression plasmid p423TEF-mEDEM. The Δhtm1 deletion delays the degradation rate of CFTR. Expression of mEDEM either from the low- or high-copy plasmid in the Δhtm1 strain restores the degradation rate of CFTR to wild-type levels. A total of 3 A₆₀₀ cells per time point were labeled with 20 μCi/A₆₀₀ TRANS-Label. Pulse-chases and quantification were done as described under Materials and Methods. Samples were separated by 7% SDS-PAGE and quantified with PhosphorImager cassettes. The protein quantity of time point t = 0 min was set to 100%. The graphs show average degradation rates of three experiments in wild-type (HTM1 + p413TEF) and deletion strains (Δ htm1) without (+ p413TEF) or with mEDEM (+ p413TEF-mEDEM/+ 423TEF-mEDEM) in a 90-min chase and average half life of CFTR protein. Error bars indicate the variations of the different experiments. As long as error bars do not overlap we can speak of statistically significant differences between the tested strains. The autoradiographs of 7% SDS gels show typical results.

Figure 3.

Expression and membrane localization of mouse EDEM in yeast cells. mEDEM-HA was cloned into high (p423TEF) and low-copy (p413TEF) yeast expression vectors and expressed in a Δ htm1 PDR1-3 yeast strain carrying human CFTR. Cells were grown to an A₆₀₀ of ∼1.2 and lysed with glass beads. Membranes were separated from supernatant and solubilized in buffer containing SDS and Triton X-100. Samples were separated by SDS-PAGE and blotted on nitrocellulose. Immunodetection was carried out with HA antibodies (Babco) and ECL (Amersham Biosciences UK). The empty vector p413 TEF was taken as a control. The protein was solely detectable in pellet fractions (P). The supernatant (S) was clear.

Figure 4.

Influence of mEDEM on glycosylated yeast ERAD substrates in Δhtm1 strains. Pulse-chase analysis was done as described in text. The graphs show average kinetics of three experiments of 90-min pulse-chases and average half-life in wild-type (HTM1 + p423TEF) and Δ htm1 deletion strains with (p423TEF-mEDEM) or without (p423TEF) mouse EDEM. Error bars indicate the variations of the different experiments. The autoradiographs show typical results. (A) Pdr5^*p degradation shows a barely significant increase in a strain with mEDEM expression. (B) mEDEM does not show a significant increase of CPY^* degradation.

Figure 5.

E3 ubiquitin ligases Der3p/Hrd1p and Doa10p are involved in CFTR degradation. Pulse-chase analysis was carried out as described in text. The mean values of four experiments were taken to create the kinetics and half-life graphs of CFTR protein for each, wild-type (HRD1/DOA10) and deletion strains Δ hrd1, Δ doa10 and the double deletion strain Δ hrd1/Δ doa10. The autoradiographs show typical results.

Figure 6.

AAA ATPase Cdc48 is involved in both yeast and mammalian polytopic transmembrane protein degradation. With shift to starvation media, cells were grown at the semipermissive temperature of 23°C. A total of 3 A₆₀₀ per time point were labeled with 20 μCi/A₆₀₀ TRANS-Label. Pulse-chase experiments and CFTR quantification were done as described in text. (A) CFTR is strongly stabilized in a cdc48-1 mutant. The graphs show average kinetics of four experiments of degradation in the 90-min pulse-chase and average half-life of CFTR in wild-type (CDC48) and mutant strains (cdc48-1). Error bars indicate the variations of the different experiments. The autoradiograph shows a typical result. (B) same as in A but for the mutated yeast ABC transporter protein Pdr5^*p, which is also strongly stabilized.

Figure 7.

An ufd1-1 mutant strain shows significant slow down of CFTR and Pdr5^*p degradation. Strains were treated as described in text. (A) The graphs show average kinetics of three experiments of degradation in the 100-min pulse-chase and average half-life of CFTR in wild-type (UFD1) and mutant strains (ufd1-1). Error bars indicate the variations of the different experiments. The autoradiograph shows a typical result. (B) Same as in A but for the mutated yeast ABC transporter protein Pdr5^*p.