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Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.

Fu L, Sztul E - J. Cell Biol. (2003)

Bottom Line: Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER.We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway.These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

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EGFP-CFTR represents yeast ERAD substrate. (A) Yeast transformed with pCU426CUP1 (EGFP-CFTR, − lane) or pCU426CUP1/EGFP-CFTR (EGFP-CFTR, + lanes) were induced, labeled with [35S]methionine for 20 min, and lysed. Lysates were immunoprecipitated with anti-CFTR, anti-F1β, or anti-GFP pAbs. A band of 175 kD, appropriate for a chimera of EGFP and CFTR, can be immunoprecipitated with anti-CFTR and anti-GFP antibodies from cells expressing EGFP-CFTR. (B) Wild-type, pre1–1, and ubc6Δ yeast were transformed with pCU426CUP1/EGFP-CFTR, induced, and pulse-labeled with [35S]methionine for 20 min. An equal amount of culture was taken at each chase time, lysed, and the lysates were immunoprecipitated with anti-CFTR antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR is stabilized in pre1–1 and ubc6Δ strains.
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fig1: EGFP-CFTR represents yeast ERAD substrate. (A) Yeast transformed with pCU426CUP1 (EGFP-CFTR, − lane) or pCU426CUP1/EGFP-CFTR (EGFP-CFTR, + lanes) were induced, labeled with [35S]methionine for 20 min, and lysed. Lysates were immunoprecipitated with anti-CFTR, anti-F1β, or anti-GFP pAbs. A band of 175 kD, appropriate for a chimera of EGFP and CFTR, can be immunoprecipitated with anti-CFTR and anti-GFP antibodies from cells expressing EGFP-CFTR. (B) Wild-type, pre1–1, and ubc6Δ yeast were transformed with pCU426CUP1/EGFP-CFTR, induced, and pulse-labeled with [35S]methionine for 20 min. An equal amount of culture was taken at each chase time, lysed, and the lysates were immunoprecipitated with anti-CFTR antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR is stabilized in pre1–1 and ubc6Δ strains.

Mentions: To provide a substrate for our studies, we generated a construct containing EGFP fused in frame to the NH2 terminus of human CFTR in a yeast expression vector under a copper-inducible promoter. As shown in Fig. 1 A, EGFP-CFTR is detected after immunoprecipitation with anti-CFTR antibody and anti-GFP antibody after copper induction of wild-type yeast carrying the EGFP-CFTR plasmid (Fig. 1 A, lanes 1 and 4), but not from yeast carrying an empty vector (Fig. 1 A, lane 3). EGFP-CFTR is not seen in a sample precipitated with anti-F1β antibody. The mobility of EGFP-CFTR (∼175 kD) is appropriate for a chimera of EGFP (∼27 kD) and full-length human CFTR (∼145 kD).


Traffic-independent function of the Sar1p/COPII machinery in proteasomal sorting of the cystic fibrosis transmembrane conductance regulator.

Fu L, Sztul E - J. Cell Biol. (2003)

EGFP-CFTR represents yeast ERAD substrate. (A) Yeast transformed with pCU426CUP1 (EGFP-CFTR, − lane) or pCU426CUP1/EGFP-CFTR (EGFP-CFTR, + lanes) were induced, labeled with [35S]methionine for 20 min, and lysed. Lysates were immunoprecipitated with anti-CFTR, anti-F1β, or anti-GFP pAbs. A band of 175 kD, appropriate for a chimera of EGFP and CFTR, can be immunoprecipitated with anti-CFTR and anti-GFP antibodies from cells expressing EGFP-CFTR. (B) Wild-type, pre1–1, and ubc6Δ yeast were transformed with pCU426CUP1/EGFP-CFTR, induced, and pulse-labeled with [35S]methionine for 20 min. An equal amount of culture was taken at each chase time, lysed, and the lysates were immunoprecipitated with anti-CFTR antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR is stabilized in pre1–1 and ubc6Δ strains.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172646&req=5

fig1: EGFP-CFTR represents yeast ERAD substrate. (A) Yeast transformed with pCU426CUP1 (EGFP-CFTR, − lane) or pCU426CUP1/EGFP-CFTR (EGFP-CFTR, + lanes) were induced, labeled with [35S]methionine for 20 min, and lysed. Lysates were immunoprecipitated with anti-CFTR, anti-F1β, or anti-GFP pAbs. A band of 175 kD, appropriate for a chimera of EGFP and CFTR, can be immunoprecipitated with anti-CFTR and anti-GFP antibodies from cells expressing EGFP-CFTR. (B) Wild-type, pre1–1, and ubc6Δ yeast were transformed with pCU426CUP1/EGFP-CFTR, induced, and pulse-labeled with [35S]methionine for 20 min. An equal amount of culture was taken at each chase time, lysed, and the lysates were immunoprecipitated with anti-CFTR antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR is stabilized in pre1–1 and ubc6Δ strains.
Mentions: To provide a substrate for our studies, we generated a construct containing EGFP fused in frame to the NH2 terminus of human CFTR in a yeast expression vector under a copper-inducible promoter. As shown in Fig. 1 A, EGFP-CFTR is detected after immunoprecipitation with anti-CFTR antibody and anti-GFP antibody after copper induction of wild-type yeast carrying the EGFP-CFTR plasmid (Fig. 1 A, lanes 1 and 4), but not from yeast carrying an empty vector (Fig. 1 A, lane 3). EGFP-CFTR is not seen in a sample precipitated with anti-F1β antibody. The mobility of EGFP-CFTR (∼175 kD) is appropriate for a chimera of EGFP (∼27 kD) and full-length human CFTR (∼145 kD).

Bottom Line: Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER.We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway.These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell Biology, University of Alabama at Birmingham, Birmingham, AL 35294, USA.

ABSTRACT
Newly synthesized proteins that do not fold correctly in the ER are targeted for ER-associated protein degradation (ERAD) through distinct sorting mechanisms; soluble ERAD substrates require ER-Golgi transport and retrieval for degradation, whereas transmembrane ERAD substrates are retained in the ER. Retained transmembrane proteins are often sequestered into specialized ER subdomains, but the relevance of such sequestration to proteasomal degradation has not been explored. We used the yeast Saccharomyces cerevisiae and a model ERAD substrate, the cystic fibrosis transmembrane conductance regulator (CFTR), to explore whether CFTR is sequestered before degradation, to identify the molecular machinery regulating sequestration, and to analyze the relationship between sequestration and degradation. We report that CFTR is sequestered into ER subdomains containing the chaperone Kar2p, and that sequestration and CFTR degradation are disrupted in sec12ts strain (mutant in guanine-nucleotide exchange factor for Sar1p), sec13ts strain (mutant in the Sec13p component of COPII), and sec23ts strain (mutant in the Sec23p component of COPII) grown at restrictive temperature. The function of the Sar1p/COPII machinery in CFTR sequestration and degradation is independent of its role in ER-Golgi traffic. We propose that Sar1p/COPII-mediated sorting of CFTR into ER subdomains is essential for its entry into the proteasomal degradation pathway. These findings reveal a new aspect of the degradative mechanism, and suggest functional crosstalk between the secretory and the degradative pathways.

Show MeSH
Related in: MedlinePlus