Limits...
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.

Show MeSH

Related in: MedlinePlus

EGFP-CFTR degradation depends on functional Sar1p/COPII machinery. Wild-type (A), sec12–4ts (B), sec23–1ts (C), sec13–1ts (D), and sec18–1ts (E) yeast were transformed with pCU426CUP1/EGFP-CFTR. Yeast were grown to log phase and induced at permissive temperature. An equal amount of culture was then pulse-labeled with [35S]methionine for 20 min at either 24°C or 39°C. An equal amount of culture was taken after indicated chase times and used to prepare cell lysates. Lysates were immunoprecipitated with anti-CFTR or anti-CPY (for sec18–1ts) antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR degradation is significantly inhibited by inactivation of Sec12p, Sec23p, and Sec13p.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172646&req=5

fig3: EGFP-CFTR degradation depends on functional Sar1p/COPII machinery. Wild-type (A), sec12–4ts (B), sec23–1ts (C), sec13–1ts (D), and sec18–1ts (E) yeast were transformed with pCU426CUP1/EGFP-CFTR. Yeast were grown to log phase and induced at permissive temperature. An equal amount of culture was then pulse-labeled with [35S]methionine for 20 min at either 24°C or 39°C. An equal amount of culture was taken after indicated chase times and used to prepare cell lysates. Lysates were immunoprecipitated with anti-CFTR or anti-CPY (for sec18–1ts) antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR degradation is significantly inhibited by inactivation of Sec12p, Sec23p, and Sec13p.

Mentions: To uncover whether the Sar1p/COPII machinery is also involved in CFTR degradation, we compared the degradation rate of EGFP-CFTR at permissive (24°C) and restrictive (39°C) temperature in yeast mutant in Sar1p/COPII components. In wild-type strain, EGFP-CFTR is degraded rapidly at both temperatures, with a half-life of 10–15 min (Fig. 3 A). The temperature-sensitive strain, sec12–4ts, is defective in catalyzing GDP/GTP exchange on Sar1p at the restrictive temperature due to a P73L mutation (Barlowe and Schekman, 1993). EGFP-CFTR is degraded rapidly in the sec12–4ts strain at permissive temperature; with a half-life (∼10 min) analogous to that in wild-type strain (Fig. 3 B). In contrast, the degradation rate is delayed significantly when the yeast is shifted to the restrictive temperature, with the half-life extending to 40–45 min, a threefold increase compared with the permissive temperature. The temperature-sensitive strains sec23–1ts and sec13–1ts (Salama et al., 1997) are defective at the restrictive temperature due to an S382L mutation (Yoshihisa et al., 1993), and a mutation that has not yet been identified, respectively. In both cases, COPII function is compromised. EGFP-CFTR is degraded rapidly in both sec23–1ts and sec13–1ts strains at the permissive temperature, with half-lives of <15 min (Fig. 3, C and D). In contrast, the degradation rates are significantly delayed in both strains at the restrictive temperatures, with a half-life of ∼50 min. The significant inhibition of EGFP-CFTR degradation suggests that functional COPII machinery is required for proteasomal degradation of EGFP-CFTR.


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 degradation depends on functional Sar1p/COPII machinery. Wild-type (A), sec12–4ts (B), sec23–1ts (C), sec13–1ts (D), and sec18–1ts (E) yeast were transformed with pCU426CUP1/EGFP-CFTR. Yeast were grown to log phase and induced at permissive temperature. An equal amount of culture was then pulse-labeled with [35S]methionine for 20 min at either 24°C or 39°C. An equal amount of culture was taken after indicated chase times and used to prepare cell lysates. Lysates were immunoprecipitated with anti-CFTR or anti-CPY (for sec18–1ts) antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR degradation is significantly inhibited by inactivation of Sec12p, Sec23p, and Sec13p.
© Copyright Policy
Related In: Results  -  Collection

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

fig3: EGFP-CFTR degradation depends on functional Sar1p/COPII machinery. Wild-type (A), sec12–4ts (B), sec23–1ts (C), sec13–1ts (D), and sec18–1ts (E) yeast were transformed with pCU426CUP1/EGFP-CFTR. Yeast were grown to log phase and induced at permissive temperature. An equal amount of culture was then pulse-labeled with [35S]methionine for 20 min at either 24°C or 39°C. An equal amount of culture was taken after indicated chase times and used to prepare cell lysates. Lysates were immunoprecipitated with anti-CFTR or anti-CPY (for sec18–1ts) antibody. Relative intensities of EGFP-CFTR bands were quantitated. EGFP-CFTR degradation is significantly inhibited by inactivation of Sec12p, Sec23p, and Sec13p.
Mentions: To uncover whether the Sar1p/COPII machinery is also involved in CFTR degradation, we compared the degradation rate of EGFP-CFTR at permissive (24°C) and restrictive (39°C) temperature in yeast mutant in Sar1p/COPII components. In wild-type strain, EGFP-CFTR is degraded rapidly at both temperatures, with a half-life of 10–15 min (Fig. 3 A). The temperature-sensitive strain, sec12–4ts, is defective in catalyzing GDP/GTP exchange on Sar1p at the restrictive temperature due to a P73L mutation (Barlowe and Schekman, 1993). EGFP-CFTR is degraded rapidly in the sec12–4ts strain at permissive temperature; with a half-life (∼10 min) analogous to that in wild-type strain (Fig. 3 B). In contrast, the degradation rate is delayed significantly when the yeast is shifted to the restrictive temperature, with the half-life extending to 40–45 min, a threefold increase compared with the permissive temperature. The temperature-sensitive strains sec23–1ts and sec13–1ts (Salama et al., 1997) are defective at the restrictive temperature due to an S382L mutation (Yoshihisa et al., 1993), and a mutation that has not yet been identified, respectively. In both cases, COPII function is compromised. EGFP-CFTR is degraded rapidly in both sec23–1ts and sec13–1ts strains at the permissive temperature, with half-lives of <15 min (Fig. 3, C and D). In contrast, the degradation rates are significantly delayed in both strains at the restrictive temperatures, with a half-life of ∼50 min. The significant inhibition of EGFP-CFTR degradation suggests that functional COPII machinery is required for proteasomal degradation of EGFP-CFTR.

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