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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 localization depends on functional Sar1p/COPII machinery. (A) Yeast expressing EGFP-CFTR was subjected to membrane fractionation. A sample of each fraction was processed by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-CFTR antibody. T; total lysate. S; supernatant after high speed centrifugation. P1, P2, P3; pellets after low speed, medium speed, and high speed centrifugation, respectively. EGFP-CFTR is recovered exclusively in the membrane fraction. (B) Wild-type yeast expressing EGFP-CFTR was grown to log phase and processed for immunofluorescence using anti-Kar2p or anti-Kex2p antibodies, or was incubated with the fluorescent dye FM 4–64 for 45 min at 0°C, followed by 1 h at 24°C to stain the vacuole. EGFP-CFTR colocalizes only with the ER marker Kar2p. (C) Wild-type yeast transformed with pCU426CUP1 (− EGFP-CFTR) or with pCU426CUP1/EGFP-CFTR (+ EGFP-CFTR) was grown to log phase, induced, and processed for immunofluorescence with anti-Kar2p antibodies (insets) and for electron microscopy. Kar2p shows typical ER localization in − EGFP-CFTR cells, but distributes to punctate structures in + EGFP-CFTR cells. Cells without EGFP-CFTR contain normal ER (arrows), but cells expressing EGFP-CFTR show accumulation of membranous elements (arrowheads). Bar, 0.5 μm. (D) Wild-type, ubc6Δ, sec18–1ts, and sec23–1ts yeast transformed with pCU426CUP1/EGFP-CFTR was grown to log phase and induced at permissive (24°C) or restrictive (39°C) temperature. Live yeast were then imaged by fluorescence microscopy. EGFP-CFTR localizes to ER subdomains in wild-type, ubc6Δ, and sec18–1ts yeast grown at permissive or restrictive temperatures. EGFP-CFTR localizes to ER subdomains in sec23–1ts yeast grown at permissive temperature, but is diffusely distributed throughout the ER in sec23–1ts yeast grown under restrictive temperature.
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fig2: EGFP-CFTR localization depends on functional Sar1p/COPII machinery. (A) Yeast expressing EGFP-CFTR was subjected to membrane fractionation. A sample of each fraction was processed by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-CFTR antibody. T; total lysate. S; supernatant after high speed centrifugation. P1, P2, P3; pellets after low speed, medium speed, and high speed centrifugation, respectively. EGFP-CFTR is recovered exclusively in the membrane fraction. (B) Wild-type yeast expressing EGFP-CFTR was grown to log phase and processed for immunofluorescence using anti-Kar2p or anti-Kex2p antibodies, or was incubated with the fluorescent dye FM 4–64 for 45 min at 0°C, followed by 1 h at 24°C to stain the vacuole. EGFP-CFTR colocalizes only with the ER marker Kar2p. (C) Wild-type yeast transformed with pCU426CUP1 (− EGFP-CFTR) or with pCU426CUP1/EGFP-CFTR (+ EGFP-CFTR) was grown to log phase, induced, and processed for immunofluorescence with anti-Kar2p antibodies (insets) and for electron microscopy. Kar2p shows typical ER localization in − EGFP-CFTR cells, but distributes to punctate structures in + EGFP-CFTR cells. Cells without EGFP-CFTR contain normal ER (arrows), but cells expressing EGFP-CFTR show accumulation of membranous elements (arrowheads). Bar, 0.5 μm. (D) Wild-type, ubc6Δ, sec18–1ts, and sec23–1ts yeast transformed with pCU426CUP1/EGFP-CFTR was grown to log phase and induced at permissive (24°C) or restrictive (39°C) temperature. Live yeast were then imaged by fluorescence microscopy. EGFP-CFTR localizes to ER subdomains in wild-type, ubc6Δ, and sec18–1ts yeast grown at permissive or restrictive temperatures. EGFP-CFTR localizes to ER subdomains in sec23–1ts yeast grown at permissive temperature, but is diffusely distributed throughout the ER in sec23–1ts yeast grown under restrictive temperature.

Mentions: Previous studies have shown that CFTR tagged at the COOH terminus with GFP (Kiser et al., 2001) or HA (Zhang et al., 2001) is degraded as an integral membrane protein and colocalizes with the ER chaperone Kar2p in punctate ER structures before degradation. To analyze our EGFP-CFTR construct, we first tested its association with membranes. As shown in Fig. 2 A, EGFP-CFTR is detected exclusively in the membrane pellet fraction after medium speed centrifugation, indicating that it is degraded as a membrane-associated form.


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 localization depends on functional Sar1p/COPII machinery. (A) Yeast expressing EGFP-CFTR was subjected to membrane fractionation. A sample of each fraction was processed by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-CFTR antibody. T; total lysate. S; supernatant after high speed centrifugation. P1, P2, P3; pellets after low speed, medium speed, and high speed centrifugation, respectively. EGFP-CFTR is recovered exclusively in the membrane fraction. (B) Wild-type yeast expressing EGFP-CFTR was grown to log phase and processed for immunofluorescence using anti-Kar2p or anti-Kex2p antibodies, or was incubated with the fluorescent dye FM 4–64 for 45 min at 0°C, followed by 1 h at 24°C to stain the vacuole. EGFP-CFTR colocalizes only with the ER marker Kar2p. (C) Wild-type yeast transformed with pCU426CUP1 (− EGFP-CFTR) or with pCU426CUP1/EGFP-CFTR (+ EGFP-CFTR) was grown to log phase, induced, and processed for immunofluorescence with anti-Kar2p antibodies (insets) and for electron microscopy. Kar2p shows typical ER localization in − EGFP-CFTR cells, but distributes to punctate structures in + EGFP-CFTR cells. Cells without EGFP-CFTR contain normal ER (arrows), but cells expressing EGFP-CFTR show accumulation of membranous elements (arrowheads). Bar, 0.5 μm. (D) Wild-type, ubc6Δ, sec18–1ts, and sec23–1ts yeast transformed with pCU426CUP1/EGFP-CFTR was grown to log phase and induced at permissive (24°C) or restrictive (39°C) temperature. Live yeast were then imaged by fluorescence microscopy. EGFP-CFTR localizes to ER subdomains in wild-type, ubc6Δ, and sec18–1ts yeast grown at permissive or restrictive temperatures. EGFP-CFTR localizes to ER subdomains in sec23–1ts yeast grown at permissive temperature, but is diffusely distributed throughout the ER in sec23–1ts yeast grown under restrictive temperature.
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Related In: Results  -  Collection

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fig2: EGFP-CFTR localization depends on functional Sar1p/COPII machinery. (A) Yeast expressing EGFP-CFTR was subjected to membrane fractionation. A sample of each fraction was processed by SDS-PAGE, transferred to nitrocellulose membrane, and immunoblotted with anti-CFTR antibody. T; total lysate. S; supernatant after high speed centrifugation. P1, P2, P3; pellets after low speed, medium speed, and high speed centrifugation, respectively. EGFP-CFTR is recovered exclusively in the membrane fraction. (B) Wild-type yeast expressing EGFP-CFTR was grown to log phase and processed for immunofluorescence using anti-Kar2p or anti-Kex2p antibodies, or was incubated with the fluorescent dye FM 4–64 for 45 min at 0°C, followed by 1 h at 24°C to stain the vacuole. EGFP-CFTR colocalizes only with the ER marker Kar2p. (C) Wild-type yeast transformed with pCU426CUP1 (− EGFP-CFTR) or with pCU426CUP1/EGFP-CFTR (+ EGFP-CFTR) was grown to log phase, induced, and processed for immunofluorescence with anti-Kar2p antibodies (insets) and for electron microscopy. Kar2p shows typical ER localization in − EGFP-CFTR cells, but distributes to punctate structures in + EGFP-CFTR cells. Cells without EGFP-CFTR contain normal ER (arrows), but cells expressing EGFP-CFTR show accumulation of membranous elements (arrowheads). Bar, 0.5 μm. (D) Wild-type, ubc6Δ, sec18–1ts, and sec23–1ts yeast transformed with pCU426CUP1/EGFP-CFTR was grown to log phase and induced at permissive (24°C) or restrictive (39°C) temperature. Live yeast were then imaged by fluorescence microscopy. EGFP-CFTR localizes to ER subdomains in wild-type, ubc6Δ, and sec18–1ts yeast grown at permissive or restrictive temperatures. EGFP-CFTR localizes to ER subdomains in sec23–1ts yeast grown at permissive temperature, but is diffusely distributed throughout the ER in sec23–1ts yeast grown under restrictive temperature.
Mentions: Previous studies have shown that CFTR tagged at the COOH terminus with GFP (Kiser et al., 2001) or HA (Zhang et al., 2001) is degraded as an integral membrane protein and colocalizes with the ER chaperone Kar2p in punctate ER structures before degradation. To analyze our EGFP-CFTR construct, we first tested its association with membranes. As shown in Fig. 2 A, EGFP-CFTR is detected exclusively in the membrane pellet fraction after medium speed centrifugation, indicating that it is degraded as a membrane-associated form.

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