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Interplay of substrate retention and export signals in endoplasmic reticulum quality control.

Kawaguchi S, Hsu CL, Ng DT - PLoS ONE (2010)

Bottom Line: The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways.These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them.Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Endoplasmic reticulum (ER) quality control mechanisms are part of a comprehensive system to manage cell stress. The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways. The mechanisms of sorting remain unclear. While some proteins are retained statically, the classical model substrate CPY* is found in COPII transport vesicles, suggesting a retrieval mechanism for retention. However, its management can be even more dynamic. If ERAD is saturated under stress, excess CPY* traffics to the vacuole for degradation. These observations suggest that misfolded proteins might display different signals for their management.

Methodology/principal findings: Here, we report the existence of a functional ER exit signal in the pro-domain of CPY*. Compromising its integrity causes ER retention through exclusion from COPII vesicles. The signal co-exists with other signals used for retention and degradation. Physiologically, the export signal is important for stress tolerance. Disabling it converts a benign protein into one that is intrinsically cytotoxic.

Conclusions/significance: These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them. Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis.

Show MeSH

Related in: MedlinePlus

ER transport deficient CPY* variants are cytotoxic.(A) D1 and D2 variants are defective in ER vesicle budding. In vitro vesicle budding assays were performed using permeabilized cells from wild-type cells highly expressing CPY* and deletion variants. Total membranes and budded vesicles (Figure S2. “Sup”) were collected from each reaction containing cytosol/Sar1p or buffer only. Cargo packaging efficiency was analyzed by immunoblotting and quantified using the LI-COR fluorescence imaging system. A representative fluorograph is shown in Figure S2. Detection of the endogenous COPII vesicle protein, Erv25p, was included as a positive control. Three independent assays were performed for each experiment with error bars reflecting the standard deviation. Statistical significance was determined using Student's paired t-test (D1 or D2 vs. CPY* control, p<0.01). (B) Expression of D1 and D2 variants do not cause a general block in transport from the ER. Wild-type cells highly expressing CPY* and the D1 and D2 variants were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for the indicated times. Endogenous Gas1p was immunoprecipitated from detergent lysate, separated by SDS-PAGE, and visualized by phosphorimaging. (C) Wild-type cells highly expressing CPY* or its variants were grown overnight in culture medium with 3% raffinose (pre-induction). Cells were spotted on SC plates containing 2% glucose (Glc, repressed) or 2% galactose (Gal, induced) as serial dilutions of each cell culture. Plates were incubated for 2 days at 30°C.
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pone-0015532-g003: ER transport deficient CPY* variants are cytotoxic.(A) D1 and D2 variants are defective in ER vesicle budding. In vitro vesicle budding assays were performed using permeabilized cells from wild-type cells highly expressing CPY* and deletion variants. Total membranes and budded vesicles (Figure S2. “Sup”) were collected from each reaction containing cytosol/Sar1p or buffer only. Cargo packaging efficiency was analyzed by immunoblotting and quantified using the LI-COR fluorescence imaging system. A representative fluorograph is shown in Figure S2. Detection of the endogenous COPII vesicle protein, Erv25p, was included as a positive control. Three independent assays were performed for each experiment with error bars reflecting the standard deviation. Statistical significance was determined using Student's paired t-test (D1 or D2 vs. CPY* control, p<0.01). (B) Expression of D1 and D2 variants do not cause a general block in transport from the ER. Wild-type cells highly expressing CPY* and the D1 and D2 variants were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for the indicated times. Endogenous Gas1p was immunoprecipitated from detergent lysate, separated by SDS-PAGE, and visualized by phosphorimaging. (C) Wild-type cells highly expressing CPY* or its variants were grown overnight in culture medium with 3% raffinose (pre-induction). Cells were spotted on SC plates containing 2% glucose (Glc, repressed) or 2% galactose (Gal, induced) as serial dilutions of each cell culture. Plates were incubated for 2 days at 30°C.

Mentions: We next sought to determine how loss of the D1 and D2 segments disrupt CPY* trafficking. The simplest explanation posits that elements contained within them are required for ER export. Alternatively, the deletions might disrupt a vacuolar sorting signal resulting in secretion and/or retrieval of substrates. Pulse-chase analysis is consistent with the first scenario. Compared with CPY*, the D1 and D2 variants displayed little of the heterogeneous outer chain glycosylation characteristic of CPY* molecules trafficking through the Golgi (Figure 2, “hyperglycosylated” forms) [22]. To address the question directly, we applied an in vitro assay to test the packaging of CPY* variants into COPII vesicles. Semi-intact cells were prepared from wild-type strains expressing the appropriate CPY* variant. To initiate the reaction, cytosol from wild-type yeast cells and recombinant Sar1p were added along with GTP, GDP-mannose, and an ATP regeneration system [37]. Budded vesicles were recovered in the supernatant fraction, purified, and concentrated. Recovery of Erv25p (a constituent of COPII vesicles) in the vesicle fraction in the defined system and absent from control membranes demonstrates the efficacy of the assay (Average packaging efficiency, 8.9%. Figure S2A). Similarly, CPY* and the D3 through D6 variants can be packaged into COPII vesicles (Figures 3A and S2A). Likely due to high substrate levels, packaging efficiency of CPY*/variants was modest, yet nevertheless similar to previous studies using a purified microsome system [16]. By contrast, CPY*-D1 and CPY*-D2 variants were largely absent in the budded vesicle fraction (Figure 3A). These data provide an independent line of evidence that traces their transport defect to the ER vesicle budding step. To determine if CPY*-D1 and CPY*-D2 interferes with general vesicle trafficking, transport of the plasma membrane protein Gas1p was analyzed following their induction [38]. As shown in Figure 3B, Gas1p is transported from the ER efficiently in all situations after substrate induction indicating that the variants do not generally affect protein trafficking. These data show that the D1 and D2 lesions prevent the trafficking of excess CPY* to the vacuole through selective exclusion from COPII vesicles. The transport defects indicate that an ER export signal(s) was compromised by the D1 and D2 deletions.


Interplay of substrate retention and export signals in endoplasmic reticulum quality control.

Kawaguchi S, Hsu CL, Ng DT - PLoS ONE (2010)

ER transport deficient CPY* variants are cytotoxic.(A) D1 and D2 variants are defective in ER vesicle budding. In vitro vesicle budding assays were performed using permeabilized cells from wild-type cells highly expressing CPY* and deletion variants. Total membranes and budded vesicles (Figure S2. “Sup”) were collected from each reaction containing cytosol/Sar1p or buffer only. Cargo packaging efficiency was analyzed by immunoblotting and quantified using the LI-COR fluorescence imaging system. A representative fluorograph is shown in Figure S2. Detection of the endogenous COPII vesicle protein, Erv25p, was included as a positive control. Three independent assays were performed for each experiment with error bars reflecting the standard deviation. Statistical significance was determined using Student's paired t-test (D1 or D2 vs. CPY* control, p<0.01). (B) Expression of D1 and D2 variants do not cause a general block in transport from the ER. Wild-type cells highly expressing CPY* and the D1 and D2 variants were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for the indicated times. Endogenous Gas1p was immunoprecipitated from detergent lysate, separated by SDS-PAGE, and visualized by phosphorimaging. (C) Wild-type cells highly expressing CPY* or its variants were grown overnight in culture medium with 3% raffinose (pre-induction). Cells were spotted on SC plates containing 2% glucose (Glc, repressed) or 2% galactose (Gal, induced) as serial dilutions of each cell culture. Plates were incubated for 2 days at 30°C.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2991357&req=5

pone-0015532-g003: ER transport deficient CPY* variants are cytotoxic.(A) D1 and D2 variants are defective in ER vesicle budding. In vitro vesicle budding assays were performed using permeabilized cells from wild-type cells highly expressing CPY* and deletion variants. Total membranes and budded vesicles (Figure S2. “Sup”) were collected from each reaction containing cytosol/Sar1p or buffer only. Cargo packaging efficiency was analyzed by immunoblotting and quantified using the LI-COR fluorescence imaging system. A representative fluorograph is shown in Figure S2. Detection of the endogenous COPII vesicle protein, Erv25p, was included as a positive control. Three independent assays were performed for each experiment with error bars reflecting the standard deviation. Statistical significance was determined using Student's paired t-test (D1 or D2 vs. CPY* control, p<0.01). (B) Expression of D1 and D2 variants do not cause a general block in transport from the ER. Wild-type cells highly expressing CPY* and the D1 and D2 variants were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for the indicated times. Endogenous Gas1p was immunoprecipitated from detergent lysate, separated by SDS-PAGE, and visualized by phosphorimaging. (C) Wild-type cells highly expressing CPY* or its variants were grown overnight in culture medium with 3% raffinose (pre-induction). Cells were spotted on SC plates containing 2% glucose (Glc, repressed) or 2% galactose (Gal, induced) as serial dilutions of each cell culture. Plates were incubated for 2 days at 30°C.
Mentions: We next sought to determine how loss of the D1 and D2 segments disrupt CPY* trafficking. The simplest explanation posits that elements contained within them are required for ER export. Alternatively, the deletions might disrupt a vacuolar sorting signal resulting in secretion and/or retrieval of substrates. Pulse-chase analysis is consistent with the first scenario. Compared with CPY*, the D1 and D2 variants displayed little of the heterogeneous outer chain glycosylation characteristic of CPY* molecules trafficking through the Golgi (Figure 2, “hyperglycosylated” forms) [22]. To address the question directly, we applied an in vitro assay to test the packaging of CPY* variants into COPII vesicles. Semi-intact cells were prepared from wild-type strains expressing the appropriate CPY* variant. To initiate the reaction, cytosol from wild-type yeast cells and recombinant Sar1p were added along with GTP, GDP-mannose, and an ATP regeneration system [37]. Budded vesicles were recovered in the supernatant fraction, purified, and concentrated. Recovery of Erv25p (a constituent of COPII vesicles) in the vesicle fraction in the defined system and absent from control membranes demonstrates the efficacy of the assay (Average packaging efficiency, 8.9%. Figure S2A). Similarly, CPY* and the D3 through D6 variants can be packaged into COPII vesicles (Figures 3A and S2A). Likely due to high substrate levels, packaging efficiency of CPY*/variants was modest, yet nevertheless similar to previous studies using a purified microsome system [16]. By contrast, CPY*-D1 and CPY*-D2 variants were largely absent in the budded vesicle fraction (Figure 3A). These data provide an independent line of evidence that traces their transport defect to the ER vesicle budding step. To determine if CPY*-D1 and CPY*-D2 interferes with general vesicle trafficking, transport of the plasma membrane protein Gas1p was analyzed following their induction [38]. As shown in Figure 3B, Gas1p is transported from the ER efficiently in all situations after substrate induction indicating that the variants do not generally affect protein trafficking. These data show that the D1 and D2 lesions prevent the trafficking of excess CPY* to the vacuole through selective exclusion from COPII vesicles. The transport defects indicate that an ER export signal(s) was compromised by the D1 and D2 deletions.

Bottom Line: The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways.These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them.Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis.

View Article: PubMed Central - PubMed

Affiliation: Temasek Life Sciences Laboratory, Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

ABSTRACT

Background: Endoplasmic reticulum (ER) quality control mechanisms are part of a comprehensive system to manage cell stress. The flux of molecules is monitored to retain folding intermediates and target misfolded molecules to ER-associated degradation (ERAD) pathways. The mechanisms of sorting remain unclear. While some proteins are retained statically, the classical model substrate CPY* is found in COPII transport vesicles, suggesting a retrieval mechanism for retention. However, its management can be even more dynamic. If ERAD is saturated under stress, excess CPY* traffics to the vacuole for degradation. These observations suggest that misfolded proteins might display different signals for their management.

Methodology/principal findings: Here, we report the existence of a functional ER exit signal in the pro-domain of CPY*. Compromising its integrity causes ER retention through exclusion from COPII vesicles. The signal co-exists with other signals used for retention and degradation. Physiologically, the export signal is important for stress tolerance. Disabling it converts a benign protein into one that is intrinsically cytotoxic.

Conclusions/significance: These data reveal the remarkable interplay between opposing signals embedded within ERAD substrate molecules and the mechanisms that decipher them. Our findings demonstrate the diversity of mechanisms deployed for protein quality control and maintenance of protein homeostasis.

Show MeSH
Related in: MedlinePlus