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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.

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Related in: MedlinePlus

CPY* variants D1 and D2 are degradation defective.Wild type and Δpep4 cells expressing CPY* and variants following galactose induction were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for times indicated. Substrate proteins were immunoprecipitated from detergent lysates, separated by SDS-PAGE, and visualized and quantified by phosphorimager analysis. Representative gel scans are shown on the left. The position of substrate proteins and hyperglycosylated species are indicated. Data plots reflect three independent experiments with standard deviations indicated by the error bars.
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pone-0015532-g002: CPY* variants D1 and D2 are degradation defective.Wild type and Δpep4 cells expressing CPY* and variants following galactose induction were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for times indicated. Substrate proteins were immunoprecipitated from detergent lysates, separated by SDS-PAGE, and visualized and quantified by phosphorimager analysis. Representative gel scans are shown on the left. The position of substrate proteins and hyperglycosylated species are indicated. Data plots reflect three independent experiments with standard deviations indicated by the error bars.

Mentions: We combined in vitro and in vivo approaches to analyze COPII vesicle packaging and vacuolar transport of CPY* variants, respectively. At moderate expression levels, CPY* is efficiently degraded by ERAD, with some molecules packaged into COPII vesicles and retrieved from the Golgi beforehand [16]. Expression of CPY* under the control of the strong GAL1 promoter saturates ERAD and activates the UPR [22]. The UPR controls the ER-to-vacuole transport pathway, which is used to dispose excess CPY* under these conditions. Consistent with our previous observations, indirect immunofluorescence confocal imaging localizes CPY* to the ER of wild type cells (Figure 1B, upper panels). Vacuolar staining is absent because of rapid substrate degradation there. To visualize the fraction that traffics to the vacuole, substrates were also expressed in the vacuolar protease deficient Δpep4 strain. Here, CPY* is detected strongly in compartments outside the ER that were previously determined to be vacuoles (Figure 1B, lower panels) [22]. All CPY* deletion variants display a similar pattern, except two (Figures 1C and S1). The CPY*-D1 and CPY*-D2 variants exhibited no detectable extra-endoplasmic reticulum staining indicating defects in ER export. In line with this view, the steady state levels of these substrates are significantly higher than CPY* and other variants in wild type cells (Figure S3A). Indeed, in pulse chase experiments, CPY*-D1 and CPY*-D2 constructs are stable proteins in wild type cells with no further stabilization in Δpep4 cells (Figure 2). This result contrasts with transport competent CPY*, where a fraction is degraded by ERAD in Δpep4 cells (Figure 2A) [22]. Because the portions deleted in CPY*-D1 and CPY*-D2 are not required for ERAD when moderately expressed [46], the data suggest that transport-defective CPY* variants can interfere with ERAD functions if highly expressed.


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

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

CPY* variants D1 and D2 are degradation defective.Wild type and Δpep4 cells expressing CPY* and variants following galactose induction were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for times indicated. Substrate proteins were immunoprecipitated from detergent lysates, separated by SDS-PAGE, and visualized and quantified by phosphorimager analysis. Representative gel scans are shown on the left. The position of substrate proteins and hyperglycosylated species are indicated. Data plots reflect three independent experiments with standard deviations indicated by the error bars.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015532-g002: CPY* variants D1 and D2 are degradation defective.Wild type and Δpep4 cells expressing CPY* and variants following galactose induction were pulse-labeled for 10 min with [35S]methionine/cysteine and chased for times indicated. Substrate proteins were immunoprecipitated from detergent lysates, separated by SDS-PAGE, and visualized and quantified by phosphorimager analysis. Representative gel scans are shown on the left. The position of substrate proteins and hyperglycosylated species are indicated. Data plots reflect three independent experiments with standard deviations indicated by the error bars.
Mentions: We combined in vitro and in vivo approaches to analyze COPII vesicle packaging and vacuolar transport of CPY* variants, respectively. At moderate expression levels, CPY* is efficiently degraded by ERAD, with some molecules packaged into COPII vesicles and retrieved from the Golgi beforehand [16]. Expression of CPY* under the control of the strong GAL1 promoter saturates ERAD and activates the UPR [22]. The UPR controls the ER-to-vacuole transport pathway, which is used to dispose excess CPY* under these conditions. Consistent with our previous observations, indirect immunofluorescence confocal imaging localizes CPY* to the ER of wild type cells (Figure 1B, upper panels). Vacuolar staining is absent because of rapid substrate degradation there. To visualize the fraction that traffics to the vacuole, substrates were also expressed in the vacuolar protease deficient Δpep4 strain. Here, CPY* is detected strongly in compartments outside the ER that were previously determined to be vacuoles (Figure 1B, lower panels) [22]. All CPY* deletion variants display a similar pattern, except two (Figures 1C and S1). The CPY*-D1 and CPY*-D2 variants exhibited no detectable extra-endoplasmic reticulum staining indicating defects in ER export. In line with this view, the steady state levels of these substrates are significantly higher than CPY* and other variants in wild type cells (Figure S3A). Indeed, in pulse chase experiments, CPY*-D1 and CPY*-D2 constructs are stable proteins in wild type cells with no further stabilization in Δpep4 cells (Figure 2). This result contrasts with transport competent CPY*, where a fraction is degraded by ERAD in Δpep4 cells (Figure 2A) [22]. Because the portions deleted in CPY*-D1 and CPY*-D2 are not required for ERAD when moderately expressed [46], the data suggest that transport-defective CPY* variants can interfere with ERAD functions if highly expressed.

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