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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

Export deficient mutants are efficiently degraded by ERAD.CPY* and the D1, D2, and D3 variants were moderately expressed under the control of its native promoter in wild type or Δcue1 cells. Equal cell numbers were harvested at the indicated times after the addition of cycloheximide (100 µg/ml) and detergent lysates prepared. Proteins were separated by SDS-PAGE and detected by immunoblotting. Quantification was performed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Endogenous Sec61p was detected from the same filters as a loading control.
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pone-0015532-g006: Export deficient mutants are efficiently degraded by ERAD.CPY* and the D1, D2, and D3 variants were moderately expressed under the control of its native promoter in wild type or Δcue1 cells. Equal cell numbers were harvested at the indicated times after the addition of cycloheximide (100 µg/ml) and detergent lysates prepared. Proteins were separated by SDS-PAGE and detected by immunoblotting. Quantification was performed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Endogenous Sec61p was detected from the same filters as a loading control.

Mentions: CPY*, expressed at moderate levels from its native promoter, is degraded exclusively by ERAD [45]. Under these conditions, some molecules are packaged into COPII vesicles indicating that they are degraded after their retrieval from the Golgi apparatus [16]. The stabilization of CPY* in COPII and COPI vesicle transport mutants suggested that trafficking might be a requirement for its degradation [15], [16]. However, it was proposed that the strong stabilization observed might be due to secondary effects of the transport mutants on ERAD [21]. The CPY*-D1 and CPY*-D2 variants can be used to resolve this issue because they carry the CPY ERAD determinant and are unable to exit via the COPII pathway [46]. To determine if transport and retrieval is coincidental or a requirement for ERAD, the D1, D2, and D3 variants were expressed moderately from the PRC1 (CPY) promoter and turnover was analyzed by cycloheximide chase and immunoblotting. The CPY* control is degraded rapidly in wild type cells and stabilized in the Δcue1 ERAD mutant as expected (Figure 6, upper left). The transport competent CPY*-D3 variant behaves identically showing that a large internal deletion has no effect on degradation as long as its ERAD determinant is present (Figure 6, lower right). Applying the same assay to CPY*-D1 and CPY*-D2, their turnover profile is identical to CPY*. These data show that substrate transport and retrieval are not requirements for ERAD. Instead, the observed transport and retrieval of misfolded proteins likely reflects a mechanism of ER retention, analogous to that of ER resident proteins bearing C-terminal HDEL retention sequences [47].


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

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

Export deficient mutants are efficiently degraded by ERAD.CPY* and the D1, D2, and D3 variants were moderately expressed under the control of its native promoter in wild type or Δcue1 cells. Equal cell numbers were harvested at the indicated times after the addition of cycloheximide (100 µg/ml) and detergent lysates prepared. Proteins were separated by SDS-PAGE and detected by immunoblotting. Quantification was performed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Endogenous Sec61p was detected from the same filters as a loading control.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991357&req=5

pone-0015532-g006: Export deficient mutants are efficiently degraded by ERAD.CPY* and the D1, D2, and D3 variants were moderately expressed under the control of its native promoter in wild type or Δcue1 cells. Equal cell numbers were harvested at the indicated times after the addition of cycloheximide (100 µg/ml) and detergent lysates prepared. Proteins were separated by SDS-PAGE and detected by immunoblotting. Quantification was performed using an Odyssey infrared imaging system (LI-COR Biosciences, Lincoln, NE). Endogenous Sec61p was detected from the same filters as a loading control.
Mentions: CPY*, expressed at moderate levels from its native promoter, is degraded exclusively by ERAD [45]. Under these conditions, some molecules are packaged into COPII vesicles indicating that they are degraded after their retrieval from the Golgi apparatus [16]. The stabilization of CPY* in COPII and COPI vesicle transport mutants suggested that trafficking might be a requirement for its degradation [15], [16]. However, it was proposed that the strong stabilization observed might be due to secondary effects of the transport mutants on ERAD [21]. The CPY*-D1 and CPY*-D2 variants can be used to resolve this issue because they carry the CPY ERAD determinant and are unable to exit via the COPII pathway [46]. To determine if transport and retrieval is coincidental or a requirement for ERAD, the D1, D2, and D3 variants were expressed moderately from the PRC1 (CPY) promoter and turnover was analyzed by cycloheximide chase and immunoblotting. The CPY* control is degraded rapidly in wild type cells and stabilized in the Δcue1 ERAD mutant as expected (Figure 6, upper left). The transport competent CPY*-D3 variant behaves identically showing that a large internal deletion has no effect on degradation as long as its ERAD determinant is present (Figure 6, lower right). Applying the same assay to CPY*-D1 and CPY*-D2, their turnover profile is identical to CPY*. These data show that substrate transport and retrieval are not requirements for ERAD. Instead, the observed transport and retrieval of misfolded proteins likely reflects a mechanism of ER retention, analogous to that of ER resident proteins bearing C-terminal HDEL retention sequences [47].

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