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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

Analysis of CPY* export signals.(A) Schematic representation of CPY* and its deletion variants (D1–D6). Carbohydrate chains are shown by branched symbols, asterisks indicate the position of the G255R mutation, dark shaded boxes indicate signal sequences, and the HA epitope tag is shaded light gray. (B) Intracellular localization of highly expressed CPY* deletion variants in wild type and Δpep4 strains. CPY* variants was detected using anti-HA antibody and visualized in the green channel. ER and nuclear envelope was visualized in the red channel using anti-Kar2p antiserum. (C) Intracellular localization of D1 and D2 variants in Δpep4 cells. Substrates and ER/nuclei are visualized as in panel B. Localization of all CPY* deletion variants in both wild type and Δpep4 cells are shown in Figure S1. Arrowhead indicates the accumulated CPY* or its variant in vacuole in a representative cell. Scale bars, 5 µm.
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pone-0015532-g001: Analysis of CPY* export signals.(A) Schematic representation of CPY* and its deletion variants (D1–D6). Carbohydrate chains are shown by branched symbols, asterisks indicate the position of the G255R mutation, dark shaded boxes indicate signal sequences, and the HA epitope tag is shaded light gray. (B) Intracellular localization of highly expressed CPY* deletion variants in wild type and Δpep4 strains. CPY* variants was detected using anti-HA antibody and visualized in the green channel. ER and nuclear envelope was visualized in the red channel using anti-Kar2p antiserum. (C) Intracellular localization of D1 and D2 variants in Δpep4 cells. Substrates and ER/nuclei are visualized as in panel B. Localization of all CPY* deletion variants in both wild type and Δpep4 cells are shown in Figure S1. Arrowhead indicates the accumulated CPY* or its variant in vacuole in a representative cell. Scale bars, 5 µm.

Mentions: We sought to understand the mechanism and physiological significance of misfolded protein export from the ER. Previously, this phenomenon was studied using transport defective mutant strains [15], [16], [21]. However, indirect effects caused by impairment of normal cargo proteins could not be ruled out [21]. This drawback could be mitigated by modifying substrates to disable transport. To test the feasibility of the approach, CPY* deletion variants were created systematically to eliminate a potential export signal (Fig. 1A). It should be noted that no ER export signal is known for CPY* nor even wild type CPY. To facilitate analysis, all constructs contain an HA epitope-tag at their carboxy-termini, which does not affect ERAD nor transport [22]. In addition, the C-terminal glycan of CPY* (previously termed the “D-glycan”) is maintained in all variants because it is required for recognition by ERAD [36].


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

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

Analysis of CPY* export signals.(A) Schematic representation of CPY* and its deletion variants (D1–D6). Carbohydrate chains are shown by branched symbols, asterisks indicate the position of the G255R mutation, dark shaded boxes indicate signal sequences, and the HA epitope tag is shaded light gray. (B) Intracellular localization of highly expressed CPY* deletion variants in wild type and Δpep4 strains. CPY* variants was detected using anti-HA antibody and visualized in the green channel. ER and nuclear envelope was visualized in the red channel using anti-Kar2p antiserum. (C) Intracellular localization of D1 and D2 variants in Δpep4 cells. Substrates and ER/nuclei are visualized as in panel B. Localization of all CPY* deletion variants in both wild type and Δpep4 cells are shown in Figure S1. Arrowhead indicates the accumulated CPY* or its variant in vacuole in a representative cell. Scale bars, 5 µm.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0015532-g001: Analysis of CPY* export signals.(A) Schematic representation of CPY* and its deletion variants (D1–D6). Carbohydrate chains are shown by branched symbols, asterisks indicate the position of the G255R mutation, dark shaded boxes indicate signal sequences, and the HA epitope tag is shaded light gray. (B) Intracellular localization of highly expressed CPY* deletion variants in wild type and Δpep4 strains. CPY* variants was detected using anti-HA antibody and visualized in the green channel. ER and nuclear envelope was visualized in the red channel using anti-Kar2p antiserum. (C) Intracellular localization of D1 and D2 variants in Δpep4 cells. Substrates and ER/nuclei are visualized as in panel B. Localization of all CPY* deletion variants in both wild type and Δpep4 cells are shown in Figure S1. Arrowhead indicates the accumulated CPY* or its variant in vacuole in a representative cell. Scale bars, 5 µm.
Mentions: We sought to understand the mechanism and physiological significance of misfolded protein export from the ER. Previously, this phenomenon was studied using transport defective mutant strains [15], [16], [21]. However, indirect effects caused by impairment of normal cargo proteins could not be ruled out [21]. This drawback could be mitigated by modifying substrates to disable transport. To test the feasibility of the approach, CPY* deletion variants were created systematically to eliminate a potential export signal (Fig. 1A). It should be noted that no ER export signal is known for CPY* nor even wild type CPY. To facilitate analysis, all constructs contain an HA epitope-tag at their carboxy-termini, which does not affect ERAD nor transport [22]. In addition, the C-terminal glycan of CPY* (previously termed the “D-glycan”) is maintained in all variants because it is required for recognition by ERAD [36].

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