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Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation.

Spear ED, Ng DT - J. Cell Biol. (2005)

Bottom Line: Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway.The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway.These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.

ABSTRACT
The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

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Related in: MedlinePlus

CPYΔ1 is a misfolded protein recognized by ER quality control but poorly degraded by ERAD. (A) Schematic representation of CPY, CPY*, and CPYΔ1. Carbohydrates are represented by branched symbols, asterisk indicates the CPY* G255R mutation, dark gray boxes indicate signal sequences, and light gray boxes represent HA-epitope tags. (B) CPYΔ1 remains unmodified by Golgi and vacuolar enzymes. Wild-type cells expressing CPYΔ1 (pES57) were pulsed labeled for 10 min and chased for 0 (lane P) or 30 min (lane C). Immunoprecipitated CPY and CPYΔ1 were resolved by SDS-PAGE and visualized by autoradiography. CPYΔ1, ER proCPY (p1), Golgi carbohydrate-modified proCPY (p2), and vacuolar protease-processed mature CPY (m) are indicated. (C) Intracellular localization of CPYΔ1. CPY* and CPYΔ1 were localized by indirect immunofluorescence as described in Materials and methods (C, panels a and c, respectively). Simultaneous localization of Kar2p was performed as a marker of the ER (C, panels b and d). (D) CPYΔ1 induces the UPR. Wild-type cells carrying an integrated UPRE-LacZ reporter gene (ESY39) and expressing HA epitope-tagged CPY, CPY*, or CPYΔ1 were assayed for β-galactosidase activity. The data reflect three independent experiments with the SD of the mean indicated. (E) Wild-type and Δcue1 cells expressing CPY* were pulse labeled for 10 min and chased for the times indicated. CPY* was immunoprecipitated from detergent lysates and resolved by SDS-PAGE. CPY* decay was quantified by phosphorimager analysis and plotted to the right of autoradiograms. The data reflect three independent experiments with the SD of the mean indicated. (F) Wild-type and Δcue1 cells expressing CPYΔ1 were analyzed by pulse-chase analysis as described in E.
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fig1: CPYΔ1 is a misfolded protein recognized by ER quality control but poorly degraded by ERAD. (A) Schematic representation of CPY, CPY*, and CPYΔ1. Carbohydrates are represented by branched symbols, asterisk indicates the CPY* G255R mutation, dark gray boxes indicate signal sequences, and light gray boxes represent HA-epitope tags. (B) CPYΔ1 remains unmodified by Golgi and vacuolar enzymes. Wild-type cells expressing CPYΔ1 (pES57) were pulsed labeled for 10 min and chased for 0 (lane P) or 30 min (lane C). Immunoprecipitated CPY and CPYΔ1 were resolved by SDS-PAGE and visualized by autoradiography. CPYΔ1, ER proCPY (p1), Golgi carbohydrate-modified proCPY (p2), and vacuolar protease-processed mature CPY (m) are indicated. (C) Intracellular localization of CPYΔ1. CPY* and CPYΔ1 were localized by indirect immunofluorescence as described in Materials and methods (C, panels a and c, respectively). Simultaneous localization of Kar2p was performed as a marker of the ER (C, panels b and d). (D) CPYΔ1 induces the UPR. Wild-type cells carrying an integrated UPRE-LacZ reporter gene (ESY39) and expressing HA epitope-tagged CPY, CPY*, or CPYΔ1 were assayed for β-galactosidase activity. The data reflect three independent experiments with the SD of the mean indicated. (E) Wild-type and Δcue1 cells expressing CPY* were pulse labeled for 10 min and chased for the times indicated. CPY* was immunoprecipitated from detergent lysates and resolved by SDS-PAGE. CPY* decay was quantified by phosphorimager analysis and plotted to the right of autoradiograms. The data reflect three independent experiments with the SD of the mean indicated. (F) Wild-type and Δcue1 cells expressing CPYΔ1 were analyzed by pulse-chase analysis as described in E.

Mentions: The most extensively studied model ERAD substrate is CPY*, a mutant of the vacuolar protease carboxypeptidase Y (CPY). CPY* is a product of the prc1-1 allele (glycine to arginine replacement at position 255) that misfolds irreversibly (Finger et al., 1993). To extend its versatility, we intended to create a substrate that permitted simultaneous monitoring of endogenous CPY as an internal control for secretory function and gel loading. A CPY variant designated CPYΔ1 was constructed that differs by a 154–amino acid deletion near the COOH terminus and migrates distinctly on SDS gels (Fig. 1, A and B). Guided by the crystal structure, we predicted that the deletion would disrupt folding due to the extensive loss of intramolecular interactions (Endrizzi et al., 1994). Three lines of evidence supported this view.


Single, context-specific glycans can target misfolded glycoproteins for ER-associated degradation.

Spear ED, Ng DT - J. Cell Biol. (2005)

CPYΔ1 is a misfolded protein recognized by ER quality control but poorly degraded by ERAD. (A) Schematic representation of CPY, CPY*, and CPYΔ1. Carbohydrates are represented by branched symbols, asterisk indicates the CPY* G255R mutation, dark gray boxes indicate signal sequences, and light gray boxes represent HA-epitope tags. (B) CPYΔ1 remains unmodified by Golgi and vacuolar enzymes. Wild-type cells expressing CPYΔ1 (pES57) were pulsed labeled for 10 min and chased for 0 (lane P) or 30 min (lane C). Immunoprecipitated CPY and CPYΔ1 were resolved by SDS-PAGE and visualized by autoradiography. CPYΔ1, ER proCPY (p1), Golgi carbohydrate-modified proCPY (p2), and vacuolar protease-processed mature CPY (m) are indicated. (C) Intracellular localization of CPYΔ1. CPY* and CPYΔ1 were localized by indirect immunofluorescence as described in Materials and methods (C, panels a and c, respectively). Simultaneous localization of Kar2p was performed as a marker of the ER (C, panels b and d). (D) CPYΔ1 induces the UPR. Wild-type cells carrying an integrated UPRE-LacZ reporter gene (ESY39) and expressing HA epitope-tagged CPY, CPY*, or CPYΔ1 were assayed for β-galactosidase activity. The data reflect three independent experiments with the SD of the mean indicated. (E) Wild-type and Δcue1 cells expressing CPY* were pulse labeled for 10 min and chased for the times indicated. CPY* was immunoprecipitated from detergent lysates and resolved by SDS-PAGE. CPY* decay was quantified by phosphorimager analysis and plotted to the right of autoradiograms. The data reflect three independent experiments with the SD of the mean indicated. (F) Wild-type and Δcue1 cells expressing CPYΔ1 were analyzed by pulse-chase analysis as described in E.
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fig1: CPYΔ1 is a misfolded protein recognized by ER quality control but poorly degraded by ERAD. (A) Schematic representation of CPY, CPY*, and CPYΔ1. Carbohydrates are represented by branched symbols, asterisk indicates the CPY* G255R mutation, dark gray boxes indicate signal sequences, and light gray boxes represent HA-epitope tags. (B) CPYΔ1 remains unmodified by Golgi and vacuolar enzymes. Wild-type cells expressing CPYΔ1 (pES57) were pulsed labeled for 10 min and chased for 0 (lane P) or 30 min (lane C). Immunoprecipitated CPY and CPYΔ1 were resolved by SDS-PAGE and visualized by autoradiography. CPYΔ1, ER proCPY (p1), Golgi carbohydrate-modified proCPY (p2), and vacuolar protease-processed mature CPY (m) are indicated. (C) Intracellular localization of CPYΔ1. CPY* and CPYΔ1 were localized by indirect immunofluorescence as described in Materials and methods (C, panels a and c, respectively). Simultaneous localization of Kar2p was performed as a marker of the ER (C, panels b and d). (D) CPYΔ1 induces the UPR. Wild-type cells carrying an integrated UPRE-LacZ reporter gene (ESY39) and expressing HA epitope-tagged CPY, CPY*, or CPYΔ1 were assayed for β-galactosidase activity. The data reflect three independent experiments with the SD of the mean indicated. (E) Wild-type and Δcue1 cells expressing CPY* were pulse labeled for 10 min and chased for the times indicated. CPY* was immunoprecipitated from detergent lysates and resolved by SDS-PAGE. CPY* decay was quantified by phosphorimager analysis and plotted to the right of autoradiograms. The data reflect three independent experiments with the SD of the mean indicated. (F) Wild-type and Δcue1 cells expressing CPYΔ1 were analyzed by pulse-chase analysis as described in E.
Mentions: The most extensively studied model ERAD substrate is CPY*, a mutant of the vacuolar protease carboxypeptidase Y (CPY). CPY* is a product of the prc1-1 allele (glycine to arginine replacement at position 255) that misfolds irreversibly (Finger et al., 1993). To extend its versatility, we intended to create a substrate that permitted simultaneous monitoring of endogenous CPY as an internal control for secretory function and gel loading. A CPY variant designated CPYΔ1 was constructed that differs by a 154–amino acid deletion near the COOH terminus and migrates distinctly on SDS gels (Fig. 1, A and B). Guided by the crystal structure, we predicted that the deletion would disrupt folding due to the extensive loss of intramolecular interactions (Endrizzi et al., 1994). Three lines of evidence supported this view.

Bottom Line: Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway.The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway.These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Pennsylvania State University, University Park, PA 16802, USA.

ABSTRACT
The endoplasmic reticulum (ER) maintains an environment essential for secretory protein folding. Consequently, the premature transport of polypeptides would be harmful to the cell. To avert this scenario, mechanisms collectively termed "ER quality control" prevent the transport of nascent polypeptides until they properly fold. Irreversibly misfolded molecules are sorted for disposal by the ER-associated degradation (ERAD) pathway. To better understand the relationship between quality control and ERAD, we studied a new misfolded variant of carboxypeptidase Y (CPY). The molecule was recognized and retained by ER quality control but failed to enter the ERAD pathway. Systematic analysis revealed that a single, specific N-linked glycan of CPY was required for sorting into the pathway. The determinant is dependent on the putative lectin-like receptor Htm1/Mnl1p. The discovery of a similar signal in misfolded proteinase A supported the generality of the mechanism. These studies show that specific signals embedded in glycoproteins can direct their degradation if they fail to fold.

Show MeSH
Related in: MedlinePlus