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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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A single, specific N-linked oligosaccharide is required for CPY* degradation. (A) Schematic representation of CPY* N-linked glycosylation mutants. Glycans at positions 124, 198, 279, and 479, are denoted by the upper case letters A, B, C, and D, respectively. Lower case letters designate mutant sites. (B) CPY* and mutant variants were pulse labeled for 10 min, immunoprecipitated, and treated (or mock treated) with Endo H to remove N-linked oligosaccharides. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Positions of CPY*, −1 CPY* (altered at one of four glycosylation sites), −4 CPY* (all four glycosylation sites altered), and deglycosylated CPY* are indicated. (C) Degradation of CPY* glycosylation mutants analyzed by metabolic pulse chase. Cells were pulse labeled for 10 min with [35S]methionine/cysteine and chase for times indicated. Proteins were resolved by SDS-PAGE and quantified by phosphorimager analysis. The data reflect two independent experiments with the SD of the mean indicated. Representative autoradiograms are shown for each experiment. (D–F) Turnover of CPY* glycosylation mutants in wild-type and Δhtm1/mnl1 cells performed as in C. ABCd-CPY* turnover was also analyzed in Δcue1 cells for comparison (E).
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fig4: A single, specific N-linked oligosaccharide is required for CPY* degradation. (A) Schematic representation of CPY* N-linked glycosylation mutants. Glycans at positions 124, 198, 279, and 479, are denoted by the upper case letters A, B, C, and D, respectively. Lower case letters designate mutant sites. (B) CPY* and mutant variants were pulse labeled for 10 min, immunoprecipitated, and treated (or mock treated) with Endo H to remove N-linked oligosaccharides. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Positions of CPY*, −1 CPY* (altered at one of four glycosylation sites), −4 CPY* (all four glycosylation sites altered), and deglycosylated CPY* are indicated. (C) Degradation of CPY* glycosylation mutants analyzed by metabolic pulse chase. Cells were pulse labeled for 10 min with [35S]methionine/cysteine and chase for times indicated. Proteins were resolved by SDS-PAGE and quantified by phosphorimager analysis. The data reflect two independent experiments with the SD of the mean indicated. Representative autoradiograms are shown for each experiment. (D–F) Turnover of CPY* glycosylation mutants in wild-type and Δhtm1/mnl1 cells performed as in C. ABCd-CPY* turnover was also analyzed in Δcue1 cells for comparison (E).

Mentions: Another outcome of the CPYΔ1 deletion was the elimination of the N-linked glycosylation site nearest the COOH terminus (Fig. 4 A, glycan D). Because CPYΔ1 retains three other N-linked glycans (Fig. 4 A, sites A–C), it was not apparent how its loss alone could so severely disrupt ERAD. To test whether the carbohydrate is a critical determinant, a CPY* glycan D mutant (Fig. 4 A, ABCd-CPY*) was created and its turnover analyzed. Indeed, ABCd-CPY* was degraded poorly compared with CPY* (Fig. 4 C). This result showed that glycan D is an important determinant of CPY* degradation and explains, at least in part, why CPYΔ1 is not a substrate for ERAD.


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

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

A single, specific N-linked oligosaccharide is required for CPY* degradation. (A) Schematic representation of CPY* N-linked glycosylation mutants. Glycans at positions 124, 198, 279, and 479, are denoted by the upper case letters A, B, C, and D, respectively. Lower case letters designate mutant sites. (B) CPY* and mutant variants were pulse labeled for 10 min, immunoprecipitated, and treated (or mock treated) with Endo H to remove N-linked oligosaccharides. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Positions of CPY*, −1 CPY* (altered at one of four glycosylation sites), −4 CPY* (all four glycosylation sites altered), and deglycosylated CPY* are indicated. (C) Degradation of CPY* glycosylation mutants analyzed by metabolic pulse chase. Cells were pulse labeled for 10 min with [35S]methionine/cysteine and chase for times indicated. Proteins were resolved by SDS-PAGE and quantified by phosphorimager analysis. The data reflect two independent experiments with the SD of the mean indicated. Representative autoradiograms are shown for each experiment. (D–F) Turnover of CPY* glycosylation mutants in wild-type and Δhtm1/mnl1 cells performed as in C. ABCd-CPY* turnover was also analyzed in Δcue1 cells for comparison (E).
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171888&req=5

fig4: A single, specific N-linked oligosaccharide is required for CPY* degradation. (A) Schematic representation of CPY* N-linked glycosylation mutants. Glycans at positions 124, 198, 279, and 479, are denoted by the upper case letters A, B, C, and D, respectively. Lower case letters designate mutant sites. (B) CPY* and mutant variants were pulse labeled for 10 min, immunoprecipitated, and treated (or mock treated) with Endo H to remove N-linked oligosaccharides. Proteins were resolved by SDS-PAGE and visualized by autoradiography. Positions of CPY*, −1 CPY* (altered at one of four glycosylation sites), −4 CPY* (all four glycosylation sites altered), and deglycosylated CPY* are indicated. (C) Degradation of CPY* glycosylation mutants analyzed by metabolic pulse chase. Cells were pulse labeled for 10 min with [35S]methionine/cysteine and chase for times indicated. Proteins were resolved by SDS-PAGE and quantified by phosphorimager analysis. The data reflect two independent experiments with the SD of the mean indicated. Representative autoradiograms are shown for each experiment. (D–F) Turnover of CPY* glycosylation mutants in wild-type and Δhtm1/mnl1 cells performed as in C. ABCd-CPY* turnover was also analyzed in Δcue1 cells for comparison (E).
Mentions: Another outcome of the CPYΔ1 deletion was the elimination of the N-linked glycosylation site nearest the COOH terminus (Fig. 4 A, glycan D). Because CPYΔ1 retains three other N-linked glycans (Fig. 4 A, sites A–C), it was not apparent how its loss alone could so severely disrupt ERAD. To test whether the carbohydrate is a critical determinant, a CPY* glycan D mutant (Fig. 4 A, ABCd-CPY*) was created and its turnover analyzed. Indeed, ABCd-CPY* was degraded poorly compared with CPY* (Fig. 4 C). This result showed that glycan D is an important determinant of CPY* degradation and explains, at least in part, why CPYΔ1 is not a substrate for ERAD.

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