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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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Degradation of the misfolded protein PrA* requires a single NH2-terminal glycan. (A) Schematic representation of PrA* and glycosylation site mutant derivatives. Asparagines modified by glycosylation at positions 107 and 308 are indicated with A and B, respectively. Lower case designations indicate mutant sites. (B) Cells expressing PrA*, aB-PrA*, or Ab-PrA* were pulse labeled for 10 min with [35S]methionine/cysteine. Proteins were immunoprecipitated from detergent lysates using polyclonal anti-PrA antibodies, mock treated (−) or treated (+) with Endo H, and resolved by SDS-PAGE. Positions of PrA*, −1 PrA*, and deglycosylated PrA* are indicated. (C) Wild-type cells expressing PrA*, aB-PrA*, or Ab-PrA* were analyzed by pulse-chase analysis as in Fig. 1 E. The data reflect two independent experiments with the SD of the mean indicated.
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fig5: Degradation of the misfolded protein PrA* requires a single NH2-terminal glycan. (A) Schematic representation of PrA* and glycosylation site mutant derivatives. Asparagines modified by glycosylation at positions 107 and 308 are indicated with A and B, respectively. Lower case designations indicate mutant sites. (B) Cells expressing PrA*, aB-PrA*, or Ab-PrA* were pulse labeled for 10 min with [35S]methionine/cysteine. Proteins were immunoprecipitated from detergent lysates using polyclonal anti-PrA antibodies, mock treated (−) or treated (+) with Endo H, and resolved by SDS-PAGE. Positions of PrA*, −1 PrA*, and deglycosylated PrA* are indicated. (C) Wild-type cells expressing PrA*, aB-PrA*, or Ab-PrA* were analyzed by pulse-chase analysis as in Fig. 1 E. The data reflect two independent experiments with the SD of the mean indicated.

Mentions: We wondered if the specificity of the glycan was unique to CPY* or a general feature of lectin-dependent ERAD. For this, we analyzed the ERAD substrate PrA* (Finger et al., 1993). PrA* is a mutant version of the endogenous vacuolar enzyme, proteinase A. PrA* contains two N-linked glycans, one near its NH2 terminus and the other near the COOH terminus at a position similar to CPY*'s glycan D. Each site was disrupted singly by replacing asparagine codons with glutamine. The mutant variants, Ab-PrA* and aB-PrA* (Fig. 5 A, follows the nomenclature of CPY* glycan mutants), were expressed in wild-type cells (deleted of endogenous PEP4 gene for detection of PrA*) and their turnover measured. As shown in Fig. 6, the Ab-PrA* was degraded indistinguishably from PrA*. Because PrA* is glycosylated at only two sites, its degradation depends on a single, specific glycan signal like CPY* or is carbohydrate independent. The question was answer by the results of two experiments. First, Ab-PrA* degradation is dependent on Htm1/Mnl1p to a similar extent as PrA* (Fig. 6, A and B). Second, turnover of reciprocal mutant, aB-PrA*, was strongly defective and little affected by the loss of Htm1/Mnl1p (Fig. 6 C). These data support the idea that single ERAD glycan determinants are preembedded in glycoproteins. However, a wider range of substrates must be tested to determine whether other configurations are used. Serendipitously, analysis of the PrA* model also ruled out COOH-terminal positioning being a requirement because its sole determinant is closer to the NH2 terminus.


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

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

Degradation of the misfolded protein PrA* requires a single NH2-terminal glycan. (A) Schematic representation of PrA* and glycosylation site mutant derivatives. Asparagines modified by glycosylation at positions 107 and 308 are indicated with A and B, respectively. Lower case designations indicate mutant sites. (B) Cells expressing PrA*, aB-PrA*, or Ab-PrA* were pulse labeled for 10 min with [35S]methionine/cysteine. Proteins were immunoprecipitated from detergent lysates using polyclonal anti-PrA antibodies, mock treated (−) or treated (+) with Endo H, and resolved by SDS-PAGE. Positions of PrA*, −1 PrA*, and deglycosylated PrA* are indicated. (C) Wild-type cells expressing PrA*, aB-PrA*, or Ab-PrA* were analyzed by pulse-chase analysis as in Fig. 1 E. The data reflect two independent experiments with the SD of the mean indicated.
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Related In: Results  -  Collection

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

fig5: Degradation of the misfolded protein PrA* requires a single NH2-terminal glycan. (A) Schematic representation of PrA* and glycosylation site mutant derivatives. Asparagines modified by glycosylation at positions 107 and 308 are indicated with A and B, respectively. Lower case designations indicate mutant sites. (B) Cells expressing PrA*, aB-PrA*, or Ab-PrA* were pulse labeled for 10 min with [35S]methionine/cysteine. Proteins were immunoprecipitated from detergent lysates using polyclonal anti-PrA antibodies, mock treated (−) or treated (+) with Endo H, and resolved by SDS-PAGE. Positions of PrA*, −1 PrA*, and deglycosylated PrA* are indicated. (C) Wild-type cells expressing PrA*, aB-PrA*, or Ab-PrA* were analyzed by pulse-chase analysis as in Fig. 1 E. The data reflect two independent experiments with the SD of the mean indicated.
Mentions: We wondered if the specificity of the glycan was unique to CPY* or a general feature of lectin-dependent ERAD. For this, we analyzed the ERAD substrate PrA* (Finger et al., 1993). PrA* is a mutant version of the endogenous vacuolar enzyme, proteinase A. PrA* contains two N-linked glycans, one near its NH2 terminus and the other near the COOH terminus at a position similar to CPY*'s glycan D. Each site was disrupted singly by replacing asparagine codons with glutamine. The mutant variants, Ab-PrA* and aB-PrA* (Fig. 5 A, follows the nomenclature of CPY* glycan mutants), were expressed in wild-type cells (deleted of endogenous PEP4 gene for detection of PrA*) and their turnover measured. As shown in Fig. 6, the Ab-PrA* was degraded indistinguishably from PrA*. Because PrA* is glycosylated at only two sites, its degradation depends on a single, specific glycan signal like CPY* or is carbohydrate independent. The question was answer by the results of two experiments. First, Ab-PrA* degradation is dependent on Htm1/Mnl1p to a similar extent as PrA* (Fig. 6, A and B). Second, turnover of reciprocal mutant, aB-PrA*, was strongly defective and little affected by the loss of Htm1/Mnl1p (Fig. 6 C). These data support the idea that single ERAD glycan determinants are preembedded in glycoproteins. However, a wider range of substrates must be tested to determine whether other configurations are used. Serendipitously, analysis of the PrA* model also ruled out COOH-terminal positioning being a requirement because its sole determinant is closer to the NH2 terminus.

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