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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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COOH-terminal lysines are not required for CPY* degradation. (A) CPY* (pDN436) or K(9)R CPY* (pES115) degradation in wild-type cells was determined by pulse-chase analysis performed in Fig. 1 E. (B) Turnover of CPYΔ1 (pES57) or R(3)K CPYΔ1 (pES95) was determined using wild-type cells as in A. Plots reflect two independent experiments with the SD of the mean indicated.
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fig3: COOH-terminal lysines are not required for CPY* degradation. (A) CPY* (pDN436) or K(9)R CPY* (pES115) degradation in wild-type cells was determined by pulse-chase analysis performed in Fig. 1 E. (B) Turnover of CPYΔ1 (pES57) or R(3)K CPYΔ1 (pES95) was determined using wild-type cells as in A. Plots reflect two independent experiments with the SD of the mean indicated.

Mentions: We next tested a second possibility. Inspection of the CPYΔ1 sequence revealed a striking consequence of the deletion. A lysine rich domain was eliminated, leaving behind a 116–amino acid stretch devoid of lysines at the COOH terminus. Because CPY* ubiquitylation is required for its dislocation (Shamu et al., 2001; Jarosch et al., 2002), we wondered whether loss of putative ubiquitylation sites crucial for ERAD could explain the defect. To test the possibility, nine lysine residues corresponding to the CPYΔ1 deleted region were changed to arginine in full-length CPY*. The resulting molecule, K(9)R-CPY*, was degraded as efficiently as CPY* demonstrating that the COOH-terminal lysines are not required for ERAD (Fig. 3 A). Conversely, the addition of lysine residues to the COOH-terminal domain of CPYΔ1 failed to destabilize it (Fig. 3 B). Together, these data show that the COOH-terminal lysine residues of CPY do not constitute critical determinants for its turnover by ERAD.


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

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

COOH-terminal lysines are not required for CPY* degradation. (A) CPY* (pDN436) or K(9)R CPY* (pES115) degradation in wild-type cells was determined by pulse-chase analysis performed in Fig. 1 E. (B) Turnover of CPYΔ1 (pES57) or R(3)K CPYΔ1 (pES95) was determined using wild-type cells as in A. Plots reflect two independent experiments with the SD of the mean indicated.
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Related In: Results  -  Collection

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fig3: COOH-terminal lysines are not required for CPY* degradation. (A) CPY* (pDN436) or K(9)R CPY* (pES115) degradation in wild-type cells was determined by pulse-chase analysis performed in Fig. 1 E. (B) Turnover of CPYΔ1 (pES57) or R(3)K CPYΔ1 (pES95) was determined using wild-type cells as in A. Plots reflect two independent experiments with the SD of the mean indicated.
Mentions: We next tested a second possibility. Inspection of the CPYΔ1 sequence revealed a striking consequence of the deletion. A lysine rich domain was eliminated, leaving behind a 116–amino acid stretch devoid of lysines at the COOH terminus. Because CPY* ubiquitylation is required for its dislocation (Shamu et al., 2001; Jarosch et al., 2002), we wondered whether loss of putative ubiquitylation sites crucial for ERAD could explain the defect. To test the possibility, nine lysine residues corresponding to the CPYΔ1 deleted region were changed to arginine in full-length CPY*. The resulting molecule, K(9)R-CPY*, was degraded as efficiently as CPY* demonstrating that the COOH-terminal lysines are not required for ERAD (Fig. 3 A). Conversely, the addition of lysine residues to the COOH-terminal domain of CPYΔ1 failed to destabilize it (Fig. 3 B). Together, these data show that the COOH-terminal lysine residues of CPY do not constitute critical determinants for its turnover by 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