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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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CPYΔ1 does not form detergent insoluble aggregates. Microsomes were prepared from Δire1 cells overexpressing CPY* (A), wild-type cells expressing CPY* (B), and wild-type cell expressing CPYΔ1 (C). Membranes were solubilized in 1% Triton X-100 and separated into pellet and supernatant fractions by centrifugation at 100,000 g. Detergent-soluble (S), detergent-insoluble (P), and total (T) fractions were resolved by SDS-PAGE, followed by immunoblotting to detect CPY* and CPYΔ1 using anti-HA antibodies. The extent of membrane solubilization was determined by reprobing blots for Sec61p, an integral membrane protein control. Asterisks indicate underglycosylated and cytosolic CPY* that form when overexpressed in Δire1 cells (Spear and Ng, 2003).
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fig2: CPYΔ1 does not form detergent insoluble aggregates. Microsomes were prepared from Δire1 cells overexpressing CPY* (A), wild-type cells expressing CPY* (B), and wild-type cell expressing CPYΔ1 (C). Membranes were solubilized in 1% Triton X-100 and separated into pellet and supernatant fractions by centrifugation at 100,000 g. Detergent-soluble (S), detergent-insoluble (P), and total (T) fractions were resolved by SDS-PAGE, followed by immunoblotting to detect CPY* and CPYΔ1 using anti-HA antibodies. The extent of membrane solubilization was determined by reprobing blots for Sec61p, an integral membrane protein control. Asterisks indicate underglycosylated and cytosolic CPY* that form when overexpressed in Δire1 cells (Spear and Ng, 2003).

Mentions: Nonetheless, we envisioned other equally plausible explanations that could account for CPYΔ1's unusual behavior. Because the deletion is large, the severity of the lesion might increase the tendency of the remaining polypeptide to aggregate. Substrate solubility is an important prerequisite for ERAD. CPY* aggregates caused by faulty chaperone function were shown to degrade inefficiently (Nishikawa et al., 2001). To determine whether aggregate formation contributes to CPYΔ1 stability, microsomal membranes containing CPY* or CPYΔ1 were prepared from logarithmically growing cells. The membranes were solubilized in nonionic detergent under physiological conditions and subjected to centrifugation. Under these conditions, large protein aggregates sediment rapidly and separate from soluble proteins remaining in the supernatant. Detergent-insoluble (pellet) and detergent-soluble (supernatant) fractions were collected, proteins resolved by SDS-PAGE, and analyzed by immunoblotting. To control for the procedure, we specifically generated CPY* aggregates from cells severely limiting for ER chaperones (Δire1; Spear and Ng, 2003). This species was analyzed in parallel. As shown in Fig. 2, CPY* and CPYΔ1 were both recovered entirely from the supernatant fraction (B and C), whereas CPY* aggregates from Δire1 cells were found predominantly in the pellet fraction (A). In every case, the ER integral membrane protein Sec61p was recovered from the soluble fraction showing that membranes were completely solubilized. This experiment showed that the formation of detergent-insoluble aggregates was not a root cause of the CPYΔ1 ERAD defect. Furthermore, CPYΔ1 puncta, which would be characteristic of intracellular aggregates, were never observed in immunolocalization experiments (DePace et al., 1998). Instead, CPYΔ1 was always found to be evenly distributed throughout the ER in patterns indistinguishable from CPY* and Kar2p (Fig. 1 C).


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

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

CPYΔ1 does not form detergent insoluble aggregates. Microsomes were prepared from Δire1 cells overexpressing CPY* (A), wild-type cells expressing CPY* (B), and wild-type cell expressing CPYΔ1 (C). Membranes were solubilized in 1% Triton X-100 and separated into pellet and supernatant fractions by centrifugation at 100,000 g. Detergent-soluble (S), detergent-insoluble (P), and total (T) fractions were resolved by SDS-PAGE, followed by immunoblotting to detect CPY* and CPYΔ1 using anti-HA antibodies. The extent of membrane solubilization was determined by reprobing blots for Sec61p, an integral membrane protein control. Asterisks indicate underglycosylated and cytosolic CPY* that form when overexpressed in Δire1 cells (Spear and Ng, 2003).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171888&req=5

fig2: CPYΔ1 does not form detergent insoluble aggregates. Microsomes were prepared from Δire1 cells overexpressing CPY* (A), wild-type cells expressing CPY* (B), and wild-type cell expressing CPYΔ1 (C). Membranes were solubilized in 1% Triton X-100 and separated into pellet and supernatant fractions by centrifugation at 100,000 g. Detergent-soluble (S), detergent-insoluble (P), and total (T) fractions were resolved by SDS-PAGE, followed by immunoblotting to detect CPY* and CPYΔ1 using anti-HA antibodies. The extent of membrane solubilization was determined by reprobing blots for Sec61p, an integral membrane protein control. Asterisks indicate underglycosylated and cytosolic CPY* that form when overexpressed in Δire1 cells (Spear and Ng, 2003).
Mentions: Nonetheless, we envisioned other equally plausible explanations that could account for CPYΔ1's unusual behavior. Because the deletion is large, the severity of the lesion might increase the tendency of the remaining polypeptide to aggregate. Substrate solubility is an important prerequisite for ERAD. CPY* aggregates caused by faulty chaperone function were shown to degrade inefficiently (Nishikawa et al., 2001). To determine whether aggregate formation contributes to CPYΔ1 stability, microsomal membranes containing CPY* or CPYΔ1 were prepared from logarithmically growing cells. The membranes were solubilized in nonionic detergent under physiological conditions and subjected to centrifugation. Under these conditions, large protein aggregates sediment rapidly and separate from soluble proteins remaining in the supernatant. Detergent-insoluble (pellet) and detergent-soluble (supernatant) fractions were collected, proteins resolved by SDS-PAGE, and analyzed by immunoblotting. To control for the procedure, we specifically generated CPY* aggregates from cells severely limiting for ER chaperones (Δire1; Spear and Ng, 2003). This species was analyzed in parallel. As shown in Fig. 2, CPY* and CPYΔ1 were both recovered entirely from the supernatant fraction (B and C), whereas CPY* aggregates from Δire1 cells were found predominantly in the pellet fraction (A). In every case, the ER integral membrane protein Sec61p was recovered from the soluble fraction showing that membranes were completely solubilized. This experiment showed that the formation of detergent-insoluble aggregates was not a root cause of the CPYΔ1 ERAD defect. Furthermore, CPYΔ1 puncta, which would be characteristic of intracellular aggregates, were never observed in immunolocalization experiments (DePace et al., 1998). Instead, CPYΔ1 was always found to be evenly distributed throughout the ER in patterns indistinguishable from CPY* and Kar2p (Fig. 1 C).

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