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

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.

Figure 1.

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.

Figure 2.

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).

Figure 3.

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.

Figure 4.

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).

Figure 5.

Degradation of the misfolded protein PrA* requires a single NH ₂ -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.

Figure 6.

The ER lectin Htm1p/Mnl1p mediates PrA* degradation through the NH₂-terminal glycan. Wild-type or Δhtm1/mnl1 cells expressing PrA* (A), Ab-PrA* (B), or aB-PrA* (C) were analyzed by pulse-chase analysis. The data reflect two independent experiments with the SD of the mean indicated.