Defining the glycan destruction signal for endoplasmic reticulum-associated degradation

Abstract

The endoplasmic reticulum (ER) must target potentially toxic misfolded proteins for retrotranslocation and proteasomal degradation while avoiding destruction of productive folding intermediates. For luminal proteins, this discrimination typically depends not only on the folding status of a polypeptide, but also on its glycosylation state. Two putative sugar binding proteins, Htm1p and Yos9p, are required for degradation of misfolded glycoproteins, but the nature of the glycan degradation signal and how such signals are generated and decoded remains unclear. Here we characterize Yos9p's oligosaccharide-binding specificity and find that it recognizes glycans containing terminal alpha1,6-linked mannose residues. We also provide evidence in vivo that a terminal alpha1,6-linked mannose-containing oligosaccharide is required for degradation and that Htm1p acts upstream of Yos9p to mediate the generation of such sugars. This strategy of marking potential substrates by Htm1p and decoding the signal by Yos9p is well suited to provide a proofreading mechanism that enhances substrate specificity.

Figure 1. Purification of biochemical amounts of Yos9p from E.coli

(A) Degradation of CPY* in (■)wild-type (WT), and (●) a yos9Δ strain harboring an empty vector, and yos9Δ strain covered with a plasmid expressing YOS9-flag (△) or a Yos9p variant that is missing glycosylation sites (◇) was monitored by cycloheximide chase. Equal amounts of log phase cells were removed at the indicated times following addition of cycloheximide. Samples were resolved by SDS-PAGE and detected by Western analysis using anti-HA and anti-hexokinase antibodies. Each time point represents the average and +/- standard error of the mean (SEM) of 4 measurements (two independent experiments done in duplicate) and normalized to the hexokinase loading control. (B) Refolded recombinantly expressed Yos9p (1) and Yos9p R200A (2) was purified and analyzed by SDS-PAGE with sample buffer containing either DTT or N-ethylmaleimide (-DTT) and stained with Coomassie. (C) Gel Filtration analysis of Yos9p (—), Yos9p R200A (- - -) and molecular size standards (gray). (D) Circular Dichroism spectra were acquired of Yos9p (top) and Yos9p R200A (bottom) as described in the experimental procedures.

Figure 2. Yos9 recognizes glycans containing a terminal α1,6-linked mannose

(A) A schematic representation of the initial Glc₃Man₉GlcNac₂ N-linked sugar and the legend for each sugar moiety represented. (B) FAC analysis of Yos9p sugar binding specificity. Indicated PA-oligasscharides were tested for binding to Yos9p by FAC analysis. Ka values were determined as described in the supplemental experimental procedures and are mean ± S.D. of three independent experiments. Each glycan structure is detailed and given a code name beneath the chart. (C-D) Elution profiles over time of fluorescently labeled (PA)-oligosaccharides applied over immobilized histidine tagged Yos9p (C, red) or R200A mutant (D, red) in comparison to a negative control sugar (black). PA-glycans are schematically represented next to the corresponding elution profile.

Figure 3. Production of Man₇GlcNac₂ sugars in vivo results in ERAD dependent degradation and bypass of HTM1

(A) Schematic representation of a portion of the asparagine linked glycosylation (ALG) pathway. Shown are the glycans produced in the wild-type (top) pathway and an alg9Δ over-expressing ALG12 (upward arrow indicates TDH3 driven expression) strain (bottom). Mannose residues are represented as blue circles and N-acetylglucosamine is represented by blue squares. (B) Degradation of CPY* in a (■) wild-type (WT), (△) alg9↑ALG12Δ, and (●) alg9Δ strains in this and the following panels were monitored as in Figure 1A except that each time point represents the average and +/- SEM of at least 8 measurements (4 independent experiments done in duplicate). (C) Degradation of CPY* in (■) wild-type (WT) and (△) alg9Δ↑ALG12 (upward arrow represents TDH3 driven expression), or CPY*0000 in (●) wild-type (WT) and (◇) alg9Δ↑ALG12 cells. CPY* is represented as a * and non-glycosylatable CPY* is represented as *0000. (D) Degradation of CPY* in (■) wild-type (WT), (△) alg9Δ↑ALG12, (◇) yos9Δalg9Δ↑ALG12 and (●) yos9Δ cells. (E) Degradation of CPY* in (■) wild-type (WT), (△) alg9Δ↑ALG12, (◇) der1Δalg9Δ↑ALG12 and (●) der1Δcells. (F) Degradation of CPY* in (■) wild-type (WT), (△) alg9Δ↑ALG12, (◇) htm1Δalg9Δ↑ALG12 and (●) htm1Δ cells.

Figure 4. Model of dual recognition of substrates by ERAD

Glycan processing from the initial N-linked Glc₃Man₉GlcNac₂ to Man₈GlcNac₂ occurs by Glucosidase I, II and Manosidase I, respectively. Htm1p marks potential substrates by playing a role in the generation of Man₇GlcNac₂ (upper panel). Misfolded proteins are recruited to the Hrd1p complex by recognition of misfolded domains by Hrd3p. Yos9p queries the N-linked glycan and substrates are committed for degradation after Yos9p has identified the presence of a terminal α1,6-linked mannose. Note: Whether Htm1p is an enzyme or cofactor remains to be determined.