Identification of endoplasmic reticulum localized degrons.

A) Cartoon depicting the ER-tFT and KHN-tFT constructs, which contain an ER-targeting signal sequence (SS), mCherry protein (red), superfastGFP (green), and the HDEL ER-retention sequence. KHN-tFT functions as a positive control for a quickly degraded ERAD substrate.

B) Yeast strains expressing the constructs described in (A) were treated with cycloheximide (CHX) for 0, 30, 60, or 90 minutes, harvested, and protein levels were assessed by immunoblotting against GFP and normalized against total protein in gel using stain-free technology (Loading).

C) Flow cytometry of yeast strains expressing the constructs in (A) treated with cycloheximide for 2 hours. The mCherry/GFP fluorescence intensity ratio of each cell was calculated and plotted.

D) Quantification of the mean mCherry/GFP ratio of four biological replicates as in (C).

E) Overview of pentapeptide library generation and isolation of unstable variants by FACS. A DNA fragment containing the pentapeptide-ER-tFT library was electroporated with digested ER-tFT plasmid. The resulting yeast library contains a mixture of more less stable variants, which can be separated from one another by FACS, with more stable variants having increased mCherry fluorescence intensity compared to unstable variants.

F) Heatmap of amino acid enrichments at each position within the unstable pentapeptide library. Values are displayed relative to either codon usage or relative to the input library.

G) Flow cytometry of yeast strains expressing either ER-tFT, KHN-tFT, unstable selected pentapeptide-ER-tFT sequences or randomly selected pentapeptide-ER-tFT sequences. The cells were analyzed after treatment with cycloheximide for 2 hours.

H) Quantification of 3 biological replicates conducted as in (G).

I) As in (C) but with yeast strains expressing ER-tFT, KHN-tFT, and IHPYW (1X), 2x repeat of IHPYW (2X), or 4x repeat of IHPYW (4X) at the N-terminus of ER-tFT.

J) Quantification of 3 biological replicates of (I).

DegV1 is an ERAD-dependent degron degraded by the cytosolic proteasome.

(A) Molecular weight of anti-GFP nanobodies (NbGFP-Flag) either with or without signal sequence in the presence or absence of DegV1, were assessed by SDS-PAGE electrophoresis followed by immunoblotting with anti-Flag antibody. This panel is representative of two biological replicates.

(B) The degradation of ER-targeted anti-GFP nanobodies (ER-NbGFP-Flag) either with, or without, DegV1 were monitored following addition of cycloheximide (CHX). Loading controls were visualized by stain-free technology.

(C) The degradation of ER-NbGFP-Flag with DegV1 replacing the CDR3 region was analyzed as in (B).

(D) The degradation of a nanobody with DegV1 located either directly preceding the C-terminal ER-retention signal (HDEL) or directly at the C-terminus of the nanobody was analyzed as in (B).

(E) The degradation of ER-targeted GFP proteins (top panel), ER-DegV1-GFP (middle panel), or ER-ConV1-GFP (bottom) were analyzed in a pdr5Δ by flow cytometry following either ethanol (EtOH) or cycloheximide (CHX) treatment for 2 hours. Where indicated, cells were pretreated with bortezomib (Btz) for 2 hours.

(F) As in (E), but in a hrd1Δpdr5Δ strain.

(G) Degradation of ER-DegV1-NbGFP was followed in pdr5Δ or hrd1Δpdr5Δ strain using a cycloheximide chase. Where indicated, cells were pretreated with bortezomib (Btz) for 2 hours.

(H) Quantification of (G) with error bars representing the standard deviation.

(I) Degradation of ER-ConV1-NbGFP (top panel) or ER-DegV1-NbGFP (bottom panel) was followed in the strains containing known ERAD component deletions using a cycloheximide chase. This panel is representative of two biological replicates.

(J) The degradation of ER-ConV-GFP (left), or ER-DegV-GFP (right) were analyzed in the indicated ERAD-component deletion strains by flow cytometry following cycloheximide treatment for 2 hours.

All panels in this figure are representative of at least three independent biological replicates, unless otherwise indicated.

DegV1 targets endogenous ER proteins for degradation.

(A) The degradation of an endogenous secretory protein with a C-terminal Flag (ER-Suc2-Flag) containing either DegV1 or ConV1 was monitored following addition of cycloheximide (CHX). Loading controls were visualized by stain-free technology.

(B) The degradation of a single membrane spanning ER resident protein (Big1) containing either DegV1 or ConV1 was followed as in (A).

(C) The degradation of polytopic integral membrane ER resident protein (Elo1) containing either DegV1 or ConV1 was followed as in (A).

(D) The degradation of Big1 with DegV1 or ConV1 was followed as in (A) but after 2 hour pretreatment with either ethanol (EtOH) or bortezomib (Btz) in a pdr5Δ strain.

(E) The degradation of Elo1 with DegV1 or ConV1 was followed as in (D).

All panels in this figure are representative of at least three independent biological replicates..

DegV1 functions as a degron in mammalian cells.

(A) ER-targeted mNeonGreen (ER-HA-mNG) was expressed alone (-), or with a ConV1, or a DegV1 in U2OS cells by transient transfection.The degradation of ER-mNG was followed by immunoblotting with anti-HA antibody after treatment with 50μM emetine. β-actin was used as a loading control.

(B) Anti-HA band intensities from (A) were quantified and normalized to the corresponding β-actin level.

(C) As in (A) but after treatment with 50nM bortezomib (Btz) for the indicated times.

(D) Quantification of (C) normalized to the control protein (ER-HA-mNG).

(E) As in (A) but after treatment with 1µM CB5083, a p97 inhibitor, for the indicated times.

(F) Quantification of (E) normalized to the control protein (ER-HA-mNG).

(G) ER-HA-mNG with either ConV1 (left panel) or DegV1 (right panel) were expressed in U2OS pretreated with either 50nM bortezomib or 1µM CB5083 for 16 hours prior to an emetine chase.

(H) The degradation of ER-HA-mNG with either ConV1 or DegV1 was followed in HEK293T cells or HRD1-/- cells using an emetine chase.

(I) Quantification of (H).

All panels in this figure are representative of at least three independent biological replicates and the quantification is presented as the mean +/-standard deviation.

(A) We targeted the tFT to the ER with two different signal peptides, either that of mating factor alpha or of Ost1. The GFP and mCherry fluorescence were plotted for each cell in the left panel. The mCherry/GFP fluorescence intensity ratio of each cell was calculated and plotted in the right panel. Based on the superior brightness of cells expressing the mating factor alpha signal sequence, we selected this signal peptide for further experimentation.

(B) Flow cytometry of yeast strains expressing an ER-tFT and ERAD-substrate KHN-tFT treated with cycloheximide (CHX) for 2 hours.

(C) Schematic of the PCR-mediated library generation (left) using degenerate primers (upper right) and homologous recombination in yeast (lower right).

(D) Heatmap of input library amino acid enrichments at each position displayed relative to codon frequency.

(E) Gating strategy for analyzing single cells by flow cytometry.

(F) Sorting bins defined relative to ER-tFT and KHN-tFT.

(G) Comparison of mCherry/GFP ratios for alternate signal sequences on ER-tFT and IHPYW-tFT.

(A) Linear diagram of degradation constructs used in this work. SS, mating factor alpha signal sequence; AS, alanine and serine dipeptide linker; NbGFP, LaG16 anti-GFP nanobody; HDEL, ER retention signal. Superscript indicates the position of DegV1.

(B) Localization of ER-targeted GFP proteins (ER-ConV1-GFP and ER-DegV1-GFP) in wild-type yeast cells. We observed both ER and vacuolar localization. The scale bar is 5 µm.

(C) The degradation of ER-targeted GFP proteins (ER-GFP (top), ER-DegV1-GFP (middle), or ER-ConV1-GFP (bottom)) were analyzed by flow cytometry following either ethanol (EtOH) or with bortezomib (Btz) for 2 hours.

(D) As in (C), but in a hrd1Δpdr5Δ strain.

(E) Degradation of ER-targeted GFP with an ER retention signal (HDEL) with, or without DegV1 was followed in a hrd1Δ strain complemented with either an empty vector or with Hrd1 on a centromeric plasmid. Using a cycloheximide chase and immunoblotting, we found in the absence of Hrd1, we observe transport of the proteins to the vacuole, where free GFP accumulates. The asterisk indicates the vacuolar localized GFP fragment.

(F) As in (E), except with CPY*-GFP-HDEL. The asterisk indicates the vacuolar localized GFP fragment.

(G) Degradation of ER-DegV1-NbAlfa, ER-DegV1-NbGFP or ER-NbGFP was followed using a cycloheximide chase in the presence or absence of bortezomib (Btz) in a pdr5Δ or hrd1Δpdr5Δ strain.

(A) Degradation of a cytosolically-localized anti-GFP nanobody (cytosolic NbGFP) or with DegV1 (cytosolic DegV1-NbGFP) was followed in pdr5Δ or hrd1Δpdr5Δ strains with, or without bortezomib (Btz) using a cycloheximide (CHX) chase. Note that Hrd1 is not required for the degradation of cytosolic DegV1-NbGFP.

(B) Quantification of three independent biological replicates from (A). The error bars represent the standard deviation.