S‐ 1 ‐  

Supporting Information

(Contains Figures S1, S2, S3, and S4 with Legends)

A technique for delineating the unfolding requirements for substrate entry into retrotranslocons during endoplasmic reticulum–associated degradation

Junfen Shi1,2,#, Xianyan Hu1,2,#, Yuan Guo1,2, Linhan Wang1,2, Jia Ji1,2, Jiqiang Li1,2 and Zai-Rong

Zhang1 *

From the 1 Interdisciplinary Research Center on Biology and Chemistry, Shanghai Institute of

Organic Chemistry, Chinese Academy of Sciences, Shanghai, 201210, China; 2 University of

Chinese Academy of Sciences, 19A Yuquan Road, Shijingshan District, Beijing, 100049, China

Running title: Unfolding requirements for substrates retrotranslocation

*To whom correspondence should be addressed: Interdisciplinary Research Center on Biology and

Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai,

201210, China; zrzhang@sioc.ac.cn; Tel.: 86-21-68582263 # These authors contributed equally to this work.

Key words: endoplasmic reticulum-associated degradation (ERAD), E3 ubiquitin ligase, membrane

protein, protein misfolding, ER quality control, retrotranslocation, Vpu, dihydrofolate reductase,

destabilizing domain, unfolded substrate, tunable folding domain, ER-to-cytosol transport

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Figure S1. Hrd1 mediates DHFR-NHK degradation.

(A) Analyze turnover of DHFR-NHK in the Hrd1 knock out (Hrd1KO) 293T cells by CHX chase.

DHFR-NHK and GFP were coexpressed in WT or Hrd1KO cells followed by CHX chase, cell lysate

preparation, and western blot analysis. The asterisk denotes non-specific bands reacting with -Hrd1

antibody in the long exposure image. (B) Analyze interaction of DHFR-NHK with ERAD factors in

the absence or presence of TMX. Cells expressing FLAG tagged DHFR-NHK or empty vectors for

24 hours were treated with TMX, CB5083 or both before harvesting, lyzed and subjected to

immunoprecipitation with -FLAG agarose. Total cell lysate and bead-bound materials were

analyzed by immunoblotting for indicated proteins.

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Figure S2. Hrd1 and gp78 are not required for Vpu-mediated CD4 retrotranslocation and

degradation.

(A) Schematic diagram of steps and factors involved in Vpu-mediated ERAD of CD4. Critical steps

include CD4 ubiquitination, retrotranslocation and proteasomal degradation. (B) Analysis of CD4

degradation in wild-type (WT), Hrd1KO, gp78KO, or Hrd1/gp78-double knockout (DKO) 293T cells.

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CD4 was cotransfected with Vpu, phosphorylation mutant Vpu-SN, or empty vector.Tthe cell lysates

were analyzed for CD4, Vpu, Hrd1, and gp78 levels by immunoblotting. Note that the Vpu is

phosphorylated, while the S52/56N (SN) mutant is not. (C) Schematic representation of CD4 and

CD4-derivated substrates used throughout this study. The CD4 variants includes CD4, superfolder

GFP-fused small-CD4 (sfGFP-sCD4), mini-CD4 (mCD4), and DHFR fused mini-CD4 (DHFR-

mCD4). (D-F) Analyze degradation of CD4 variants, including sfGFP-sCD4 (D), mCD4 (E), and

DHFR-mCD4 (F). Twenty-four hours after cotransfection of Vpu or Vpu-SN and CD4 variants, cells

were lyzed and the resulting cell lysates were analyzed for levels of Vpu and various CD4 variants.

In (D) and (F), WT 293T cells were used to perform the experiments. In (E), the experiments were

performed in WT, Hrd1KO, gp78KO, and Hrd1/gp78-DKO 293T cells where indicated. In (F), TMX

or DMSO (as a control) was added to incubate with the cells for 4 hours before harvesting. (G)

DHFR-mCD4 retrotranslocation is independent of Hrd1 and gp78. CHX chase with or without

MG132 was performed for analyzing DHFR-mCD4 retrotranslocation in Hrd1/gp78 DKO cells. The

deglycosylated substrate represents retrotranslocated species accumulated in the cytoplasm due to

proteasome inhibition. (H) Analyze DHFR-mCD4 retrotranslocation in cells. Cells expressing Vpu

and DHFR-mCD4 were treated with indicated individual or combination of chemicals, followed by

lysis and immunoblotting analysis for indicated proteins. The red arrow indicates deglycosylated,

retrotranslocated substrate, which disappeared in the presence of CB5083, a p97 ATPase inhibitor.

Relevant bands were quantified and plotted (right panel).

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Figure S3. Construction of DHFR-Bio-mCD4 and the hydrophilicity analysis.

(A) Schematic diagram of DHFR-Bio-mCD4 for capturing partially retrotranslocated intermediate.

Prol. SS: the signal sequence from bovine Prolactin; CHO: glycosylation site; TMD: transmembrane

segment; D4: the immunoglobin-like D4 domain of CD4 (amino acids 316-396 of homo sapiens

CD4). The positions of glycosylation motif and insertion site for BioTag on mCD4 were indicated.

(B) Mature DHFR-Bio-mCD4 protein sequence was analyzed for hydrophilicity using the Kyte-

Doolittle scale. (https://fasta.bioch.virginia.edu/fasta_www2/fasta_www.cgi?rm=misc1&pgm=tkd)

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Figure S4. Degradation of mTAP2 and DD-mTAP2 from the ER membrane.

(A) A CHX chase experiment for analyzing mTAP2 turn over in WT, gp78KO, Hrd1/gp78-DKO and

Hrd1KO 293T cells. The construct for transfection contains GFP and mTAP2 separated by a viral 2A

“self-cleaving” peptide. Thus, GFP and mTAP2 were produced in equimolar amounts as independent

polypeptides. The arrowheads and asterisk denote non-specific bands reacting with -FLAG

polyclonal and -gp78 antibodies, respectively. (B) A CHX chase experiment for measuring DD-

mTAP2 turnover rate in the presence of Shield-1 or CB5083. DMSO was used as a control. Cells

were pre-incubated with either Shield-1 for 5 hours or CB5083 for 1 hour before co-incubation with

CHX for indicated times. EGFP was expressed as a control.