1. Cell Biology
  2. Human Biology and Medicine
Download icon

The cargo receptor SURF4 promotes the efficient cellular secretion of PCSK9

  1. Brian T Emmer
  2. Geoffrey G Hesketh
  3. Emilee Kotnik
  4. Vi T Tang
  5. Paul J Lascuna
  6. Jie Xiang
  7. Anne-Claude Gingras
  8. Xiao-Wei Chen
  9. David Ginsburg  Is a corresponding author
  1. University of Michigan, Michigan
  2. Lunenfeld-Tanenbaum Research Institute, Sinai Health System, Canada
  3. University of Toronto, Canada
  4. Peking University, China
Research Article
Cite this article as: eLife 2018;7:e38839 doi: 10.7554/eLife.38839
5 figures, 1 table, 2 data sets and 6 additional files

Figures

Proximity-dependent biotinylation with a PCSK9-BirA* fusion.

(A) Proximity detection by mass spectrometry of streptavidin-purified prey proteins biotinylated by a fusion of BirA* to a bait protein of interest. (B) Immunoblotting of lysates of cells expressing various BirA*-fusion proteins. (C) Spectral counts of prey proteins identified from lysates of cells expressing PCSK9-BirA* relative to A1AT-BirA*. (D) Spectral counts of prey proteins purified from lysates of cells expressing PCSK9-BirA* relative to the maximum spectral count from lysates of cells expressing either SAR1A-BirA* or SAR1B-BirA*. (E) Venn diagram of identified prey proteins from lysates of cells expressing BirA* fusions with PCSK9, A1AT, or the maximum for either Sar1A or Sar1B. (F) Heat map of spectral counts for candidate proteins demonstrating interaction with both PCSK9-BirA* and either SAR1A-BirA* or SAR1B-BirA*. Spectral count values represent averages of 2 biologic replicates. Only prey proteins that exhibit BFDR ≤0.01 for one or more bait proteins are displayed. Source data is provided in Supplementary file 1.

https://doi.org/10.7554/eLife.38839.002
Figure 2 with 3 supplements
Whole genome CRISPR mutagenesis screen for PCSK9 secretion modifiers.

(A) Strategy for whole genome screen. (B) Flow cytometry of reporter cells stably expressing PCSK9-eGFP-2A-A1AT-mCherry, treated with 1 µg/mL brefeldin A or a sgRNA targeting LMAN1. (C) Normalized abundance of each sgRNA in the library in eGFP high and eGFP low populations. (D) MAGeCK gene-level enrichment scores for each gene targeted by the library arranged by chromosome number and transcription start site. The diameter of the bubble is proportional to the number of unique sgRNAs targeting each gene that demonstrate significant enrichment in GFP high cells. Source data is provided in Supplementary files 2 and 3.

https://doi.org/10.7554/eLife.38839.003
Figure 2—figure supplement 1
Analysis of PCSK9-eGFP-2A-A1AT-mCherry reporter cell clones.

(A) Construct for CMV-promoter-driven expression of PCSK9-eGFP-2A-A1AT-mCherry expression. (B) Immunoblotting of HEK293T cells transfected with the PCSK9-eGFP-2A-A1AT-mCherry vector, and four individual drug-resistant clonal cell lines, relative to parental untransfected cells.

https://doi.org/10.7554/eLife.38839.004
Figure 2—figure supplement 2
Whole genome screen analysis.

(A) Cumulative distribution function of mapped reads for each sgRNA in the whole genome library. (B) Comparison of read counts for every pairwise combination of 4 biologic replicates with Pearson correlation coefficient.

https://doi.org/10.7554/eLife.38839.005
Figure 2—figure supplement 3
Validation experiments for additional candidate genes.

The top-scoring sgRNA for each of the next 21 most enriched genes after SURF4 was individually cloned into lentiCRISPRv2 and tested in PCSK9-eGFP-2A-A1AT-mCherry reporter cells. Flow cytometry was performed for each of 3 biologic replicates. The mean fluorescent intensity of PCSK9-eGFP and A1AT-mCherry for sgRNA against each candidate gene relative to nontargeting sgRNA controls is shown. Error bars represent standard deviations.

https://doi.org/10.7554/eLife.38839.006
Figure 3 with 2 supplements
SURF4 mutagenesis causes an accumulation of intracellular PCSK9-eGFP.

(A) Individual sgRNA sequencing counts for SURF4-targeting sgRNA in eGFP high and eGFP low populations for each of 4 biologic replicates. Adjusted p values were calculated using DESeq2. (B) Flow cytometry histograms of PCSK9-eGFP and A1AT-mCherry fluorescence in reporter cells transfected with plasmids delivering Cas9 and SURF4-targeting sgRNA or empty vector. (C) Quantification of intracellular fluorescence for cells treated with empty vector (n = 3) or unique SURF4-targeting sgRNAs (n = 6). (D) Quantification of intracellular fluorescence for clonal cell lines each containing frameshift-causing indels at two different SURF4 target sites (n = 7 wild-type clones, n = 3 clones generated from each SURF4-targeting sgRNA). (E) Flow cytometry histograms for cells expressing PCSK9-eGFP-2A-A1AT-mCherry and deleted for SURF4 with or without stable expression of a wild-type or FLAG-tagged SURF4 cDNA. (F) Time course of intracellular accumulation of tetracycline-inducible PCSK9-eGFP on WT, SURF4-deficient, or SURF4 rescue background (n = 3 biologic replicates for each cell line at each time point). *p<0.05 by Student’s t-test. Error bars represent standard deviations.

https://doi.org/10.7554/eLife.38839.007
Figure 3—figure supplement 1
ER stress markers.

Clonal cell lines with a stably integrated tetracycline-inducible PCSK9 cDNA on either WT or SURF4 mutant background were treated with tetracycline or vehicle control and analyzed by immunoblotting for various markers of ER stress (A), quantified by densitometry (B). Error bars represent standard deviations.

https://doi.org/10.7554/eLife.38839.008
Figure 3—figure supplement 2
SURF4-deficient genotypes.

Clonal cell lines were genotyped by PCR amplification of sgRNA target site and Sanger sequencing of the amplicon with either TIDE decomposition of individual alleles from the amplicon, or ligation of the amplicon into cloning plasmids and Sanger sequencing of multiple individual clones. Sequence corresponding to sgRNA is underlined and expected double-strand break site is indicated (^).

https://doi.org/10.7554/eLife.38839.009
SURF4 localizes to the early secretory pathway where it physically interacts with PCSK9.

(A) Immunofluorescence of FLAG-SURF4 together with markers of the ER (calnexin), ERGIC (LMAN1), and Golgi (GM130). Scale bar = 10 µm. (B) Quantification of colocalization (n = 8 cells analyzed for each combination of antibody staining). (C) Spectral counts for SURF4 in streptavidin-purified eluates from cells expressing various BirA* fusion proteins. (D) Immunoprecipitations were performed using antibodies directed against FLAG or GFP from lysates of cells expressing FLAG-SURF4, PCSK9-eGFP, both, or neither. Error bars represent standard deviations.

https://doi.org/10.7554/eLife.38839.010
Figure 5 with 2 supplements
SURF4 mutagenesis causes a decrease in PCSK9 extracellular secretion and an accumulation of PCSK9 in the ER.

(A) Fluorescence detection of PCSK9-eGFP in extracellular conditioned media relative to cellular lysate in WT (n = 7) and clonal SURF4-deficient (n = 5) fluorescent reporter cell lines. (B) Immunoblotting of tetracycline-inducible PCSK9 in extracellular conditioned media and cellular lysates from WT, SURF4-deficient, or SURF4 rescued cells. (C) Quantification of densitometry of native PCSK9 relative in conditioned media and cellular lysates, normalized to GAPDH, for WT, SURF4-deficient, or SURF4 rescued cells (n = 4 biologic replicates for each cell line). (D) Quantitative PCR of SURF4 and PCSK9 transcript levels from RNA isolated from a SURF4 WT or mutant fluorescent reporter cell line (n = 3 measurement replicates). (E) Live cell fluorescence microscopy of fluorescent reporter cells, either WT or SURF4-deficient, transfected with the ER marker ERoxBFP. Scale bar = 10 µm. (F) Quantification of PCSK9-eGFP signal intensity in pixels positive for ERoxBFP fluorescence (n = 14 for each cell line). (G) EndoH-sensitivity of PCSK9 expressed in WT, SURF4-deficient, or SURF4 rescued cells. (H) Quantification of endoH-sensitivity, normalized to average intensity of average WT band intensity (n = 4 biologic replicates for each cell line). *p<0.05 by Student’s t-test. Error bars represent standard deviations.

https://doi.org/10.7554/eLife.38839.011
Figure 5—figure supplement 1
Pulse-chase labeling of PCSK9-eGFP secretion.

A SURF4 mutant cell line with a tetracycline-inducible PCSK9-eGFP construct and with (SURF4 rescue) or without (SURF4 mutant) a stably integrated SURF4 cDNA were radiolabeled with tracer containing 35S-Met and 35S-Cys. Conditioned media and cellular lysates were harvested at the indicated time points and PCSK9-eGFP immunoprecipitated and analyzed by SDS-PAGE electrophoresis and autoradiography.

https://doi.org/10.7554/eLife.38839.012
Figure 5—figure supplement 2
EndoH-sensitivity of PCSK9 expressed in WT, SURF4-deficient, or SURF4 rescued cells for each of 4 independent biologic replicates.
https://doi.org/10.7554/eLife.38839.013

Tables

Key resources table
Reagent
type (species)
or resource
DesignationSource or referenceIdentifiersAdditional

information
Gene (Homo
sapiens)
PCSK9NAUniprot Q8NBP7
Gene (Homo
sapiens)
A1ATNAUniprot P01009
Gene (Homo
sapiens)
SURF4NAUniprot O15260
Cell line (Homo
sapiens)
HEK293TATCCRRID:CVCL_1926
Cell line (Homo
sapiens)
T-Rex-293InvitrogenRRID:CVCL_D585
Antibodyanti-PCSK9 (rabbit
polyclonal)
Cayman ChemicalRRID:AB_569536(1:1000)
Antibodyanti-GFP (rabbit
monoclonal)
AbcamRRID:AB_303395(1:5000)
Antibodyanti-mCherry (rabbit
polyclonal)
AbcamRRID:AB_2571870(1:1000)
Antibodyanti-GAPDH (rabbit
monoclonal)
AbcamRRID:AB_2630358(1:10,000)
Antibodyanti-β-actin (mouse
monoclonal)
Santa CruzRRID:AB_2714189(1:10,000)
Antibodyanti-LMAN1 (rabbit
monoclonal)
AbcamRRID:AB_10973984(1:5000)
Antibodyanti-calnexin (rabbit
monoclonal)
Cell SignalingRRID:AB_2228381(1:2000)
Antibodyanti-GM130 (rabbit
monoclonal)
AbcamRRID:AB_880266(1:1000)
Antibodyanti-BiP (rabbit
monoclonal)
AbcamRRID:AB_10859806(1:5000)
Antibodyanti-PDI (rabbit
monoclonal)
Cell SignalingRRID:AB_2156433(1:1000)
AntibodyHRP-conjugated anti-
FLAG (goat
polyclonal)
AbcamRRID:AB_299061(1:10,000)
AntibodyHRP-conjugated anti-
mouse secondary (goat
polyclonal)
BioRadRRID:AB_11125547(1:5000)
AntibodyHRP-conjugated anti-
rabbit IgG (goat
polyclonal)
BioRadRRID:AB_11125142(1:5000)
AntibodyFITC-conjugated anti-
FLAG (mouse
monoclonal)
SigmaRRID:AB_439701(1:500)
AntibodyAlexa647-conjugated
anti-rabbit secondary
(donkey polyclonal)
Abcamab150075(1:500)
Recombinant DNA reagentpLentiCRISPRv2Addgene52961
Recombinant DNA reagentpX459Addgene62988
Recombinant DNA reagentpNLF-C1PromegaE1361
Recombinant DNA reagentpDEST-pcDNA5-BirA-FLAG-C-termPMID 24255178
Recombinant DNA reagentpENTR/D-TOPOInvitrogenK2400-20
Chemical compound, drugbrefeldin ABioLegend420601
Chemical compound, drugdithiobis(succinimidyl propionate)Pierce22586

Data availability

Proteomic data has been deposited to the MassIVE database under accession MSV000082222. Sequencing data has been uploaded to the Sequence Read Archive under accession SRP149835. Source data files are included in the supplementary information.

The following data sets were generated
  1. 1
  2. 2

Additional files

Supplementary file 1

BioID of PCSK9-interacting proteins.

Spectral counts are listed for prey proteins identified from streptavidin-purified lysates of cells expressing fusions of BirA* with GFP, PCSK9, A1AT, SAR1A, and SAR1B. Gene Ontology enrichment analysis (Ashburner et al., 2000; The Gene Ontology Consortium, 2017) is displayed for candidate PCSK9 cargo receptors.

https://doi.org/10.7554/eLife.38839.014
Supplementary file 2

CRISPR screen sgRNA-level analysis.

DESeq2 output for each sgRNA showing normalized mapped reads in control (GFP-low) populations and log2 fold-change in experiment (GFP-high) populations with significance testing for enrichment or depletion. A copy of the reference library showing the corresponding gene target and target sequence and gene for each sgRNA is included.

https://doi.org/10.7554/eLife.38839.015
Supplementary file 3

CRISPR screen gene-level analysis.

Enrichment for each sgRNA targeting the same gene was combined to generate a gene-level enrichment score using MAGeCK (Li et al., 2014), which is displayed in the column corresponding to the number of unique sgRNA targeting that same that demonstrated enrichment (p<0.05) by analysis with DESeq2 (Supplementary file 2).

https://doi.org/10.7554/eLife.38839.016
Supplementary file 4

Oligonucleotide sequences.

Primers used for CRISPR/Cas9 targeting and genotyping are displayed.

https://doi.org/10.7554/eLife.38839.017
Source code 1

gRNA_mapping.zip

https://doi.org/10.7554/eLife.38839.018
Transparent reporting form
https://doi.org/10.7554/eLife.38839.019

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Download citations (links to download the citations from this article in formats compatible with various reference manager tools)

Open citations (links to open the citations from this article in various online reference manager services)