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. 2009 Mar 27;323(5922):1693-7.
doi: 10.1126/science.1167983.

Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum

Martin C Jonikas  1 , Sean R Collins, Vladimir Denic, Eugene Oh, Erin M Quan, Volker Schmid, Jimena Weibezahn, Blanche Schwappach, Peter Walter, Jonathan S Weissman, Maya Schuldiner
Affiliations

Comprehensive characterization of genes required for protein folding in the endoplasmic reticulum

Martin C Jonikas et al. Science. .

Abstract

Protein folding in the endoplasmic reticulum is a complex process whose malfunction is implicated in disease and aging. By using the cell's endogenous sensor (the unfolded protein response), we identified several hundred yeast genes with roles in endoplasmic reticulum folding and systematically characterized their functional interdependencies by measuring unfolded protein response levels in double mutants. This strategy revealed multiple conserved factors critical for endoplasmic reticulum folding, including an intimate dependence on the later secretory pathway, a previously uncharacterized six-protein transmembrane complex, and a co-chaperone complex that delivers tail-anchored proteins to their membrane insertion machinery. The use of a quantitative reporter in a comprehensive screen followed by systematic analysis of genetic dependencies should be broadly applicable to functional dissection of complex cellular processes from yeast to human.

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Figures

Fig. 1
Fig. 1
Quantitative screen for gene deletions that perturb UPR signaling. (A) Strategy for quantifying UPR levels in deletion strains. (B) GFP/RFP reporter levels as a function of concentration of DTT, a reducing agent that causes protein misfolding in the ER. (C) UPR reporter levels of up-regulator hits by functional category.
Fig. 2
Fig. 2
Double mutant analysis provides information on functional dependencies between genes. (A) “Double mutant” (DM) plot of Δhac1. Each point represents a gene. X-coordinate represents the reporter level of a strain deleted for that gene in a WT background. Y-coordinate represents the reporter level in a double mutant lacking the same gene and additionally deleted for a second gene (in this case HAC1). The horizontal blue line indicates the reporter level in the Δhac1 single mutant. Circled in red are up-regulators whose reporter induction is HAC1-independent, which are highly enriched for chromatin architecture factors. (B) (Top) Schematic of the lumenal steps of the N-linked glycan synthesis pathway. (Bottom) DM plot for Δdie2/alg10. (C) DM plot depicting genetic interactions between deletion mutants and over-expression (OE) of the ERAD substrate KWS.
Fig. 3
Fig. 3
Systematic identification of genetic interactions. (A) Generalized DM plot illustrating the distribution of reporter levels in double mutants Δxxx Δyfg plotted against reporter levels in single mutants Δxxx. A red curve traces the typical double mutant reporter level as a function of the single mutant reporter level. The interaction value (π-score) is determined by the difference between the expected and measured UPR levels in a double mutant. Double mutants with unusually high fluorescence (blue dots), typical fluorescence (black dots), or unusually low fluorescence (yellow dots) represent aggravating, no, or alleviating genetic interactions, respectively. Fully masking interactions are found either on the horizontal blue line (Δyfg fully masks Δxxx) or on the diagonal blue line (Δxxx fully masks Δyfg). (B) Hierarchical clustering of a genetic interaction map based on systematic π-score analysis. To the right of the map, functional clusters are labeled (Table S5). Clusters referred to in the text are highlighted in red; those containing novel components are marked in italics.
Fig. 4
Fig. 4
Genetic interactions identify functional dependencies of uncharacterized proteins. (A) DM plot of Δhrd3. (Inset) Enlargement of a region of Fig. 3B, showing genetic interactions of the ERAD cluster. (B) Selected genetic interactions of the ER Membrane Complex (EMC). (C) SDS PAGE analysis of immunoprecipitation of Emc3p -FLAG and associated proteins; protein identities were determined by mass spectrometry. *The specificity of the Por1p interaction has not been evaluated.
Fig. 5
Fig. 5
YOR164C/GET4 and MDY2/GET5 function in the pathway of tail-anchored protein insertion. (A) DM plot depicting the functional dependencies of MDY2/GET5. (B) In-vitro translocation assay. Sec22p was translated in cytosol from wild-type (WT) or Δmdy2/get5 strains. Error bars represent +/− SEM, N=3 (C) GFP-Sed5p localization defect in Δget3, Δget4 and Δmdy2/get5 strains. The images of at least 20 cells per strain with similar average fluorescence were quantified to determine the distribution of each strain’s total fluorescence across pixels of different intensities. (D) Silver stain of immunoprecipitation of Get3-FLAGp from ER microsomes and cytosol; protein identities were determined by mass spectrometry.
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