Volume 43, Issue 2, Pages 229-241 (July 2011) Global Functional Map of the p23 Molecular Chaperone Reveals an Extensive Cellular Network  Frank J. Echtenkamp, Elena Zelin, Ellinor Oxelmark, Joyce I. Woo, Brenda J. Andrews, Michael Garabedian, Brian C. Freeman  Molecular Cell  Volume 43, Issue 2, Pages 229-241 (July 2011) DOI: 10.1016/j.molcel.2011.05.029 Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 SGA Analysis Revealed a Broad Genetic Interaction Network for Sba1 that Included Factors Involved in Vesicle-Mediated Protein Transport The genes that produced an SSL phenotype with sba1Δ were categorized by a GO Slim analysis. (A) Each hit was assigned to an initial cellular process and displayed, or (B) all potential processes were considered for each gene and the enrichments in each category were determined relative to the distribution of all nonessential yeast ORFs. (C) To test if Golgi function was Sba1 dependent, parental (WT) and sba1Δ transformants carrying either an empty or an Sba1-expression vector (+ Sba1) were treated with the Golgi transport inhibitor Brefeldin A (BFA) (100 μg mL−1). (D) To check if mammalian Golgi function is p23 dependent, exponentially growing parental or p23 null MEFs were cultured in unsupplemented or BFA-supplemented (50 μg mL−1) media. Error bars represent the standard error of the mean. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 The Yeast and Mouse p23 Proteins Are Negative Modulators of Protein Transport (A) Colocalization of mouse p23 (red) with the Golgi maker protein Giantin (green) was determined by IIF. The cells were counterstained with DAPI to detect the nuclei. (B) The impact of Sba1 levels on yeast Invertase secretion was determined. The influence of Sba1 loss (sba1Δ), Sba1 overexpression, or Sba1Δ84 (chaperone mutant) expression was checked as well as the secretion-defective mutant sec18-1. The levels of intracellular Myc-Invertase were detected by immunoblot analysis. (C) The efficiency of Preprolactin processing to Prolactin during transport in parental and p23 null MEF cells was determined. (D) The effect of Sba1 overexpression on Fus-Mid-GFP transport was examined by fluorescence microscopy in WT and secretion-compromised yeast. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Cell Motility Is p23 Dependent (A) The abilities of parental and p23 null MEFs to migrate into an artificial wound was determined, and (B) the results were quantified. Error bars represent the standard error of the mean. (C) The relative levels of the focal adhesion proteins Vinculin and α-Actinin were determined in parental and p23 null cells by IIF. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 Sba1 Physically Interacts with Proteins Functioning in a Wide Variety of Cellular Processes Including Ribosome Biogenesis Yeast ProtoArrays were used to detect proteins bound by Sba1. (A) Each hit was assigned to an initial cellular process using a GO Slim analysis and displayed, or (B) all potential processes were considered for each gene and the enrichments in each category were determined relative to the distribution for all yeast ORFs. (C) The sensitivity of yeast to the ribosome inhibitor Hyg B (300 μg mL−1) fluctuates with Sba1 levels. (D) The Hyg B (100 μg mL−1) sensitivity of exponentially growing parental and p23 null MEFs was determined. Error bars represent the standard error of the mean. (E) Sba1 facilitates ATP-dependent release of ribosome biogenesis factors from Rix1-associated, nascent 60S particles. The TAP purifications were prepared from wild-type and sba1Δ yeast expressing Rix1-TAP, ATP (2 mM) was included during the Calmodulin affinity step where indicated, and following elution the proteins were analyzed by SDS-PAGE and Coomassie staining. The maturation factors known to dissociate in an ATP-dependent manner are demarked (short lines) along with the ATP-insensitive components (long lines) (Ulbrich et al., 2009). Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 The Sba1 Network Has a Significant Nuclear Component that Includes DNA Repair Activities The SGA, ProtoArray, and all curated Sba1 interactors were pooled and analyzed using GO Slim. (A) Each hit was assigned to a cellular process and displayed, or (B) all potential processes were considered for each gene and the enrichments in each category were determined relative to the distribution for all nonessential yeast ORFs. (C) The nuclear-associated Sba1 interactors were analyzed using Osprey. Genes marked in yellow are associated with multiple activities, but for clarity each hit was placed only once in the presented network. (D) Wild-type (WT) or sba1Δ yeast transformants carrying either an empty vector or SBA1 expression vector (+Sba1) were grown in the presence of the DNA mutagens MMS (300 μg mL−1) or BLEO (20 mU). Alternatively, the transformants were exposed to UV light (200 J/m2) and then cultured in selective glucose media. (E) The sensitivities of parental or p23 null MEFs to DNA mutagens were tested by exposing the cells either to MMS (500 μg mL−1), BLEO (200 mU), or UV light (7.5 J/m2). Error bars represent the standard error of the mean. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 Sba1 and Hsp82 Associate with Factors Functioning in Common Cellular Processes and Locales The composite Sba1 catalog and all curated Hsp82 interactors were categorized by a GO Slim analysis, and the relative enrichments in either cellular processes (A) or compartments (B) were determined relative to the distribution of all nonessential yeast ORFs. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 7 The Sba1 and Hsp82 Networks Share Few Mutual Direct Hits Yet Are Connected to Common Cellular Processes (A) The relative overlap in discrete ORFs between the Sba1 and Hsp82 interactors was established and displayed in a Venn diagram. (B) The stable protein complexes that have Sba1 and Hsp82 interactors were determined. The inner circle represents complexes with Sba1 hits, the outer circle has Hsp82-linked structures, and the lines connect complexes that are associated with both chaperones. (C) The ability of Sba1 and Hsp82 to interact with different protein complexes was determined using the TAP tag pull-down assay. The indicated protein complexes were precipitated using a TAP fusion subunit that was not predicted to be a chaperone target. Sba1 and Hsp82 association with the various complexes was detected by immunoblot analysis. (D) Organizational models for chaperone-associated complexes are shown. The subunits predicted to interact with Sba1 (white), Hsp82 (Black), both (dark gray), or neither (light gray) are marked. Molecular Cell 2011 43, 229-241DOI: (10.1016/j.molcel.2011.05.029) Copyright © 2011 Elsevier Inc. Terms and Conditions