Adaptor protein complex-1 (AP-1) is recruited by the HEATR5 protein Laa1 and its co-factor Laa2 in yeast

Cellular membrane trafficking mediated by the clathrin adaptor protein complex-1 (AP-1) is important for the proper composition and function of organelles of the endolysosomal system. Normal AP-1 function requires proteins of the HEAT repeat–containing 5 (HEATR5) family. Although HEATR5 proteins were first identified based on their ability to interact with AP-1, the functional significance of this interaction was unknown. We used bioinformatics-based phenotypic profiling and information from genome-wide fluorescence microscopy studies in the budding yeast Saccharomyces cerevisiae to identify a protein, Laa2, that mediates the interaction between AP-1 and the yeast HEATR5 protein Laa1. Further characterization of Laa2 revealed that it binds to both Laa1 and AP-1. Laa2 contains a motif similar to the characterized γ-ear–binding sites found in other AP-1–binding proteins. This motif in Laa2 is essential for the Laa1–AP-1 interaction. Moreover, mutation of this motif disrupted AP-1 localization and function and caused effects similar to mutations that remove the γ-ear of AP-1. These results indicate that Laa2 mediates the interaction between Laa1 and AP-1 and reveal that this interaction promotes the stable association of AP-1 with membranes in yeast.

AP-1 is a highly conserved clathrin adaptor that functions at internal organelles including the trans-Golgi network (TGN), 2 endosomes, and lysosome-related organelles (5). AP-1 plays pivotal roles in lysosomal protein sorting and recycling from the endosomes and nascent lysosome-related organelles. It is made of four subunits: ␤, ␥, , and . The N termini of the ␤and ␥-subunits together with theand -subunits form the cargo and membrane-binding "core" (6), whereas appendages on the ␤and ␥-subunits bind clathrin and several accessory factors (7). Therefore, the interaction of AP-1 with cargo sets in motion a process that assembles a coat and ultimately dictates where cargo will be directed. For this reason, AP-1 association with cargo is highly regulated.
In budding yeast, AP-1 localizes to the TGN (8,9). In yeast, the TGN is a dynamic compartment that forms from the maturation of the Golgi and dissipates through the action of recycling, secretion, and vacuolar protein sorting (10,11). In this continuum, AP-1 participates in one of the last acting events at the TGN as monitored by time-lapse microscopy (12,13). It is still unclear how the cell orchestrates the precise temporal specificity of AP-1 recruitment to this late stage.
AP-1 recruitment depends, in part, on physical interaction with cargo, the small GTPase Arf1, and phosphatidyl-inositol-4-phosphate (PI4P). Structural studies suggest that AP-1 can bind all three simultaneously, and in vitro, purified AP-1 binds synthetic liposomes containing all three factors (14,15). This cooperative binding does not completely explain the temporal specificity of AP-1 recruitment, because AP-1 is recruited after the peak of Arf1 and PI4P, suggesting additional factors may play a role in AP-1 recruitment in vivo (13). One candidate for such a factor is Laa1, a member of the conserved HEATR5 family, which was previously reported to contribute to AP-1 localization (16).
HEATR5 proteins are large proteins made of many tandem HEAT repeats. The HEAT repeat is a simple fold often found in scaffolding proteins (17). Based on this property, HEATR5 proteins may have many binding partners. The founding member of this family, HEATR5b (also known as p200a), was identified in a proteomics study of proteins that interact with the AP-1 ␥ subunit appendage domain known as the ␥-ear (18). HEATR5b and homologs in Drosophila melanogaster (CG2747), Caenorh-  This article contains Tables S1-S3. 1  abditis elegans (SOAP-1), Schizosaccharomyces pombe (Sip1), and Saccharomyces cerevisiae (Laa1) are required for many AP-1 functions (16, 19 -22). Although disruption of HEATR5 genes causes strong defects in AP-1 recruitment in flies, nematodes, fission, and budding yeast, it was unclear whether HEATR5 played a direct role in recruiting AP-1 and whether this required the interaction between HEATR5 and AP-1 (16,21,22). One challenge was that there was no way to selectively disrupt HEATR5 binding with AP-1 to test the functional significance of the interaction in AP-1 localization. This is because the interaction between AP-1 and HEATR5 proteins is thought to be indirect. HEATR5 proteins lack canonical AP-1 ␥-earbinding sites. In mammalian cells, HEATR5b binds two proteins, aftiphilin and ␥-synergin, that themselves interact directly with the ␥-ear (19,23,24). These proteins are thought to mediate the interaction between HEATR5b and AP-1. However, this has yet to be tested because HEATR5, aftiphilin, and ␥-synergin stabilize one another; thus knocking down any one destabilizes the other two (19,25). Furthermore, the functional orthologs of aftiphilin and ␥-synergin were unknown in other, more genetically and biochemically tractable organisms. Here we present the identification of the protein Ybl010c in budding yeast as the bridge between the HEATR5 protein Laa1 and AP-1. We identify a single canonical ␥-earbinding site in Ybl010c that is required for the interaction between Laa1 and AP-1. Moreover, disruption of this motif disrupts AP-1 localization and function. Together, these results argue that in budding yeast, HEATR5 cooperates with cargo, Arf1, and PI4P to recruit AP-1.

Results
Budding yeast is an excellent system to understand gene networks important for membrane traffic. It is amenable to both genetic and biochemical approaches. The genome and membrane trafficking pathways are simpler, yet many of the gene functions are conserved (26). Moreover, as the first eukaryote for which a systematic deletion/depletion system was developed, there is a wealth of genome-wide phenotypic data to inform on gene function (27). We took advantage of these data sets to search for new genes important for AP-1 function. We mined an existing database that contains information about the effect of gene deletions on growth under ϳ1800 different chemical treatments (28). These data were subjected to global two-way hierarchical clustering to identify gene deletions whose sensitivity and resistance profiles were highly correlated (28). We analyzed the gene deletions whose profiles correlated with the AP-1 ␥-subunit gene APL4. As expected, deletions that disrupted other subunits of the AP-1 complex were correlated with APL4. The top correlated genes also included deletion of MIL1, a recently identified co-factor for AP-1, LAA1, and TRS33, a core component of the TRAPP tethering complexes that function at the Golgi and endosomes (Fig. 1A) (29,30). The top hits also included one uncharacterized gene YBL010c; on the basis of the results described below, we refer to YBL010c as LAA2 (Large Adaptin Accessory 2).
In addition to unbiased phenotypic correlation with AP-1 subunit genes, Laa2-GFP was reported to co-localize with clathrin at internal structures in a high-throughput data set (31). Based on these data, we first asked whether LAA2 was important for AP-1 localization. To do this, we visualized GFPtagged ␤-subunit (Apl2) expressed from endogenous locus in WT and laa2⌬ (Fig. 1B). In WT cells, Apl2 localizes to several bright puncta per cell. In the laa2⌬ cells, Apl2 localized to fewer puncta, and these puncta were substantially dimmer. When intensity values of individual puncta were determined, those in laa2⌬ were on average half as intense as those in WT cells, and the maximum intensity of puncta in laa2⌬ cells was lower than the median intensity in WT cells. This defect was not due to a significant loss of Apl2 expression, as determined by Western blotting analysis, indicating a severe defect in AP-1 localization (Fig. 1C).
To determine whether this effect was specific to AP-1, we examined the effect of laa2⌬ on the localization of Laa1 and on Gga2, a clathrin adaptor that localizes to the TGN. Similar to AP-1, we found that Laa1 localization was disrupted in laa2⌬ cells. Like AP-1, it localized to fewer puncta, and the puncta were dimmer than in WT (Fig. 1B). In contrast, Gga2 localized to many puncta, and the puncta intensities were slightly higher in the laa2⌬ cells than in WT cells (Fig. 1B). As with Apl2, loss of Laa2 did not significantly affect the expression of either Laa1 or Gga2 (Fig. 1C). The fluorescence of Gga2 argues that the decrease in GFP fluorescence seen with AP-1 and Laa1 was not due to changes in cytosolic chemistry that reduces GFP fluorescence. Together, these results argue Laa2 is important for the localization of both AP-1 and Laa1, but based on the localization of Gga2, laa2⌬ does not disrupt the TGN, indicating a specific role in AP-1 and Laa1 localization.
The effect of Laa2 on Laa1 localization led us to investigate whether Laa2 interacts with Laa1. We found that Laa2-Myc expressed from its endogenous locus co-immunoprecipitated Laa1-Flag expressed from its endogenous locus, indicating that the two interact ( Fig. 2A). We next asked whether Laa2 localization depends on Laa1. We first established that Laa2-GFP expressed from its endogenous locus co-localized with AP-1 (Fig. 2B). We then examined Laa2 localization in cells lacking Laa1. We found that in cells lacking Laa1, Laa2 localization was disrupted. Laa2-GFP was localized to fewer, dimmer puncta in laa1⌬ compared with WT cells (Fig. 2C). Notably, this was associated with a dramatic decrease in Laa2 levels, suggesting that Laa2 requires Laa1 for stability (Fig. 2D). This indicates Laa2 is an obligate binding partner for Laa1. However, Laa1 levels were unaffected in cells lacking Laa2, suggesting that Laa1 does not require Laa2 for stability in vivo (Fig. 1C).
Based on the obligate role of Laa1 for Laa2 stability, we asked whether loss of the two genes caused similar effects on AP-1 localization and function. Using quantitative fluorescence microscopy, we found that like laa2⌬, laa1⌬ reduced the number of AP-1 puncta and reduced the intensity by ϳ50% (Fig.  3A). Next, we investigated effects of laa2⌬ and laa1⌬ on AP-1 functions. In yeast, AP-1 forms two functionally distinct complexes defined by their incorporation of one of two alternate -subunits (29). The more well-studied AP-1 complex in yeast (referred to here as AP-1C for conventional) contains the -subunit encoded by Apm1, whereas the newly characterized AP-1 complex (AP-1R) contains the -subunit encoded by The Laa complex recruits AP-1 Apm2 (29). These complexes recognize distinct cargo and can be assayed independently. To monitor AP-1C, we used calcofluor white (CFW) sensitivity in cells lacking the exomer subunit Chs6. This assay is thought to monitor the AP-1c-dependent retention of the chitin synthase Chs3 in the TGN when its normal traffic out of the TGN is disrupted (29,32). We found that laa2⌬ and laa1⌬ caused similar increases in the CFW sensitivity in cells lacking Chs6, suggesting a similar defect in AP-1C-mediated traffic (Fig. 3B). To monitor AP-1R, we used localization of Sna2 Y75A and sertraline sensitivity. Sna2 Y75A is a vacuolar membrane protein whose delivery to the vacuole depends on AP-1R (29). Sertraline is a cationic amphiphilic drug that is thought to interfere with membrane trafficking pathways by intercalating into phospholipid bilayers (33). It selectively inhibits the growth of cells lacking Apm2 (29). We found that laa2⌬ and laa1⌬ mis-sorted Sna2 Y75A to the plasma membrane to a similar extent and caused similar levels of sensitivity to sertraline. These results indicate that laa2⌬ and laa1⌬ cause similar defects in AP-1R-mediated traffic. Consistent with the partial localization of AP-1 in the laa2⌬ and laa1⌬ deletions strains, complete loss of AP-1 caused stronger effects on CFW sensitivity and Sna2 Y75A mis-sorting than The Laa complex recruits AP-1 laa2⌬ and laa1⌬. In contrast, all three strains were similarly sensitive to sertraline. However, cationic amphipathic drugs such as sertraline are thought to concentrate in cellular membranes, which may lead to nonlinear effects explaining why the three mutants appear similarly sensitive (33). Together, these results indicate that Laa2 and Laa1 are important for the function of both AP-1C and AP-1R consistent with their strong effect on the localization of Apl2 and Apl4, which are components of both complexes.
Laa2 is a small protein with no identifiable homology to known folded protein domains. However, it contains two regions of similarity to aftiphilin, a HEATR5b-binding protein that interacts with both AP-1 and clathrin. Notably, one region of similarity is in the canonical AP-1 ␥-earbinding motif found in many AP-1-binding proteins (Fig. 4A) (18,24,34,35). Based on the presence of this motif, we tested whether Laa2 is an AP-1 ␥-earbinding protein. We found that Laa2 bound the ␥-ear encoded in the C terminus of Apl4 using yeast two-hybrid and recombinant proteins (Fig. 4, B and C). In yeast lysates, recombinant AP-1 ␥-ear interacted with Laa2-3HA (Fig. 4D). Moreover, mutation of two amino acids of the ␥-earbinding motif in Laa2 abolished this interaction, suggesting that this motif is essential for the interaction. We next asked whether the interaction of Laa2 with AP-1 was important for the interaction of Laa1 with the ␥-ear. We found in cells lacking Laa2, Laa1 did not interact with AP-1 by co-immunoprecipitation (Fig. 4E). Furthermore, in lysates from cells expressing the ear-binding mutant form of Laa2 (laa2-⌬DF24AA) as the sole copy of Laa2, Laa1-Myc was unable to bind the AP-1 ␥-ear (Fig. 4F). Notably, this mutation in LAA2 did not alter the interaction of the ␥-ear with Ent3 or Ent5, clathrin adaptors that contain their own ␥-earbinding motifs (Fig. 4, D and F) (34). This indicates that Laa2 ␥-ear binding is specifically required for Laa1 ␥-ear binding.
We next tested the functional significance of the interaction between Laa2 and the ␥-ear. We found the ear-binding mutant form of Laa2 (laa2-⌬DF24AA) was unable to maintain normal levels of AP-1 localization (Fig. 5A) and function, as assessed with CFW and sertraline sensitivity (Fig. 5, A-C). The defects caused by laa2-DF24AA were nearly as strong as those caused by the complete deletion of LAA2 (compare Figs. 5, A-C, and 3,  A, B, and D). This indicates that Laa2 ␥-ear binding is required for AP-1 localization and function. Importantly, the localization of Laa1 and Laa2 were unaffected in these cells (Fig. 5, D and E). Because their localization is unaffected, these data strongly suggest that Laa2 directly mediates the interaction between Laa1 and AP-1 and that this interaction is required for AP-1 localization.
Finally, we determined whether additional ␥-earbinding proteins contribute to AP-1 localization and function. We reasoned that if additional ␥-earbinding proteins contribute to AP-1 localization, deletion of the ␥-ear would cause stronger defects than loss of laa1⌬ or laa2⌬. We first examined the localization and function of AP-1 lacking the ␥-ear. Deletion of the ␥-ear reduced AP-1 puncta number and intensity to a level similar to that of laa2-⌬DF24AA, laa2⌬, and laa1⌬ (Fig. 6A). In CFW assays, deletion of the ␥-ear reduced CFW resistance to a level similar to that of laa2-⌬DF24AA, laa2⌬, and laa1⌬ (Fig. 6B). In sertraline sensitivity assays, deletion of the ␥-ear had a weaker effect than laa2-⌬DF24AA, laa2⌬, and laa1⌬ (Fig. 6C). These data indicate that other ␥-earbinding proteins cannot compensate for the loss Laa complex in terms of AP-1 localization and function and suggest that the Laa complex is the primary ␥-earbinding factor required for AP-1 localization.

Discussion
Proteins of the HEATR5 family are important for AP-1 function in diverse organisms (16, 19 -22, 36). Despite their original identification as AP-1-associated proteins, the functional significance of their interaction with AP-1 was unclear in any system until now (19). Our identification of Laa2 as the critical factor that bridges the interaction between the HEATR5 protein Laa1 and AP-1 in yeast has allowed us to test the functional significance of the interaction for the first time in any system. We found that a point mutation that disrupts the single ␥-earbinding site in Laa2 disrupts the interaction between Laa1 and AP-1 and also disrupts AP-1 localization without altering Laa1 localization. These results indicate that binding of the Laa1-Laa2 complex to AP-1 plays a direct role in recruiting and/or stabilizing AP-1 on membranes.
Our results further indicate that like mammalian HEATR5b, Laa1 functions in the context of a protein complex. We propose that the Laa1-Laa2 protein complex cooperates with Arf1 and PI4P synthesis to confer temporal specificity to AP-1 recruitment. In this light, it is noteworthy that Laa1 depends on Arf1 for localization (16). This could explain why AP-1 recruitment peaks after the peak recruitment of the Arf-GEF Sec7 (12,13). In this case, the temporal lag of AP-1 is explained by the time required to recruit Laa1 in response to active Arf1 and then the time required for Laa1 to recruit AP-1. The concept that an additional ␥-earbinding factor would cooperate with cargo, Arf1, and PI4P is supported by early studies with overexpressed AP-1 ␥-subunits in mammalian cells, which showed a dramatic loss of membrane association in the absence of the ␥-ear (37). It is appealing to speculate that HEATR5 complexes may be the key ␥-earbinding factors important for AP-1 localization in mammalian cells.
Although it seems that the requirement for a co-factor to mediate the interaction between AP-1 and HEATR5 proteins is conserved, it is not yet clear what features of Laa2 are conserved. Like the mammalian HEATR5-binding proteins aftiphilin and ␥-synergin, Laa2 binds to AP-1 using a canonical ␥-earbinding motif (18,23,24,38). Moreover, Laa2 is similar to a section of aftiphilin known as pfam15045 or the Clba (clathrin box of aftiphilin) domain. The Clba domain is

The Laa complex recruits AP-1
an ϳ80-amino acid region that spans a highly conserved tryptophan residue that is required for clathrin binding in aftiphilin (19). This tryptophan residue and flanking residues are similar in Laa2, raising the possibility Laa2 shares activities with aftiphilin in addition to ␥-ear binding. However, aftiphilin is three times larger than Laa2. This suggests that if Laa2 is the functional ortholog of aftiphilin, then aftiphilin may have additional functions that are lacking in Laa2. Moreover, aftiphilin is required for HEATR5b stability, whereas Laa2 does not appear to stabilize Laa1 (19,25). This could indicate that Laa2 and aftiphilin bind to different regions of HEATR5 and that they are not homologs. In support of the possibility that Laa2 represents a novel type of HEATR5-binding protein, we identified several additional proteins annotated as containing the Clba domain in diverse species, which may represent Laa2 homologs (Fig. 4A). Like Laa2, these additional Clba domain-containing proteins are small proteins, with sequences that match the ␥-earbinding motif. However, it remains to be determined whether any of these proteins bind clathrin, AP-1, or HEATR5 or perform any functions similar to Laa2.
In summary, these findings reveal Laa2 as a critical factor important for AP-1 function. Laa2 provides the major ␥-earbinding activity for the HEATR5 protein Laa1 in yeast. The disruption of ␥-ear binding reveals that one major function of the Laa1 complex in yeast is to recruit or stabilize AP-1 on membranes. Further work will be needed to determine whether this complex performs additional activities that contribute to normal membrane traffic before or after AP-1 is recruited.
Imaging and analysis was performed as described previously (46). Briefly, cells were cultured to logarithmic phase in SD . Alignment of ear-binding motif (EBM) with those of ␥-synergin and aftiphilin is shown. Underlined residues were mutated to alanines to generate laa2-DF24AA (middle). Alignment of Laa2 with Clba domains of aftiphilin, uncharacterized human protein Clba1, and the consensus motif for the domain defined in Pfam is shown. Underlined residues in aftiphilin are required for clathrin binding (bottom). B, Laa2 interacts with the ␥-ear of AP-1 but not that of Gga2 in two-hybrid analysis. Cell expressing activation domain fusions to full-length Laa2 or Ent3, as a positive control, and DNA-binding domain fusions to indicated regions of Apl4 or Gga2 or empty vector were spotted on medium that selected for the plasmids (left) or medium that selected for interaction (right). C, Laa2 interacts directly with the ␥-ear of AP-1. Recombinant Laa2 was subjected to GST pulldown using recombinant ␥-ear of AP-1 or GST. Input is 10%. The data are representative of three repeats. D, Laa2 ear-binding motif is required for interaction with the ␥-ear of AP-1 in GST pulldown experiments. Asterisks denote nonspecific binding of antibodies to GST. E, Laa2 is required for Laa1 interaction with AP-1. Laa1-Myc was immunoprecipitated from WT or laa2⌬ cells. Endogenous AP-1 or Gga2 were detected with polyclonal antibodies. Lysates are 4% of immunoprecipitations. Data shown are representative of at least three repeats. F, Laa2 binding to AP-1 is required for Laa1 interaction with the ␥-ear of AP-1. Cells expressing indicated allele of Laa2 and Laa1 from their endogenous loci were subjected to GST pulldown using the ␥-ear of AP-1, or the ␥-ear of Gga2, or GST as a negative control. Samples were probed for GST, HA, Myc, and the adaptor-interacting proteins Ent5 or Ent3, as a positive control. Lysates are 5 and 6% of pulldown without any GST protein for D and E, respectively. Locations of molecular weight standards are indicated. The data shown are representative of at least three repeats.
Measurement of fluorescence intensities of puncta was conducted using a previously described method using a custom graphical user interface written in MATLAB (47). Briefly, the signal region was a 6 ϫ 6-pixel box centered on the brightest four pixels of a manually selected spot in the micrograph. The background fluorescence was defined by a concentric 8 ϫ 8-pixel box surrounding signal region. The background intensity was the median value of pixel intensities in the outermost ring of this box. The fluorescence signal was the sum of all 36 pixels in the 6 ϫ 6 box. Intensity analysis was performed on structures from a minimum of 50 cells. The data shown come from analysis performed on a single day; intensity variance between days was minimal. To determine co-localization of Laa2 and AP-1, images were denoised using SpatTrackV2 (48). From the denoised images, ImageJ.v5.1K (National Institutes of Health) was used to threshold the images using Otsu's method and identify particles (49). Co-localization was defined as a particle where at least 20% of the area of a particle defined in one channel was also positive for the second channel.
Whole-cell yeast extracts were generated using glass bead lysis as previously described (50), with the exception of Fig. 2D. For Fig. 2D, cell pellets were resuspended in 0.2 M NaOH and incubated on ice for 10 min. TCA was added to 10%, and cells were disrupted by glass bead lysis. Precipitated proteins were pelleted, rinsed in ice cold acetone, and then resuspended in Laemmli sample buffer with 0.5 M Tris, pH 7, and boiled before loading. For immunoprecipitation, spheroplasts were first gen- The Laa complex recruits AP-1 erated by resuspending cells in 100 mM Tris-SO4, pH 9.5, 2% glucose, and 5 mM DTT for 10 min. The cells were then resuspended in YP medium supplemented with 0.5% glucose, 10 mM Tris-HCl, 1.2 M sorbitol, and 120 units of lyticase (Sigma). The cells were gently agitated for 15 min at 30°C and resuspended in HEKG5 (20 mM Hepes, pH 7.5, 1 mM EDTA, 150 mM KCl, 5% glycerol) with protease inhibitor mixture without EDTA (Promega). The cells were disrupted by glass-bead lysis, followed by the addition of 0.3% CHAPS. The lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4°C. The lysates were incubated overnight at 4°C with 50 l of 20% protein A-Sepharose slurry (GE LifeSciences) and 0.5 l of antibody. The beads were washed three times with ice-cold HEKG5 with CHAPS, and the bound proteins were eluted with SDS sample buffer.
Cell lysates for GST-tagged protein interaction studies were generated by liquid nitrogen blending as previously described (44). Powder was thawed at a ratio of 0.15 g of lysate to 0.85 ml of HEKG5 with protease inhibitor mixture without EDTA (Promega). The lysate was clarified by centrifugation at 13,000 rpm for 10 min, and the resulting supernatants were used for binding studies. Purification of GST and ␥-ear constructs were described previously (44). His-tagged Laa2 was purified using Talon resin according to the manufacturer's instructions (Clontech). For binding studies, GST-tagged proteins were prebound to GSH-Sepharose (GE LifeSciences) in 1 ml of PBS with 0.1 mg/ml BSA for 1 h at room temperature, washed twice with PBS, and resuspended in 1 ml lysate or in 1 ml of PBS containing purified His-tagged Laa2. Protein binding pro-ceeded for 2 h at 4°C, and beads were washed. For Fig. 4 (C and  D), beads were washed three times in PBS. For Fig. 4E, beads were wash twice with HEKG5 with 0.3% CHAPS and once with HEKG5 without CHAPS. The beads were resuspended in 100 mM Tris, pH 9, 200 mM NaCl, 5 mM DTT, 20 mM reduced GSH and incubated for 20 min at room temperature. The supernatants were taken as the bound fraction. Two-hybrid interactions were monitored as previously described (34).
For immunoblotting, after SDS-PAGE, samples were transferred to nitrocellulose, blocked with 5% milk in TBS-T (137 mM NaCl, 15.2 mM Tris-HCl, 4.54 mM Tris, 0.896 mM Tween 20), and then probed with primary and fluorescent secondary antibodies. Fluorescence and chemiluminescent signals were detected on an Azure c600 imaging system (Azure Biosciences). For chemiluminescence, Immobilon Western reagents were used (Millipore).
CFW and sertraline sensitivity assays were performed as previously described (51). Briefly, a log phase culture medium was diluted to 0.02 (A 600 ) in yeast/peptone and transferred to a sterile reagent reservoir. The diluted culture was distributed into the wells of a sterile 96-well assay plate. An equal volume of chemical in yeast/peptone media was added to the prepared 96-well plate. An assay plate with lid was placed in a Spectra Max 340PC plate reader (Molecular Devices). The cells were incubated at 30°C without shaking. Absorbance at 600 nm was collected every 30 min for 18 h. IC 50 values, halfway between the maximal and the minimal inhibition, were calculated from a sigmoidal dose-response curve (variable slope, four parameters) using Prism 7 (GraphPad). All other statistical analyses were performed using Prism 7 (GraphPad).  The Laa complex recruits AP-1