Endocytosis mutants have related genetic interaction profiles. (A) Hierarchical clustering was used to analyze the invertase activity values of double mutant strains generated by crossing top-scoring mutants from the primary screen (y axis) to 81 diverse trafficking mutants (x axis). Yellow indicates lower than expected levels of GSS at the cell surface, whereas blue indicates higher than expected levels (see Methods and materials for details). (B) Detailed view of clusters showing names of genes and highly represented pathways/complexes. Clusters are numbered to indicate their relative position on the heat map in A. Red bar indicates gene clusters on the y axis enriched in endocytosis or recycling genes. See Fig. S1 for a detailed view of gene names and cluster relationships.

Yeast AP180 homologues have a conserved role in Snc1 internalization. (A) Quantification of cell surface GSS levels in wild-type (WT) and candidate endocytic mutants by the liquid invertase assay. Invertase activity represents relative surface levels of the GSS reporter, quantified as nanomole glucose released per OD₆₀₀ (mean of at least three experiments ± SD). The red line indicates the baseline invertase activity of the wild-type strain. (B) Localization of GFP-tagged Snc1 and NPF-Sso1 in WT and mutant strains. Bars, 2 µm. (C) Schematic of the GFP-Snc1-Suc2 (GSS) and 3xNPF-GFP-Sso1-Suc2 (NGSS) reporters. (D) Relative cell surface levels of GSS and NGSS reporters measured by liquid invertase assay in mutant strains (mean of three experiments ± SD). Results for each reporter are expressed as the percentage of invertase activity relative to WT. Different endocytosis defective controls (*EN-) were used for each reporter. GSS, endocytosis-defective GSS (snc1^V40AM43A) reporter in WT cells; NGSS, NGSS in sla1 mutant strain.

Ldb17 is required for proper coat and actin dynamics. (A–C) Colocalization of Ldb17-GFP with Sla1-RFP, Myo5-RFP, and Abp1-RFP. Representative single frames from GFP and RFP channels are shown together with merged images. Arrows indicate cortical patches containing both Ldb17 and RFP-tagged proteins. (D) Schematic diagram, adapted from Kaksonen et al., 2005, illustrating the timing of Ldb17 recruitment to cortical patches relative to markers of the late coat (Sla1), WASP/myo (Myo5), and actin (Abp1) modules, based on kymograph analysis of 10 patches for each GFP/RFP pair (see Fig. S3, A–C). The mean difference in the timing of Ldb17 arrival or disappearance (±SD, in seconds) relative to other markers is indicated, together with the total mean lifetime of Ldb17, Myo5, and Abp1 patches. (E) Localization of Ldb17-GFP and Abp1-RFP in cells treated with DMSO (left), or 200 µM LatA (right) for 30 min at 30°C. (F) Lifetime of GFP-tagged Ldb17 and/or Ldb17ΔPRD at cortical patches in wild type (WT), bzz1, and sla1. Lifetimes were measured for at least 10 patches per strain (mean ± SD). (G) Reduced number and increased brightness of actin patches in ldb17 cells, as assessed by microscopy of Abp1-GFP. (H) Kymograph representation of time-lapse wide-field fluorescence microscopy showing extended lifetime of Sla1-RFP at endocytic patches in ldb17 mutants, as compared with wild-type cells over 120 s. Bars, 2 µm.

Genome-wide screen for endocytic recycling mutants. (A) Schematic of the GFP-Snc1-Suc2 (GSS) reporter. At the plasma membrane (PM), the Suc2 (invertase) portion is accessible to the extracellular space. (B) Localization of GFP-Snc1, GSS, and an endocytosis-defective form of GSS containing snc1^V40A,M43A mutations. Bar, 2 µm. (C) Representative portion of the yeast knockout array tested using the invertase activity overlay assay. Mutants defective in Snc1 endocytosis exhibit increased surface invertase activity relative to wild-type cells, and appear dark. Mutants with high (tpm1 and rvs161) or low (snx4 and irs4) levels of cell surface invertase activity are indicated. (D) Relative cell surface invertase activities of the top 1,000 mutants. The red line indicates threshold used to designate top hits.

Integration of genome-wide genetic and physical interaction data identifies pathways and complexes. Significant gene clusters contributing to GSS localization were mapped based on integrated physical and genetic interaction networks. Array genes are represented as nodes (where relative size is proportional to GSS surface levels in the corresponding deletion mutant), and are connected by edges that represent either a physical interaction or a genetic correlation extracted from genome-wide data. Clusters were assigned to subcellular compartments based on the localization of their constituent proteins.

Cargo specificity of genes required for Snc1 endocytosis. (A) Array-based analysis of cargo specificity. The difference (DIFF) in cell surface levels of GSS and NGSS reporters was determined for each mutant by subtracting normalized invertase activity scores measured in parallel analyses of mutant arrays. Mutants with differences >2 SD (P < 0.05) from the mean are shown. Asterisks indicate loss of gene function by deletion of an overlapping ORF. (B) Relative cell surface levels of GSS and NGSS reporters measured by liquid assay in inp52 and ldb17 mutants. Results for each reporter are expressed as the percentage of invertase activity relative to appropriate wild-type (WT) control (mean of three experiments ± SD; P < 0.05). (C) Yap180 and Sla2 are present on internal puncta in inp52 strains. Wild type and inp52 mutants expressing Sla1-GFP, Sla2-GFP, or both Yap1801-GFP and Yap1802-GFP were examined by live cell microscopy. Arrows indicate internal patches. (D) Localization of GFP-tagged Snc1 and NPF-Sso1 in WT, ldb17, and end3 strains. Bars, 2 µm. (E) Ldb17 shares homology with the mammalian SPIN90 and contains a PRD. The conserved PTHR13357 domain is indicated (blue region).