Supplement Material for Wrasman et al., 2018

<b>Figure S1: Endocytic function quantified using flow cytometry</b>. Mup1-pHl fluorescence was assayed in WT and CME- cells using flow cytometry after 45 min methionine treatment. Forward scatter corresponds to cell size, and fluorescent signal corresponds to relative Mup1-pHl intensity. Measurement gates were defined/applied as follows: Four quadrants (Q1 - Q4) resulted from two perpendicular selection criteria. Q3 in the lower left represents the majority of inviable cells. P1 is the sum of Q1 plus Q3. Red points indicate "NOT P1" (the sum of Q2 plus Q4), and correspond to cells with a higher fluorescent signal than the measurement gate. The percentage of total gated cells is shown on the scatter plots.<br><br><b>Figure S2: Sorting of cells during an EMS mutagenesis screen using Mup1-pHl fluorescence to select for potential endocytic mutants.</b> WT Mup1-pHl cells were sorted without EMS exposure (left) after 45 min methionine treatment to optimize gating in order to limit the number of WT cells sorted. Cells treated with EMS (right) were sorted under the same gating conditions 45 min after the addition of 20 μg/ml methionine. Forward scatter corresponds to cell size, and fluorescent signal corresponds to relative Mup1-pHl intensity. Red points indicate cells with a higher fluorescent signal than the sorting gate.<br><br><b>Figure S3: Categorization of mutant alleles as dominant or recessive. </b>Mutants were mated with non-mutagenized parental strain of opposite mating type to generate diploids. Fluorescence intensity was assessed for both the haploid mutant and diploid mutant strains using flow cytometry 45 min after the addition of 20 μg/ml methionine. Representative plots of dominant and recessive mutants are shown. Forward scatter corresponds to cell size, and fluorescent signal corresponds to relative 1 Mup1-pHl intensity. Red points indicate cells with a higher fluorescent signal than the measurement gate.<br><br><b>Figure S4:</b> <b><i>LDB19</i>/<i>ART1</i> is a suppressor of seven mutants.</b> <i>LDB19</i> was expressed from a low-copy plasmid, and complementation was assessed via fluorescence microscopy and flow cytometry of seven endocytic mutants: Mup1-selective (three mutants) or permease-selective (four mutants) classes. Representative images and plots are shown of EV (empty vector), and <i>LDB19</i> complementation. A dashed line indicates the plasma membrane of +<i>LDB19</i> cells. Forward scatter corresponds to cell size, and fluorescent signal corresponds to relative Mup1-pHl intensity. Scale bar, 2μm.<br><br><b>Figure S5: Truncations of endocytic proteins cause disruption in endocytosis.</b> Representative images of A) Syp1-GFP, and B) Pan1-GFP (green) and Abp1-RFP (red) in WT, <i>sla2<sup>W360*</sup>, sla1<sup>Q682*</sup></i>, and <i>ede1<sup>W319*</sup></i> cells were acquired by live-cell fluorescence microscopy. Scale bar, 2μm. C) Schematics of <i>Saccharomyces cerevisiae</i> Sla2, Sla1, and Ede1 protein domains showing the location of the mutation causing the truncation with a *.<br><br><b>Figure S6: Localization of CME machinery proteins in WT and arp3<sup>R346H</sup> cells.</b> A) Representative images of Ede1-GFP, B) Pan1-GFP, and Abp1-RFP in WT and <i>arp3R<sup>346H</sup></i> cells were acquired by live-cell fluorescence microscopy. Scale bar, 2μm.<br><br><b>Table S1: </b>List of yeast strains used in this study<br><br><b>Table S2: </b>List of plasmids used in this study<br><br><b>Table S3</b>: Outcome of the Mup1-pHl FACS screen<br><br><b>Table S4:</b> List of genes tested for complementation of mutant strains