Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Pulse-chase analyses of Sna3 and Sna3-GFP turnover. Representative gels used to generate the data presented in Figure 1C. The indicated strains were metabolically labeled with ³⁵S-Cys/Met, chased with an excess of unlabeled amino acids, and time points were collected to perform immunprecipitations with anti-Sna3 antibody. Immunoprecipitated species were resolved by SDS-PAGE and visualized using a phosphoimager.
• Supplemental Figure 2 - Localization of Sna3 mutants. Sna3 mutants localize to PtdIns(3)P-positive endosomes in addition to the vacuolar lumen or limiting membrane. Various forms of Sna3-GFP were co-expressed with DsRed-FYVE in sna3· cells and visualized by fluorescence microscopy. Scale bar is 5 microns.
• Supplemental Figure 3 - Rsp5 WW domain mutants. A) Wild type cells were transformed with the indicated forms of TAP-Rsp5 and expression levels were compared by probing equivalent amounts of whole cell extracts with anti-actin antibody to detect TSP-tagged Rsp5. B) Strains were generated in which the chromosomal copy of RSP5 was deleted and the indicated form of Rsp5 was expressed from a plasmid. These strains were transformed with Sna3^KallR-GFP and visualized by fluorescence microscopy. Scale bar is 5 microns.