Supplemental Material

This article contains the following supporting material:

• Supplemental Figure 1 - Localization of GFP-tagged proteins in VPS4 dominant-negative strains.

  Cells containing GFP-tagged fusion proteins were transformed with a VPS4 dominant-negative plasmid and the transformants were cultured to mid-log phase in SD media. The VPS4 dominant negative plasmid was used to create enlarged endosomes indicative of class E vps mutants and which can accumulate proteins that transiently associate with endosomes. Cells were co-labelled with the lipophilic styryl dye FM®4-64 to label endocytic compartments. Doa4-GFP, Rup1-GFP, Hua1-GFP and to a lesser extent Ubp2 were found to localize to endocytic compartments.

• Supplemental Figure 2 - Both ubp2Δ and hua1Δhse1-ΔPY exhibit no defect in ubiquitination of GFP-Cps1.

  Wild-type, ubp2Δ and hua1Δhse1-ΔPY cells containing GFP-Cps1 or GFP-Cps1 (K_8,12R) were labeled with ³⁵S Methionine for 10 min at 30ºC. GFP-Cps1 was immunoprecipitated, deglycosylated with endoglycosidase H, and resolved by SDS-PAGE. A band corresponding the the predicted molecular weight of monoubiquitianted GFP-Cps1 is indicated. This band was found only for GFP-Cps1 but not a mutant GFP-Cps1 (K_8,12R), which lacks the Cps1 ubiqutination sites and which is defective for sorting to the vacuole lumen.