Supplemental Material

This article contains the following supporting material:

• Figure 1 - The clathrin binding activity in the UBA region maps to residues 101-140 and not the UBA domain. The same experimental conditions as shown in Fig.1 were used here for the GST pull-down with the following modification. Clarified yeast lysate was further centrifuged at 130,000 x g and the supernatant was then used as the source of clathrin free triskelia to incubate with indicated GST fusions. 1% of the total unbound (U) proteins and 20% of the total bound (B) proteins were resolved by SDS-PAGE and the amount of clathrin heavy chain in both fractions were visualized by Western blot. GST-(1-100) (CB1) binds very weakly to clathrin. Both GST-(1-140)(lacking the UBA domain) and GST-(1-181)(containing the UBA domain) pull-down clathrin equally well from the high speed supernatant as compared to GST-(1-237).

• Figure 2 - Clc1-GFP puncta colocalize with the TGN marker, Sec7-RFP. Strain JX08 carrying Clc1-GFP, Sec7-RFP, and pURA3-SWA2 were selected and imaged for the Clc1-GFP and Sec7-RFP fluorescence.

• Table 1-3