Figure 4. Cos5 requires ubiquitination for its MVB sorting and its function in facilitating MVB sorting of other cargo.

A) Top, sorting of GFP fused to Cos5 lacking its lysine residues (Cos5^KR-GFP) and Cos5-GFP fused to the UL36 deubiquitinating (DUb) catalytic domain in WT cells. Bottom, Cos5-GFP localization in temperature-sensitive rsp5-1 mutant cells grown at 30°C and 37°C for 4 hrs.

B) Pulse/chase immunoprecipitation of Cos5-GFP and Cos5^KR-GFP in WT (Pep⁺) and Pep^- cells. Cells were labeled with ³⁵S~Met for 10 min followed by the indicated chase times. Cos5-GFP and Cos5^KR-GFP were immunopreciptated from cell lysates with α-GFP antibodies prior to SDS-PAGE and autoradiography.

C) Localization of Mup1-GFP in cells co-transformed with a vector control or plasmids over-expressing Cos5-HA, Cos5^KR-HA and Cos5^KR-HA-Ub from the CUP1 promoter. Cells were grown to mid-log phase in SD-Met media containing 50 µM CuCl₂.

D) Corresponding immunoblot analysis of cells in C) expressing Cos5-HA, Cos5^KR-HA and Cos5^KR-HA-Ub with α-HA and α-CPY, provided as a loading control.

E) Localization of Cos5-GFP in sna3Δ and sna3Δ bsd2Δ double null mutant cells.

F) Synthetic effects of deleting COS6 and SNA3 or BSD2. Localization of GFP-Cps1 in the indicated single and double null mutants is shown.

G) Cos5-GFP and Cos5^KR-GFP were immunoprecipitated from lysates of cells also expressing HA-Ub using α-GFP antibodies. Immunoprecipitates were immunoblotted with α-GFP and α-HA. Cells carried the pep12Δ mutation alone or in combination with sna3Δ and bsd2Δ mutations.

H) Binding of MBP-Rsp5 to fusion proteins between GST and the C-terminal domains (CTD) of Cos4, Cos5, Cos6, Sna3, and the Sna3-CTD lacking its Rsp5-binding PY motif (ΔPY). GST fusion proteins were immobilized on GSH-agarose. Beads were incubated with recombinant MBP-Rsp5, washed, and immunoblotted with α-MBP antibodies. Bar = 5 µm.