Identification of a Novel 29-Linked Polyubiquitin Binding Protein, Ufd3, Using Polyubiquitin Chain Analogues^,

Abstract

Lysine 48-linked polyubiquitin chains are the best understood form of polyubiquitin and are necessary for the function of the ubiquitin-proteasome system. However, other forms of polyubiquitin (e.g., K29- and K63-linked chains) are also present in vivo. Less is known about the functional roles of these linkages or the proteins specifically interacting with these forms of polyubiquitin. Use of native polyubiquitin chains to identify binding proteins is complicated by the difficulties of synthesis and stability. Here, we report the synthesis of a nonhydrolyzable analogue of 29-linked polyubiquitin chains on an affinity support and its use in identifying proteins that bind 29-linked polyubiquitin chains. The 29-linked Ub4 resin was stable and tightly bound recombinant human Isopeptidase T (USP5), a deubiquitinating enzyme known to bind the 29-linked polyubiquitin chains. Two high affinity interactors of the 29-linked polyubiquitin analogues were identified from Saccharomyces cerevisiae lysates. They were identified as Ubp14, the yeast ortholog of Isopeptidase T, and Ufd3, a member of the ubiquitin-fusion degradation pathway with unknown function. Purified recombinant Ufd3 bound to the resin as well, confirming that Ufd3 is a novel binding partner of polyubiquitin. These results demonstrate the efficacy of using polyubiquitin analogue affinity supports to identify novel binding partners of specifically linked polyubiquitin chains. Identification of these proteins will lead to a greater understanding of the physiological relevance of different polyubiquitin linkages.

Role of Doa1 in the Saccharomyces cerevisiae DNA Damage Response

“…1A). These results agree with those reported previously by Russell and Wilkinson, who showed that Doa1 interacts with K29-linked polyubiquitin chains (35).…”

Novel Predicted Peptidases with a Potential Role in the Ubiquitin Signaling Pathway

“…30,31 In particular, truncation and mutational data suggest that the C-terminal region of Ufd3p/Lub1 containing the PUL domain is specifically required for the Ub stabilization and interaction with poly-ubiquitin. 32,33 Taken together, these observations suggest that PUL is novel domain (Fig. 5) which interacts with Ub and Ub-like domains; fungal PPPDE proteins could bind Ub/Ub-like domains via this region, analogously to Ub-binding mediated by PUG (PUB) and Uba domains fused to other DUBs.…”

Doa1 Is a Cdc48 Adapter That Possesses a Novel Ubiquitin Binding Domain

“…Control, monoubiquitin, and 29-linked tetraubiquitin analogue-Sepharose resins were synthesized as previously described (38). For analysis of ubiquitin binding by Doa1 truncations from whole-cell yeast lysates, 300 g of yeast whole-cell lysate (150 mM NaCl, 50 mM Tris, pH 7.4, 1 mM dithiothreitol, Complete protease inhibitors, 0.05% Triton X-100) was incubated with 100 l of ubiquitin resin (8 mg/ml ubiquitin) or control resin for 3 h at 4°C, washed (three times) with lysis buffer, and eluted by boiling in 1.5ϫ sodium dodecyl sulfate (SDS) loading buffer.…”

“…doa1⌬ yeast expressing HisFlagDoa1 or wild-type yeast were grown to log phase (OD 600 of 1.0) in synthetic raffinose-galactose medium. Yeast were harvested and lysed by the liquid nitrogen/mortar and pestle method as previously described (38). Yeast lysates were subjected to Ni 2ϩ affinity chromatography to isolate Doa1 and Doa1 binding proteins.…”

“…Yeast lysates were subjected to Ni 2ϩ affinity chromatography to isolate Doa1 and Doa1 binding proteins. Prominent bands from the resulting Sypro Ruby-stained SDS-PAGE gel were excised and submitted for MALDI mass spectrometry as previously described (38).…”