Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain - PubMed (original) (raw)

Doa1 is a Cdc48 adapter that possesses a novel ubiquitin binding domain

James E Mullally et al. Mol Cell Biol. 2006 Feb.

Abstract

Cdc48 (p97/VCP) is an AAA-ATPase molecular chaperone whose cellular functions are facilitated by its interaction with ubiquitin binding cofactors (e.g., Npl4-Ufd1 and Shp1). Several studies have shown that Saccharomyces cerevisiae Doa1 (Ufd3/Zzz4) and its mammalian homologue, PLAA, interact with Cdc48. However, the function of this interaction has not been determined, nor has a physiological link between these proteins been demonstrated. Herein, we demonstrate that Cdc48 interacts directly with the C-terminal PUL domain of Doa1. We find that Doa1 possesses a novel ubiquitin binding domain (we propose the name PFU domain, for PLAA family ubiquitin binding domain), which appears to be necessary for Doa1 function. Our data suggest that the PUL and PFU domains of Doa1 promote the formation of a Doa1-Cdc48-ubiquitin ternary complex, potentially allowing for the recruitment of ubiquitinated proteins to Cdc48. DOA1 and CDC48 mutations are epistatic, suggesting that their interaction is physiologically relevant. Lastly, we provide evidence of functional conservation within the PLAA family by showing that a human-yeast chimera binds to ubiquitin and complements doa1Delta phenotypes in yeast. Combined, our data suggest that Doa1 plays a physiological role as a ubiquitin binding cofactor of Cdc48 and that human PLAA may play an analogous role via its interaction with p97/VCP.

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Figures

FIG. 1.

FIG. 1.

Localization of a novel ubiquitin binding domain in Doa1, the PFU domain. (A) Whole-cell lysates from yeast strains expressing various HA-tagged Doa1 truncations were incubated with 29-linked tetraubiquitin analogue-Sepharose (ubiquitin) or control-Sepharose (control). Load, bound, and unbound Doa1 were detected immunochemically using antibodies to the HA epitope. B, bound; U, unbound. (B) Schematic depicting the domain architecture of the PLAA family above a sequence alignment comparing the primary structure of the PFU domains from human PLAA and S. cerevisiae Doa1. Arrowheads point to conserved residues mutated in panel C. (C) Whole-cell lysates containing recombinantly expressed, His-Flag-tagged wild-type Doa1 (HisFlag-Doa1) or Doa1(F417D, F434D) (HisFlag-Doa1-FD) were incubated with monoubiquitin-Sepharose. Load, bound, and unbound Doa1 were detected immunochemically using antibodies to the Flag epitope. B, bound; U, unbound.

FIG. 2.

FIG. 2.

Ubiquitin binding by Doa1 is necessary for its function of maintaining ubiquitin homeostasis. (A) Whole-cell lysates from wild-type and doa1Δ yeast strains grown on SD medium were analyzed immunochemically for monoubiquitin and high-molecular-weight ubiquitin conjugates using ubiquitin antibodies. (B) Serial dilutions of wild-type (DOA1) and doa1Δ yeast, which were transformed with empty vector or a ubiquitin expression vector (UBI), were used to determine the effect of ubiquitin overexpression on sensitivity to canavanine and anisomycin. Growth on defined glucose medium (control) is shown to demonstrate equal loading. (C) Whole-cell lysates expressing Flag-tagged wild-type Doa1 (Flag-Doa1) or Doa1(F417D, F434D) (Flag-Doa1-FD) grown on SD medium were analyzed immunochemically for ubiquitin to determine the effect of PFU domain mutations on Doa1 function in vivo as measured by monoubiquitin levels. (D) Serial dilutions of Flag-Doa1-, doa1Δ-, and Flag-Doa1-FD-expressing yeast strains were used to determine the effect of PFU mutations on Doa1 function in vivo as measured by sensitivity to canavanine and anisomycin. Growth on defined glucose medium (control) is shown to demonstrate equal loading.

FIG. 3.

FIG. 3.

The function of the human PLAA C-terminal domain is conserved. (A) Top: schematic depicting the chimera of the yeast Doa1 WD40 domain and the human PLAA C-terminal domain. Bottom: relative size and expression levels of HA-Doa1 and the HA-Doa1-PLAA chimera in yeast whole-cell lysates. Both proteins were detected immunochemically using antibodies to the HA epitope. (B) Whole-cell lysate from yeast strains expressing HA-tagged Doa1-PLAA were incubated with monoubiquitin-, 29-linked tetraubiquitin analogue-, or control-Sepharose. Load, bound, and unbound Doa1-PLAA were detected immunochemically using antibodies to the HA epitope. B, bound; U, unbound. (C) Whole-cell lysates from wild-type parental (WT) and doa1Δ cell lines transformed with empty vector, HA-DOA1, or HA-DOA1-PLAA (chimera) were analyzed immunochemically for monoubiquitin and high-molecular-weight ubiquitin conjugates using antibodies to ubiquitin. (D) Serial dilutions of wild-type parental (DOA1 + vector) and doa1Δ cell lines transformed with empty vector (doa1Δ + vector), HA-DOA1 (doa1Δ + HA-DOA1), or HA-DOA1-PLAA (doa1Δ + HA-chimera) were used to demonstrate that HA-Doa1-PLAA complements doa1Δ sensitivity to canavanine and anisomycin. Growth on defined glucose medium (control) is shown to demonstrate equal loading.

FIG. 4.

FIG. 4.

Cdc48 is recruited to ubiquitin via direct interaction with the C-terminal PUL domain of Doa1. (A) Whole-cell lysates from wild-type parental (WT) or doa1Δ yeast expressing HisFlag-Doa1 were subjected to Ni2+ agarose affinity chromatography. Eluted proteins were separated by SDS-PAGE, visualized by staining with Sypro Ruby, and excised for identification by mass spectrometry. Arrows indicate the two most prominent proteins identified, Doa1 and Cdc48. (B) Antibodies to the Flag epitope were used to coimmunoprecipitate purified, recombinant His-Cdc48 in the presence or absence of purified, recombinant HisFlag-Doa1, BSA, and/or ATP. Load, bound, and unbound Cdc48 and Doa1 were detected immunochemically using antibodies to the His6 epitope. (C) Antibodies to the Flag epitope were used to coimmunoprecipitate purified, recombinant His-Cdc48 in the presence or absence of purified, recombinant His-Flag-tagged full-length Doa1 (HisFlag-Doa1), WD40 domain (HisFlag-Doa1WD40), C-terminal domain (HisFlag-Doa1Cterm), or PUL domain (HisFlag-Doa1PUL). The schematic depicts the location of the PUL domain within Doa1. Load, bound, and unbound Cdc48 and Doa1 were detected immunochemically using antibodies to the His6 epitope. Asterisks denote a weak cross-reaction to Flag antibody heavy and light chains. (D) Purified, recombinant His-Cdc48 and HisFlag-Doa1 were incubated individually or in combination, with or without ATP, and analyzed for binding to monoubiquitin-Sepharose. Load, bound, and unbound Cdc48 and Doa1 were detected immunochemically using antibodies to the His6 epitope. L, load; B, bound; U, unbound.

FIG. 5.

FIG. 5.

DOA1 is genetically linked to CDC48. (A) Serial dilutions were used to determine the effects of inactivating Cdc48 (cdc48-1) on canavanine sensitivity in parental wild-type (WT), doa1Δ, and doa4Δ yeast cells cultured at 30°C. Growth on defined glucose medium (control) is shown to demonstrate equal loading. (B) Whole-cell lysates from yeast strains described for panel A and cultured at 20°C were analyzed immunochemically for monoubiquitin and high-molecular-weight ubiquitin conjugates using ubiquitin antibodies. Lowest panel: immunochemical analysis using antibodies to PGK, used as a loading control.

FIG. 6.

FIG. 6.

Proposed model of Doa1's functional domains and relevance to Cdc48 function. In this model, the Doa1 PFU domain binds to ubiquitinated substrates which are acted upon by Cdc48 via its interaction with the PUL domain of Doa1. Binding to the WD40 domain of Doa1 by an as-of-yet-unidentified protein may either contribute to the actions of Cdc48 or act as a regulator of the Cdc48-Doa1 interaction.

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