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Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates - PubMed

  • ️Fri Jan 01 2010

Ubiquitin chain elongation enzyme Ufd2 regulates a subset of Doa10 substrates

Chang Liu et al. J Biol Chem. 2010.

Abstract

Ufd2 is the founding member of E4 enzymes that are specifically involved in ubiquitin chain elongation but whose roles in proteolysis remain scarce. Here, using a genome-wide screen, we identified one cellular target of yeast Ufd2 as the membrane protein Pex29. The ubiquitin chains assembled on Pex29 in vivo by Ufd2 mainly contain Lys-48 linkages. We found that the ubiquitin-protein E3 ligase for overexpressed Pex29 is Doa10, which is known to be involved in protein quality control. Interestingly, not all Doa10 substrates are regulated by Ufd2, suggesting that E4 involvement is not specific to a particular E3, but may depend on the spatial arrangement of the E3-substrate interaction. Cells lacking UFD2 elicit an unfolded protein response, expanding the physiological function of Ufd2. Our results lead to novel insights into the biological role of Ufd2 and further underscore the significance of Ufd2 in proteolysis.

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Figures

FIGURE 1.
FIGURE 1.

Identification of Pex29 as a substrate of the Ufd2-Rad23/Dsk2 pathway. A, representative images from the plasmid overexpression screen. Each of ∼5280 yeast open reading frames regulated by the GAL1 promoter was transformed separately into wild-type Y8835 and ufd2Δ cells. Transformants were grown on media containing either glucose (SD, expression off) or galactose (SG, expression on). Each open reading frame is represented by two spots on the plate to reduce false positives. After 2 days for SD plates and 3 days for SG plates, colony sizes were scored for possible hits. Boxed spots are PEX29 on each plate. Strains and growth conditions are labeled at the top of each image. B, overexpression of Pex29 leads to slower growth in ufd2Δ or rad23Δ dsk2Δ double mutant cells. GST-His6-tagged Pex29 isolated from the genome-wide screen was transformed into wild-type, or indicated mutants. These cells were grown to similar densities, and 5-fold serial dilutions were spotted onto SD or SG media. C, efficient degradation of Pex29 requires Ufd2, Rad23, and Dsk2. Wild-type (Tyr-8835) and mutant cells containing a GAL1 promoter-driven GST-His6-Pex29 were first grown in raffinose-containing medium. Expression of Pex29 was induced by the addition of galactose. Samples were taken after promoter shutoff at the time points indicated and analyzed by anti-His6 Western blots. Equal amounts of protein extracts were used and confirmed by blotting with anti-Rpt5 antibody in the expression shutoff experiments (lower panels). The identities of proteins are indicated on the left. The pull-down (IP) and Western blot (blot) are indicated to the right of the panels. D, quantification of the data in C for Pex29.

FIGURE 2.
FIGURE 2.

Ufd2 regulates Pex29 ubiquitylation and degradation. A, Pex29 is degraded by the proteasome. Wild-type yeast cells expressing Pex29 were treated with or without the proteasome inhibitor MG132 (14). Pex29 degradation was monitored as described in Fig. 1C. B, deletion of the U-box does not affect Ufd2 levels. The C-terminal U-box was replaced by a TAP tag. Wild-type and mutant Ufd2-TAP were detected by immunoblotting. C, Pex29 degradation is impaired in ufd2U-boxΔ mutant cells. GST-tagged Pex29 was transformed into wild-type and ufd2 mutants. Pex29 degradation was assayed as described above. D, reduced Pex29 multiubiquitylation in ufd2Δ. GST-His6-tagged Pex29 was co-transformed with the plasmid YEp105 expressing the CUP1-promoter regulated Myc-tagged Ub alleles into wild-type or ufd2Δ cells. Pex29 was precipitated with GST beads and analyzed by immunoblotting first with anti-Myc antibody and later with anti-His6 antibody. Ubiquitylated and non-ubiquitylated Pex29 proteins are indicated on the left. Loading was ascertained by blotting with anti-Rpt5 antibody. E, Ufd2 promotes Lys-48-linked Ub chains in vivo. Pex29 was isolated from wild-type cells through a two-step purification (i.e. nickel-nitrilotriacetic acid, glutathione beads) and resolved by SDS-PAGE. Gel regions below and above ∼140 kDa were excised and prepared for Ub-AQUA analysis (15). The amounts (picomoles) of Lys-48- and Lys-63-linked chains in both regions are shown.

FIGURE 3.
FIGURE 3.

Pex29 degradation is mediated by the Doa10 pathway. A, Pex29 overexpression leads to growth retardation in doa10Δ cells. 5-fold serial dilutions of wild-type and various isogenic mutants bearing a plasmid for inducible PEX29 expression were plated as in Fig. 1B. B and C, Doa10 and Ubc7 are involved in Pex29 degradation. Pex29 stability was determined in wild-type, doa10Δ, ubc4Δ, and ubc7Δ cells. D, Ufd1 is required for Pex29 degradation. E, Doa10 is required for Pex29 ubiquitylation. Pex29 ubiquitylation patterns in wild-type or mutant cells were determined as in Fig. 2D. The p81 plasmid bearing the GAL1-promoter-regulated Ha-tagged Ub was used. F, elevated UPR activity in cells lacking UFD2. Levels of β-galactosidase activity in yeast cells harboring a plasmid that contains the LACZ gene under the control of the KAR2 promoter. The strain genotypes are indicated at the bottom. The ire1Δ strain defective in UPR signaling is included as a negative control. Values shown are the means derived from three measurements. Bars represent ±S.D.

FIGURE 4.
FIGURE 4.

The interactions among Pex29, Doa10, and Ufd2. A and B, Pex29 binds Doa10 and Ufd2. Co-immunoprecipitation analysis of the interaction between GST-His6-tagged Pex29 and Doa10–13Myc or Myc-tagged Ufd2 was done as previously described (18). Briefly, proteins were extracted from cells expressing galactose-inducible GST-His6-tagged Pex29 and Myc-tagged Ufd2 or endogenous Doa10–13Myc and immunoprecipitated with various antibodies as indicated. Immunoprecipitates were resolved by SDS-PAGE and probed with indicated antibodies. The antibodies for immunoprecipitation (IP) and immunoblot (blot) are shown to the left of the panels. C, Doa10 and Ufd2 do not form a stable complex. The binding was done as in A.

FIGURE 5.
FIGURE 5.

Ufd2 is not required for all Doa10 or Cdc48 substrates. A and B, degradation of two commonly employed Doa10 substrates Deg1-βgal and Ubc6* in wild-type, ufd2Δ, or doa10Δ cells. The expression shut-off assays for these two substrates were preformed as previously described (32). C and D, degradation of two Cdc48 substrates Ha-tagged KHN and Sec61-2 in wild-type, or ufd2Δ. The protein stability assays were done as described above.

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