Physical and Genetic Associations of the Irc20 Ubiquitin Ligase with Cdc48 and SUMO

<div><p>A considerable percentage of the genome is dedicated to the ubiquitin-proteasome system, with the yeast genome predicted to encode approximately 100 ubiquitin ligases (or E3s), and the human genome predicted to encode more than 600 E3s. The most abundant class of E3s consists of RING finger-containing proteins. Although many insights have been obtained regarding the structure and catalytic mechanism of the E3s, much remains to be learned about the function of the individual E3s. Here we characterize <i>IRC20</i>, which encodes a dual RING- and Snf/Swi family ATPase domain-containing protein in yeast that has been implicated in DNA repair. We found that overexpression of <i>IRC20</i> causes two transcription-associated phenotypes and demonstrate that the Irc20 RING domain possesses ubiquitin E3 activity <i>in vitro</i>. Two mass spectrometry approaches were undertaken to identify Irc20-associated proteins. Wild-type Irc20 associated with Cdc48, a AAA-ATPase that serves as an intermediary in the ubiquitin-proteasome system. A second approach using a RING mutant derivative of Irc20 detected increased association of the Irc20 mutant with SUMO. These findings provide a foundation for understanding the roles of Irc20 in transcription and DNA repair.</p></div

Identifying the Cdc48-binding domain of Irc20.

<p>A) Diagram of Irc20 deletion derivatives and a summary of their ability to co-IP Cdc48. B) & C) Co-immunoprecipitation analysis. The indicated Irc20 derivatives were expressed from a 2 μ plasmid in the presence of Cdc48–3xMyc, extracts were prepared, proteins immunoprecipitated with anti-HA beads, and western blotted with anti-HA antibody to detect Irc20, or with anti-Myc antibody to detect Cdc48. The binding interface of Irc20 that physically interacts with Cdc48 lies between amino acids 883 and 1238.</p

Irc20 is SUMOylated and binds SUMO.

<p>A) Proteins that preferentially associate with the irc20-C1239A RING- mutant. GY460 (<i>his4–912δ lys2–128δ suc2Δuas(−1900/−390) ura3–52 leu2Δ1</i>) expressing either pAR11 (<i>3xHA-IRC20</i>) or pAR14 (<i>3xHA-irc20-C1239A</i>) was cross-linked <i>in vivo</i> with formaldehyde, pelleted, lysed, immunoprecipitated, and the resulting material analyzed by mass spectrometry. All proteins that were detected at least 2-fold greater in the Irc20 RING- mutant co-IPs compared to the WT Irc20 co-IPs are presented. Smt3 ( = SUMO) was detected in the Irc20 RING- mutant at 11-fold greater level than in the wild-type 3xHA-Irc20 control. B) Irc20 binds SUMO. To address whether Irc20 is binding to SUMO, co-IPs under native conditions were performed with WT HA-Irc20 and RING- mutant HA-Irc20 to test for the ability of each to bind a non-conjugatable GAL4(DBD)-6xSUMO chain fusion protein. Both WT and RING- mutant Irc20 pull down the 6xSUMO in equal amounts, ruling out the possibility that RING- mutant Irc20 is simply binding to more SUMO than WT Irc20 in the previous experiment. C) Irc20 is SUMOylated. To test whether Irc20 is SUMOylated, the indicated proteins were immunoprecipitated and Western blots probed with anti-HA and anti-SUMO antibodies. D) To determine the level of SUMOylated Irc20 when SUMO is overexpressed, untagged Irc20 (pGM7) and 3xHA-Irc20 (pAR11) were co-IPd from lysates prepared from cell expressing only endogenous SUMO (“vector”) or overexpressing SUMO from pZW61 (2 μ <i>SMT3</i>). Western blots were probed with anti-HA and anti-SUMO antibodies.</p

Overexpression of <i>IRC20</i> causes transcription phenotypes.

<p>A) Yeast strain GY2205, which contains the <i>suc2Δuas(−1900/−390)</i> reporter and endogenous <i>TAP-IRC20</i>, was transformed with either empty vector (pRS425) or a 2 μ <i>TAP-IRC20</i> plasmid (pAR41) and assayed for the Bur phenotype on a YPsucrose plate (top). A western blot to detect expression of TAP-Irc20 is shown below, along with G6PDH loading control. B) 2 μ <i>GAL1pr-IRC20</i> exhibits an Spt- phenotype. Yeast strains GY482 (<i>his4–912δ lys2–128δ</i>) and GY2203 (<i>his4–912δ lys2–128δ GAL1pr-TAP-IRC20</i>) were transformed with the indicated plasmids and transformants were assessed for their Spt phenotype on SC-His and SC-Lys plates. C) Overexpression of epitope-tagged Irc20. Irc20 was HA-tagged at its N- or C-terminus and expressed from a 2 μ plasmid. The 2 μ Bur- phenotype was disrupted by the C-terminal 6HA tag (pAR9), while N-terminal 3xHA-Irc20 fusion (pAR11) remained functional. D) N-terminally HA-tagged D534A E535A <i>irc20</i> ATPase (pAR30) and C1239A RING finger (pAR14) mutants were created and expressed from a 2 μ plasmid in yeast strain GY460. Both mutants are expressed at similar levels as wild-type Irc20, but are incapable of producing the 2 μ Bur- phenotype.</p

<i>cdc48</i> mutations can cause a Bur- phenotype.

<p>A) A heterozygous <i>CDC48–12xMYC::KAN</i> integrant diploid (GY2476) was induced to undergo meiosis and tetrads dissected. Two representative tetrads are shown, with spores labeled A-D. Perfect linkage was observed between slow growth, G418-resistance, and the Bur- phenotype in 30 four-spored tetrads. B) A plasmid shuffle screen for <i>cdc48</i> Bur- mutants uncovered an arginine-to-lysine change at amino acid 369, located in the ATPase D1 domain. This mutation confers both Bur- and cold-sensitive phenotypes (bottom panel). The strain being tested here contains the <i>suc2Δuas-HIS3</i> reporter, with growth on SC-His plate being indicative of the Bur- phenotype. C) The cold sensitivity of <i>cdc48-R369K</i> is complemented by <i>CDC48</i> on a CEN plasmid. Strains GY2387 (<i>cdc48Δ::TRP1</i> URA3 CDC48> and GY2406 (<i>cdc48Δ::TRP1</i> URA3 cdc48-R369K>) were transformed with empty vector or wild-type <i>CDC48</i> on a <i>LEU2</i>-marked CEN plasmid (pAR56). Cold sensitivity was assayed at 16°C. D) The <i>cdc48-R369K</i> mutation does not affect the ability of Cdc48 to co-immunoprecipitate with Irc20.</p

<i>S. cerevisiae</i> strains used in this study.

<p><i>S. cerevisiae</i> strains used in this study.</p

Plasmids used in this study.

<p>Plasmids used in this study.</p

Characterization of <i>irc20</i> mutants defective for binding Cdc48.

<p>A) N-terminal (pAR11) and C-terminal (pAR9) 3xHA-Irc20 were expressed from a 2 μ plasmid in the presence of Cdc48–3xMyc, extracts were prepared, proteins immunoprecipitated with anti-HA beads, and western blotted with anti-HA antibody to detect Irc20, or with anti-Myc antibody to detect Cdc48. B) Diagram of deletions created with the context of full-length 3xHA-Irc20. A summary of their results in the functional assays and co-IPs presented in panels C and D is shown to the right. C) The <i>3xHA-irc20</i> deletion derivatives were expressed from a 2 μ plasmid and tested for their Bur- phenotype and D) for their ability to co-precipitate Cdc48–3xMyc. WCE  =  whole cell extract</p

Identification of Tup1 and Cyc8 sumoylation sites.

<p>(<b>A</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing either wild-type Tup1-3HA or Tup1^K270R-3HA from the endogenous <i>TUP1</i> promoter were examined for Tup1 sumoylation at 0 or 15 minutes of hyperosmotic stress (1.2M sorbitol) as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>B</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing the indicated Cyc8-3HSV deletion mutant were subject to hyperosmotic stress (1.2M sorbitol) for 15 minutes. Cell lysates were generated and subject to the same analysis as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>C</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing either wild-type Cyc8-3HSV or Cyc8^4KtoR-3HSV from the endogenous <i>CYC8</i> promoter were examined for Cyc8 sumoylation as in (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005809#pgen.1005809.g002" target="_blank">Fig 2D</a>). (<b>D</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing wild-type Tup1-3HA from the endogenous <i>TUP1</i> promoter and either wild-type Cyc8-3HSV or Cyc8^4KtoR-3HSV from the endogenous <i>CYC8</i> promoter were examined for Tup1 sumoylation as in (A). (<b>E</b>) <i>His</i>_<i>6</i><i>-FLAG-SMT3</i> cells expressing wild-type Cyc8-3HSV from the endogenous <i>CYC8</i> promoter and either wild-type Tup1-3HA or Tup1^K270R-3HA from the endogenous <i>TUP1</i> promoter were examined for Cyc8 sumoylation as in (C). (<b>F</b>) <i>6His-FLAG-SMT3</i> wild-type or <i>tup1</i>^<i>K270R</i><i>cyc8</i>^<i>4KtoR</i> cells were subjected to hyperosmotic stress (1.2M sorbitol) for 0 or 15 minutes, and global sumoylation patterns examined by western analysis using an anti-FLAG antibody.</p

Tup1 and Cyc8 are sumoylated during hyperosmotic stress.

<p>(<b>A</b>) Scheme of the MS strategy to identify proteins sumoylated during hyperosmotic stress. (<b>B</b>) Total peptide counts identified for Tup1 and Cyc8 at 0 and 15 minutes of hyperosmotic stress. Total peptide counts for Smt3 are included to demonstrate equivalent levels of SUMO in the samples. (<b>C</b>) Total peptide counts identified for proteins where the significance of the changes between 0 and 15 minutes of hyperosmotic stress was <i>p</i>≤0.05. Gray areas represent ≥3 fold changes in the 15 minute samples compared with the 0 minute samples. Tup1 and Cyc8 are noted. (<b>D</b>) Cells expressing His₆-FLAG-Smt3 (HF-Smt3) and either a 3xHA epitope-tagged Tup1 (Tup1-3HA) or a 3xHSV epitope-tagged Cyc8 (Cyc8-3HSV) from their endogenous promoters were subject to hyperosmotic stress (1.2M sorbitol) over a 60-minute time course. Cell lysates (input) and purified sumoylated proteins (SUMO pulldown) were subject to western analyses using anti-HA, anti-HSV, or anti-Pgk1 antibodies to detect Tup1, Cyc8, or Pgk1 respectively. (<b>E</b>) Similar experiment as in (D) except cells were subject to hyperosmotic stress (1.2M sorbitol), heat shock (42°C), or high ethanol (10% v/v) for 0, 15, and 60 minutes.</p