Limits...
Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

Show MeSH

Related in: MedlinePlus

Heterologous function of human UEV genes in yeast. WXY903 (mms2Δ::HIS3) was transformed with various pUev-G4BD plasmids and the transformants were compared with HK580-10D (wild type) in a gradient plate assay for their ability to complement the yeast mms2 defect. Overnight cell cultures were printed on YPD (A) or YPD + 0.025% MMS (B) plates and incubated for 36 h before taking the photograph. The arrow points toward higher MMS concentration.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171356&req=5

fig3: Heterologous function of human UEV genes in yeast. WXY903 (mms2Δ::HIS3) was transformed with various pUev-G4BD plasmids and the transformants were compared with HK580-10D (wild type) in a gradient plate assay for their ability to complement the yeast mms2 defect. Overnight cell cultures were printed on YPD (A) or YPD + 0.025% MMS (B) plates and incubated for 36 h before taking the photograph. The arrow points toward higher MMS concentration.

Mentions: A prerequisite for our hypothesis is that both Mms2 and Uev1 form stable complexes with Ubc13. Although the physical interaction between Ubc13 and Uev has been reported in various studies (Deng et al., 2000; Hofmann and Pickart, 1999; McKenna et al., 2001), we decided to systematically test all three Mms2 homologues found in human cells for their in vitro and in vivo (in yeast cells) interactions. As shown in Fig. 2 A, bacterial cell extracts from cells expressing GST-Mms2 (lane 2) and GST-Uev1A (lane 3) were able to pull down purified recombinant Ubc13 by GST affinity. In contrast, extracts from cells expressing GST alone (not depicted) or GST-Uev1B (Fig. 2 A, lane 4) were unable to pull down detectable amounts of Ubc13. To further confirm the in vitro data, yeast two-hybrid analysis was performed by fusing Uev with the Gal4 DNA binding domain (Gal4BD) at the COOH terminus and Ubc13 with the Gal4 activation domain (Gal4AD) at the NH2 terminus. As shown in Fig. 2 B, only Mms2-Gal4BD and Uev1A-Gal4BD were able to interact with Gal4AD-Ubc13, resulting in simultaneous activation of PGAL1-HIS3 and PGAL2-ADE2, whereas Uev1B-Gal4BD was unable to interact with Gal4AD-Ubc13. To determine whether the human Ubc13–Uev interaction is related to its biological functions in yeast cells, we attempted to functionally complement the yeast mms2 mutant by expressing each of the Uev-Gal4BD constructs. Although expression of Mms2-Gal4BD and Uev1A-Gal4BD was able to fully alleviate the severe methyl methanesulfonate (MMS) sensitivity of mms2 cells to the wild-type level in a gradient plate assay, vector alone or the Uev1B-Gal4BD construct failed to rescue mms2-deficient cells from killing by MMS (Fig. 3). The lack of Uev1B activity in yeast cells is likely due to the extended NH2-terminal sequence, as deletion of the NH2-terminal 80–amino acid coding region from UEV1B restores its DNA repair function in yeast (Xiao et al., 1998) and the interaction with Ubc13 in a yeast two-hybrid assay (unpublished data). Due to the lack of detection of Uev1B–Ubc13 interaction, and our previous observation (Franko et al., 2001) that mouse cells do not express the corresponding Uev1B transcript, we decided to focus our attention on the cellular functions of Mms2 and Uev1A in this study.


Distinct regulation of Ubc13 functions by the two ubiquitin-conjugating enzyme variants Mms2 and Uev1A.

Andersen PL, Zhou H, Pastushok L, Moraes T, McKenna S, Ziola B, Ellison MJ, Dixit VM, Xiao W - J. Cell Biol. (2005)

Heterologous function of human UEV genes in yeast. WXY903 (mms2Δ::HIS3) was transformed with various pUev-G4BD plasmids and the transformants were compared with HK580-10D (wild type) in a gradient plate assay for their ability to complement the yeast mms2 defect. Overnight cell cultures were printed on YPD (A) or YPD + 0.025% MMS (B) plates and incubated for 36 h before taking the photograph. The arrow points toward higher MMS concentration.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171356&req=5

fig3: Heterologous function of human UEV genes in yeast. WXY903 (mms2Δ::HIS3) was transformed with various pUev-G4BD plasmids and the transformants were compared with HK580-10D (wild type) in a gradient plate assay for their ability to complement the yeast mms2 defect. Overnight cell cultures were printed on YPD (A) or YPD + 0.025% MMS (B) plates and incubated for 36 h before taking the photograph. The arrow points toward higher MMS concentration.
Mentions: A prerequisite for our hypothesis is that both Mms2 and Uev1 form stable complexes with Ubc13. Although the physical interaction between Ubc13 and Uev has been reported in various studies (Deng et al., 2000; Hofmann and Pickart, 1999; McKenna et al., 2001), we decided to systematically test all three Mms2 homologues found in human cells for their in vitro and in vivo (in yeast cells) interactions. As shown in Fig. 2 A, bacterial cell extracts from cells expressing GST-Mms2 (lane 2) and GST-Uev1A (lane 3) were able to pull down purified recombinant Ubc13 by GST affinity. In contrast, extracts from cells expressing GST alone (not depicted) or GST-Uev1B (Fig. 2 A, lane 4) were unable to pull down detectable amounts of Ubc13. To further confirm the in vitro data, yeast two-hybrid analysis was performed by fusing Uev with the Gal4 DNA binding domain (Gal4BD) at the COOH terminus and Ubc13 with the Gal4 activation domain (Gal4AD) at the NH2 terminus. As shown in Fig. 2 B, only Mms2-Gal4BD and Uev1A-Gal4BD were able to interact with Gal4AD-Ubc13, resulting in simultaneous activation of PGAL1-HIS3 and PGAL2-ADE2, whereas Uev1B-Gal4BD was unable to interact with Gal4AD-Ubc13. To determine whether the human Ubc13–Uev interaction is related to its biological functions in yeast cells, we attempted to functionally complement the yeast mms2 mutant by expressing each of the Uev-Gal4BD constructs. Although expression of Mms2-Gal4BD and Uev1A-Gal4BD was able to fully alleviate the severe methyl methanesulfonate (MMS) sensitivity of mms2 cells to the wild-type level in a gradient plate assay, vector alone or the Uev1B-Gal4BD construct failed to rescue mms2-deficient cells from killing by MMS (Fig. 3). The lack of Uev1B activity in yeast cells is likely due to the extended NH2-terminal sequence, as deletion of the NH2-terminal 80–amino acid coding region from UEV1B restores its DNA repair function in yeast (Xiao et al., 1998) and the interaction with Ubc13 in a yeast two-hybrid assay (unpublished data). Due to the lack of detection of Uev1B–Ubc13 interaction, and our previous observation (Franko et al., 2001) that mouse cells do not express the corresponding Uev1B transcript, we decided to focus our attention on the cellular functions of Mms2 and Uev1A in this study.

Bottom Line: In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2.Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains.Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Immunology, University of Saskatchewan, Saskatoon, Saskatchewan S7N 5E5, Canada.

ABSTRACT
Ubc13, a ubiquitin-conjugating enzyme (Ubc), requires the presence of a Ubc variant (Uev) for polyubiquitination. Uevs, although resembling Ubc in sequence and structure, lack the active site cysteine residue and are catalytically inactive. The yeast Uev (Mms2) incites noncanonical Lys63-linked polyubiquitination by Ubc13, whereas the increased diversity of Uevs in higher eukaryotes suggests an unexpected complication in ubiquitination. In this study, we demonstrate that divergent activities of mammalian Ubc13 rely on its pairing with either of two Uevs, Uev1A or Mms2. Structurally, we demonstrate that Mms2 and Uev1A differentially modulate the length of Ubc13-mediated Lys63-linked polyubiquitin chains. Functionally, we describe that Ubc13-Mms2 is required for DNA damage repair but not nuclear factor kappaB (NF-kappaB) activation, whereas Ubc13-Uev1A is involved in NF-kappaB activation but not DNA repair. Our finding suggests a novel regulatory mechanism in which different Uevs direct Ubcs to diverse cellular processes through physical interaction and alternative polyubiquitination.

Show MeSH
Related in: MedlinePlus