The loop-less tmCdc34 E2 mutant defective in polyubiquitination in vitro and in vivo supports yeast growth in a manner dependent on Ubp14 and Cka2

Agnieszka Lass,Michael Skowyra,Sergey Korolev,Kenneth M Scaglione,Dorota Skowyra,Ross Cocklin,Mark Goebl

doi:10.1186/1747-1028-6-7

Abstract

BackgroundThe S73/S97/loop motif is a hallmark of the Cdc34 family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. The inability of the loop-less Δ12Cdc34 mutant to support growth was linked to its inability to catalyze polyubiquitination. However, the loop-less triple mutant (tm) Cdc34, which not only lacks the loop but also contains the S73K and S97D substitutions typical of the K73/D97/no loop motif present in other E2s, supports growth. Whether tmCdc34 supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown.ResultstmCdc34 supports yeast viability with normal cell size and cell cycle profile despite producing fewer polyubiquitin conjugates in vivo and in vitro. The in vitro defect in Sic1 substrate polyubiquitination is similar to the defect observed in reactions with Δ12Cdc34 that cannot support growth. The synthesis of free polyubiquitin by tmCdc34 is activated only modestly and in a manner dependent on substrate recruitment to SCFCdc4. Phosphorylation of C-terminal serines in tmCdc34 by Cka2 kinase prevents the synthesis of free polyubiquitin chains, likely by promoting their attachment to substrate. Nevertheless, tmCDC34 yeast are sensitive to loss of the Ubp14 C-terminal ubiquitin hydrolase and DUBs other than Ubp14 inefficiently disassemble polyubiquitin chains produced in tmCDC34 yeast extracts, suggesting that the free chains, either synthesized de novo or recycled from substrates, have an altered structure.ConclusionsThe catalytic motif replacement compromises polyubiquitination activity of Cdc34 and alters its regulation in vitro and in vivo, but either motif can support Cdc34 function in yeast viability. Robust polyubiquitination mediated by the S73/S97/loop motif is thus not necessary for Cdc34 role in yeast viability, at least under typical laboratory conditions.

Highlights

The covalent attachment of ubiquitin to other proteins often serves as the signal for their degradation by the 26 S proteasome [1]
Either motif can support Cdc34 function in cell growth and division, with normal cell size and cell cycle profile. These findings suggest that robust polyubiquitination catalyzed in a manner dependent on the S73/S97/loop motif is not necessary for Cdc34 function in vivo, at least under typical laboratory conditions
Robust polyubiquitination activity is thought to be key to the function of the Cdc34 ubiquitin-conjugating enzyme that, together with the SCF ubiquitin ligases, promotes degradation of proteins involved in cell cycle and growth regulation

Summary

Introduction

The covalent attachment of ubiquitin to other proteins often serves as the signal for their degradation by the 26 S proteasome [1]. While the term “ubiquitin ligase” implies that all E3s are enzymes, only the HECT-type E3s contain a catalytic site cysteine that directly participates in the ubiquitin transfer. The RING-type E3s promote ubiquitination of specific substrates by the catalytic site of an E2 The significance of this difference is unclear, as both types of ubiquitin transfer cascades lead to formation of an isopeptide bond between the C-terminus of ubiquitin and a lysine residue on the substrate. Protein substrates can be modified with one or multiple ubiquitins and a chain of polyubiquitin can be synthesized when a lysine of ubiquitin serves as the isopeptide bond acceptor. The S73/S97/loop motif is a hallmark of the Cdc family of E2 ubiquitin-conjugating enzymes that together with the SCF E3 ubiquitin ligases promote degradation of proteins involved in cell cycle and growth regulation. Whether tmCdc supports growth despite defective polyubiquitination, or the S73K and S97D substitutions, directly or indirectly, correct the defect caused by the loop absence, are unknown

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Conclusion