Molecular basis for lysine specificity in the yeast ubiquitin-conjugating enzyme Cdc34

Abstract

Ubiquitin (Ub)-conjugating enzymes (E2s) and ubiquitin ligases (E3s) catalyze the attachment of Ub to lysine residues in substrates and Ub during monoubiquitination and polyubiquitination. Lysine selection is important for the generation of diverse substrate-Ub structures, which provides versatility to this pathway in the targeting of proteins to different fates. The mechanisms of lysine selection remain poorly understood, with previous studies suggesting that the ubiquitination site(s) is selected by the E2/E3-mediated positioning of a lysine(s) toward the E2/E3 active site. By studying the polyubiquitination of Sic1 by the E2 protein Cdc34 and the RING E3 Skp1/Cul1/F-box (SCF) protein, we now demonstrate that in addition to E2/E3-mediated positioning, proximal amino acids surrounding the lysine residues in Sic1 and Ub are critical for ubiquitination. This mechanism is linked to key residues composing the catalytic core of Cdc34 and independent of SCF. Changes to these core residues altered the lysine preference of Cdc34 and specified whether this enzyme monoubiquitinated or polyubiquitinated Sic1. These new findings indicate that compatibility between amino acids surrounding acceptor lysine residues and key amino acids in the catalytic core of ubiquitin-conjugating enzymes is an important mechanism for lysine selection during ubiquitination.

FIG. 1.

Amino acids proximal to lysines regulate ubiquitination of Sic1 by SCF^Cdc4/Cdc34. (A) Monoubiquitination of single-lysine Sic1 derivatives by SCF^Cdc4/Cdc34 with lysine-less Ub [Ub(K0)]. As a control, Cdc34 was omitted from the reaction mixture (lane 1). Monoubiquitinated Sic1 (Sic1-Ub₁) levels were normalized relative to monoubiquitinated Sic1 (K53). Error bars represent the SD. Gaps indicate where irrelevant lanes were removed from the same experiment. (B) Positions of CDK phosphorylation sites and the six physiologically relevant lysines of Sic1 (top) and their local amino acid sequence environment (bottom). The gray box represents the CDK-inhibitory domain. (C) The indicated single-lysine derivatives with changes to amino acids proximal to the lysine were subjected to the same analysis as that described above (A). Monoubiquitinated Sic1 (Sic1-Ub₁) was normalized relative to the levels of monoubiquitinated wild-type K32 (white bars), K50 (light gray bars), K53 (gray bars), and K88 (black bars). Error bars represent the SD. (D) Effect of Sic1 concentration (15 to 800 nM) on the rate of monoubiquitination. Monoubiquitinated Sic1 was quantified by PhosphorImager analysis to calculate the apparent V_max and K_m by nonlinear regression with GraphPad Prism software. Data in parentheses indicate standard errors.

FIG. 2.

Amino acids proximal to Sic1 lysines regulate proliferation of S. cerevisiae and degradation of Sic1. (A) Growth of yeast cells expressing the indicated Sic1 derivatives. An empty plasmid (empty) and lysine-less Sic1 K0 were included as controls. Serial 10-fold dilutions of exponentially growing cells were spotted onto plates with galactose to induce expression or without galactose as a control. (B) Cells were arrested in G₁ phase with α-factor, and the expression of Sic1 K53 and Sic1 K53 K84-like (left), or Sic1 K32 and Sic1 K32 K53-like (right), was induced with galactose. Cells were induced to enter the cell cycle, Sic1 expression was repressed by the addition of fresh medium, and samples were taken at the indicated times. Cell extracts were prepared, and Sic1, actin, and Clb5 were detected by immunoblotting. Sic1 levels were quantified and normalized according to the actin levels. The results are the means and ranges of data from two independent experiments.

FIG. 3.

Y89, S139, and A141 of Cdc34 are important for viability of S. cerevisiae. (A) Sequence alignments of the core domains of E2s reveal 11 variations of the amino acid sequence analogous to Y89, S139, and A141 of yeast Cdc34. The three sites are highlighted by boxes and listed in parentheses on the right. The active-site cysteine and the oxyanion-stabilizing asparagine are underlined. (B) The indicated Cdc34 mutants were analyzed with the Δcdc34 deletion strain. An empty tester plasmid (empty) was included as a control. (C) Survivors of the experiment in panel B were tested for growth by drop test analysis. (D) The ectopic expression of Cdc34 (lane 2) and the mutant alleles (lanes 3 to 12) from pRS415-LEU2 was analyzed by immunoblotting with polyclonal Cdc34 antibody in a derivative of cdc34 deletion strain YMS034 (MATa ura3-1 trp1-1 ade2-1 leu2-3,112 his3-11,15 can1-100 cdc34::kanMX4) (24), which was kept viable by the ectopic expression of truncated Cdc34 [pES12-ΔCdc34(1-244)] (30). Full-length Cdc34 and the truncated derivative (residues 1 to 244) are indicated on the right. Empty plasmid (pRS415-LEU2) was employed as a control (lane 1). Molecular masses (in kilodaltons) are indicated on the left.

FIG. 4.

Y89, S139, and A141 of Cdc34 are important for ubiquitination of Sic1. (A and B) Monoubiquitination of Sic1 Wt for 60 min (A) or for the indicated times (B). (C) The indicated single-lysine Sic1 mutants were assessed with the indicated Cdc34 derivatives. No E2 represents control with no Cdc34 added, or reactions were stopped at the indicated times (lanes 2 to 4, with Cdc34) or at 60 min. Gaps indicate where lanes of the same experiment were rearranged for consistency.

FIG. 5.

Y89 and S139 of Cdc34 affect different steps in Sic1 polyubiquitination. (A and B) Polyubiquitination of Sic1 Wt with Ub(Wt) for 60 min (A) or at the indicated times (B). (C) Sic1 K53 ubiquitination with Ub(K48o) was assessed with the indicated Cdc34 derivatives. Lane 1, no Cdc34 added as a control. (D) To analyze Ub K48 chain elongation without preceding Sic1 ubiquitination, Sic1 K53-Ub(K48o)₁ was used as a substrate with Ub(K0) (lanes 1 to 7). As a control, Sic1 K53 was assayed under the same conditions (lanes 8 to 14). (E) The S139 mutants of Cdc34 do not change linkage specificity for other Ub lysines. Ub chain formation was monitored with Sic1 K32-Ub(Wt) as a substrate in the presence of Ub(K48R). Gaps indicate where irrelevant lanes were removed from the images.

FIG. 6.

Mutation of residues proximal to Ub K48 reactivates YDA in Ub chain synthesis. (A) Cdc34 and YDA were tested for their abilities to generate Ub chains on Sic1 K36 with either Ub(Wt), Ub(G47Q), Ub(Q49P), Ub(L50S), or Ub(K48R). Ub(K48R), which was used as a control, has a lower MW than the other Ub derivatives, as indicated by the lower mobility of monoubiquitinated Sic1 K36. Gaps indicate where irrelevant lanes were removed. (B) The polyubiquitination of Sic1 Wt (0.4 μM) was assessed with either wild-type Cdc34 (lanes 1 to 5) or the YDA mutant (lanes 6 to 10) with 20 μM Ub(L50S) at the indicated times.

FIG. 7.

Sequence-dependent ubiquitination is intrinsic to Cdc34. (A) Cdc34 mutants were analyzed in autoubiquitination reactions with Ub(Wt). Autoubiquitinated E2 (E2-Ub_n) and unanchored di-Ub (Ub₂) are indicated. A truncated version of Cdc34 (200Δ) missing the C-terminal autoubiquitination sites and yeast Ubc4, which is inefficient in di-Ub synthesis (13), were employed as controls. The truncated Cdc34 (200Δ) efficiently synthesized unanchored di-Ub (Ub₂) and high-molecular-mass Ub chains (Ub_n) of ∼150 to 250 kDa (lane 13). Ubc4, which autoubiquitinates, generated predominantly monoubiquitinated Ubc4 (Ubc4-Ub₁) and failed to form unanchored Ub₂ (lane 14). (B) The autoubiquitination of Cdc34 and the YDA mutant with Ub(Wt), Ub(G47Q), Ub(Q49P), Ub(L50S), or Ub(K48R) was monitored as described above (A) except that Cdc34 was ³²P labeled. (C) The autoubiquitination of Cdc34 and the YDA mutant with Ub(Wt), Ub(Q49P), or Ub(L50S) was monitored as described above (B) for the indicated times. (D) Amino acid changes proximal to K48 of ubiquitin alter the V_max of Ub chain synthesis by Cdc34(YDA). The effect of the Ub concentration (0.5 to 20 μM) on the rate of Ub chain extension by wild-type Cdc34 (lanes 1 to 7) and the YDA mutant (lanes 8 to 14) was measured in autoubiquitination reaction mixtures containing the indicated concentrations of ³²P-labeled Ub(Wt) (top), Ub(Q49P) (middle), and Ub(L50S) (bottom). Autoubiquitinated Cdc34 (Cdc34-Ub_n) and unanchored di-Ub (Ub₂) and tri-Ub (Ub₃) are indicated on the right-hand side. Cdc34-Ub conjugates of wild-type Cdc34 and Cdc34(YDA) with more than four Ub moieties (number of major autoubiquitination sites) were quantified by PhosphorImager analysis to calculate the apparent V_max and K_m of the Ub chain extension on Cdc34 by nonlinear regression with GraphPad Prism software. Data in parentheses indicate standard errors.