The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4 - PubMed (original) (raw)

The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4

Gary Kleiger et al. J Biol Chem. 2009.

Abstract

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF(Cdc4) and makes a major contribution to the submicromolar K(m) of Cdc34 for SCF(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly.

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Figures

FIGURE 1.

Yeast Cdc34-Δ190 is functional but cannot be activated by SCF. Di-ubiquitin synthesis assays containing 250 n

E1, 2 μ

Cdc34-Δ270 or Cdc34-Δ190, 6 μ

32P-labeled K48R ubiquitin, and 50 μ

D77 ubiquitin were performed for the specified times both in the absence and the presence of 100 n

SCF. A, Cdc34-Δ270; B, Cdc34-Δ190; C, Cdc34-Δ270 with SCF; D, Cdc34-Δ190 with SCF. Notice that Cdc34-Δ270 and Cdc34-Δ190 form product at similar rates in the absence of SCF, demonstrating that tail deletion does not disturb the structure and catalytic function of Cdc34-Δ190. Each graphical data point represents the mean of triplicate data values from three independent experiments, and the error bars are the standard deviations.

FIGURE 2.

The acidic tail inhibits Sic1 ubiquitylation by Cdc34-SCF. Reactions containing 1.6 μ

yeast E1, 100 n

yeast SCF, 1 μ

32P-labeled Sic1, 2 μ

yeast Cdc34-Δ270, and 150 μ

ubiquitin were incubated for 1 min at 20–22 °C along with various concentrations of GST-tail (residues 171–270; GST-YACT) or GST prior to quenching with reducing SDS-PAGE buffer. Substrate conversion to product was quantified and plotted as the rate of Sic1 ubiquitylation versus the log of the GST fusion concentration. An IC50 of 16 ± 6 μ

was estimated from nonlinear curve fitting the data to a sigmoidal dose-response curve with a fixed slope of 1 (Prism).

FIGURE 3.

Progressive deletion of the Cdc34 acidic tail results in defects in both Cdc34 binding to SCF and catalysis. All of the reactions contained 1.6 μ

yeast E1, 100 n

yeast SCF, 1.2 μ

32P-labeled Sic1, 300 μ

ubiquitin, and Cdc34 (2-fold serial dilutions starting at 5 or 100 μ

). A, various concentrations (see figure) of Cdc34-Δ230 were incubated with the above reactants at 20–22 °C for 1 min and quenched by SDS-PAGE buffer. B, same as above, except Cdc34-Δ190 was the E2, and reactions were incubated for 30 min prior to quenching. C, Cdc34-Δ225. The incubation period was 1 min. D, Cdc34-Δ220. The incubation period was 1 min. E, Cdc34-Δ210. The incubation period was 1 min. F, Cdc34-Δ205. The incubation period was 2 min. G, graph plotting the rate of Sic1 ubiquitylation against the concentration of Cdc34-Δ230. The data were fit to the Michaelis-Menten equation using nonlinear curve fitting (Prism). Each graphical data point represents the mean of duplicate data values from two independent experiments, and the error bars are the standard deviations. H, summary of the results from Fig. 3. Km and _k_cat values are given for each Cdc34 construct. The units for _k_cat are min−1. The black bar encompasses residues from the distal tail, and the gray bar encompasses residues from the proximal tail.

FIGURE 4.

The distal segment of the acidic tail can be replaced entirely by poly(Glu-Asp). Reactions containing 1.6 μ

yeast E1, 100 n

yeast SCF, 1.2 μ

32P-labeled Sic1, 300 μ

ubiquitin, and a 2-fold linearly decreasing titration of Cdc34-Δ210ED-230 were incubated for 1 min and quenched by SDS-PAGE buffer. Each data point was measured in duplicate. The kinetic parameters obtained should be compared with those reported in Fig. 3 for Cdc34-Δ230 and Cdc34-Δ210.

FIGURE 5.

Fusion of human Cdc34-Δ190 to Cul1 can partially rescue the defect of Cdc34 tail deletion in ubiquitylation reactions. A, di-ubiquitin synthesis assay comparing wild type and Cdc34-Δ190 in the presence of Rbx1+Cul1. B, comparison of wild type (WT) and Cdc34-Δ190 fusions to Cul1. Reactions containing either 300 n

Rbx1+Cul1 and 300 n

Cdc34 or containing 300 n

Rbx1+Cul1-Cdc34, 0.7 μ

human E1, 6 μ

32P-labeled K48R ubiquitin, and 50 μ

D77 ubiquitin were incubated at 20–22 °C for the specified times and quenched with SDS-PAGE buffer. No products were observed when Cdc34-Δ190 was assayed as an unfused species. C and D, di-ubiquitin synthesis assay comparing Cul1 fusions to either wild type Cdc34 (C) or Δ190 Cdc34 (D). The reactions containing 300 n

Rbx1+Cul1-Cdc34, 0.7 μ

human E1, 6 μ

32P-labeled K48R ubiquitin, and 50 μ

D77 ubiquitin were incubated at 20–22 °C for the specified times and quenched with SDS-PAGE buffer. E, Ub-β-catenin (25) ubiquitylation reactions comparing unfused wild type and Cdc34-Δ190 (300 n

) in the presence of Rbx1+Cul1 (300 n

) or of wild type and Cdc34-Δ190 fusions (1.2 μ

). SCFβTrCP complex formation was accomplished by briefly mixing equimolar amounts of either Rbx1+Cul1 or the Rbx1+Cul1-Cdc34 fusions with βTrCP prior to initiation of the reaction by the addition of substrate. The reactions also contained 0.7 μ

E1, 75 μ

Ub, and 3 μ

32P-labeled Ub-β-catenin peptide. The reactions were incubated at 20–22 °C for the specified times. All of the experiments were done in duplicate.

FIGURE 6.

Proteins that contain long acidic stretches are abundant in the yeast proteome, suggesting that the paradigm of Cdc34-SCF represents a common mechanistic solution for facilitating protein assembly. Each protein sequence in the yeast proteome was searched for peptides of lengths between 15 and 25 residues that contain at least 60% acidic residues (A), 70% acidic residues (B), or 80% acidic residues (C).

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The acidic tail of the Cdc34 ubiquitin-conjugating enzyme functions in both binding to and catalysis with ubiquitin ligase SCFCdc4 - PubMed (original) (raw)