A longevity protein, Lag2, interacts with SCF complex and regulates SCF function

Abstract

SCF-type E3-ubiquitin ligases control numerous cellular processes through the ubiquitin-proteasome pathway. However, the regulation of SCF function remains largely uncharacterized. Here, we report a novel SCF complex-interacting protein, Lag2, in Saccharomyces cerevisiae. Lag2 interacts with the SCF complex under physiological conditions. Lag2 negatively controls the ubiquitylation activities of SCF E3 ligase by interrupting the association of Cdc34 to SCF complex. Overexpression of Lag2 increases unrubylated Cdc53, whereas deletion of lag2, together with the deletions of dcn1 and jab1, results in the accumulation of Rub1-modified Cdc53. In vitro rubylation assays show that Lag2 inhibits the conjugation of Rub1 to Cdc53 in competition with Dcn1, which suggest that Lag2 down-regulates the rubylation of Cdc53 rather than promoting derubylation. Furthermore, Dcn1 hinders the association of Lag2 to Cdc53 in vivo. Finally, the deletion of lag2 combined with the deletion of either dcn1 or rub1 suppresses the growth of yeast cells. These observations thus indicate that Lag2 has a significant function in regulating the SCF complex by controlling its ubiquitin ligase activities and its rubylation cycle.

Figure 1

Lag2 interacts with the SCF complex under physiological conditions. (A, B) The interaction of the indicated F-box proteins (A) and Cdc53 (B) with Lag2 was monitored with a two-hybrid assay by expression of the LacZ-reporter gene. (C) The indicated yeast strains were cultured in YPD medium, collected in the exponential growth phase, and lysed using glass beads and a multibead shocker. Lysates were subjected to immunoprecipitation (IP) with anti-HA antibodies, and the resulting precipitates and total cell lysates were subjected to immunoblot analysis (IB) with anti-HA, anti-Cdc53, anti-Cdc4, anti-Skp1, and anti-Rbx1. (D) The indicated recombinant proteins were mixed and incubated on ice for 1 h. Binding of Lag2 and SCF^Cdc4 complex was detected by immunoprecipitation (IP) with anti-FLAG, and followed immunoblot analysis (IB) with anti-FLAG, anti-Cdc53, anti-Cdc4, anti-Skp1, and anti-Rbx1. (E) Lysates of the yeast strains expressing Lag2-3HA or Saf1-3HA were prepared as described in (C) and subjected to immunoprecipitation (IP) with anti-HA. The resulting precipitates and total cell lysates were subjected to immunoblot analysis (IB) with anti-HA and anti-Cdc53. (F) Lysates of Lag2-3HA yeast cells before or after incubation with control IgG and protein A beads at 4°C for 1 h were subjected to immunoblot analysis (IB) with anti-Cdc53. (G–I) Lysates of the yeast strains expressing the indicated proteins were prepared as described in (C) and subjected to immunoprecipitation (IP) with anti-HA antibodies. The resulting precipitates and total cell lysates were subjected to immunoblot analysis (IB) with anti-HA (G, H, I), and anti-Cdc53 (G), or anti-myc (G–I).

Figure 2

Lag2 inhibits the Rub1 conjugation to Cdc53 in vivo and in vitro. (A, B) The indicated yeast cells were cultured in YPD medium, collected in the exponential growth phase, and lysed by the TCA lysis method. Lysates were then subjected to immunoblot analysis (IB) with anti-Cdc53 (A, B), anti-HA (A), and anti-EF2 (A, B). (C) Deletion of Lag2 stimulates the rubylation of Cdc53. The indicated yeast strains were cultured in YPD medium, collected in the exponential growth phase, and lysed using glass beads and a multibeads shocker. For the Dcn1-dependent in vitro Rub1 conjugation assay, the lysates were mixed with Ula1, Uba3, and His₆-Rub1 in a 6-μl reaction in the presence of ATP. Reaction mixtures were incubated at 26°C for the indicated times. Rubylated native Cdc53 was detected by immunoblot analysis (IB) using anti-Cdc53. (D) The addition of recombinant Ubc12 activates the Rub1 conjugation to Cdc53. The extracts were prepared as described in (C). For the Dcn1-independent in vitro Rub1 conjugation assay, recombinant Ubc12 was added to the reactions shown in (C). (E) Recombinant Lag2 inhibits Rub1 conjugation to Cdc53. The lysate from the rub1Δ lag2Δ strain was subjected to the Dcn1-dependent in vitro Rub1 conjugation assay as in (C) with the indicated amount of recombinant Lag2. NS: non-specific band. (F, G) Lag2 inhibits Dcn1-dependent Rub1 conjugation to Cdc53. Lysates from the indicated strains were subjected to the Dcn1-dependent in vitro Rub1 conjugation assay as in (C) with the indicated amounts of Dcn1 (F) and Dcn1 and Lag2 (G).

Figure 3

Dcn1 interrupts the association of Lag2 to Cdc53. (A) Lysates of the indicated yeast cells were prepared as described in Figure 1C and subjected to immunoprecipitation (IP) with anti-HA or anti-FLAG. The resulting precipitates and total cell lysates were subjected to immunoblot analysis (IB) with anti-HA and anti-FLAG. (B, C) Indicated yeast cell lysates were prepared as described in Figure 1C and subjected to immunoprecipitation (IP) with anti-HA. The resulting precipitates and total cell lysates were subjected to immunoblot analysis (IB) with anti-HA (B, C), anti-Cdc53 (B, C), and anti-FLAG (C).

Figure 4

Deletion of lag2 combined with the deletion of either dcn1 or rub1 inhibits the growth of W303-1A cells. (A) The indicated yeast cells were grown to log phase in YPD medium. Cells were harvested and resuspended in YPD medium to an OD₆₀₀ of 0.4; 3 μl of 10-fold serial dilutions was spotted onto YPD plates, incubated for 24 h at the indicated temperature, and photographed. (B) The indicated cells were plated onto the YPD plates, grown for 6 days at 30°C, and photographed. (C) The diameter of the colonies shown in (B) was measured. Data are expressed as a percentage of the size of W303-1A wild-type cells and are the means±s.d. from 60 independent colonies. (D) Overnight cultures of the indicated cells were inoculated into YPD medium at a density of an OD₆₀₀=0.05. Samples were taken every 1 h to measure the cell density.

Figure 5

Genetic interactions of lag2, dcn1, and jab1 in SCF ts cells. (A) Deletion of lag2 lowers the restrictive temperature of cdc34-2 cells. The growth of the indicated yeast cells was compared as described in Figure 3A at the indicated temperature for 48 h. (B) Overexpression of Lag2 lowers the permissive temperature of skp1-11 cells. The indicated yeast strains were spotted onto YPD plates as shown in Figure 3A, incubated for 48 h at the indicated temperature, and photographed. (C) The combination of deletions of lag2, dcn1, and jab1 affects the ts phenotype of cdc34-2 cells. The indicated cells were spotted onto YPD plates as described in Figure 3A at the indicated temperature for 48 h.

Figure 6

Lag2 inhibits the activity of the SCF E3 ligase complex. (A) Deletion of lag2 partially rescues the viability of rub1Δ cdc34-2 cells. The indicated yeast cells were spotted onto YPD plates and cultured as described in Figure 4A at the indicated temperature for 48 h. (B–D) Lag2 negatively regulates polyubiquitin conjugation to phosphorylated Sic1. The indicated yeast strains were cultured in YPD medium, harvested in the exponential growth phase, and lysed using glass beads and a multibeads shocker. The cell extracts were incubated with phosphorylated Sic1 (Sic1-P), Uba1, Cdc34, and His₆-ubiquitin for the indicated times in the absence (B) of endogenous Lag2 or presence (C, D) of recombinant Lag2. Ubiquitylation of Sic1 was detected by immunoblot analysis (IB) using anti-HPC4 (B–D) and anti-Cdc53 (C). (E) Deletion of Lag2 decreases the abundance of Aah1 in vivo. The indicated cells were cultured in YPD medium to an OD₆₀₀=6 and harvested. One half of the cells were lysed by the TCA lysis method. Then, extracts were subjected to immunoblot analysis (IB) with anti-HA, and the membrane was stained with Coomassie as a loading control. Total RNA was isolated from the other half of the cells. The abundance of mRNAs for Aah1 and Actin (internal standard) was determined by RT–PCR. PCR of Aah1 without RT reaction was used for negative control. (F) Deletion of Lag2 decreases the stability of Sic1 in vivo. The indicated cells were cultured in YPD medium to an OD₆₀₀=0.5, and then in YP raffinose medium for 1 h. Cells were arrested by 2 μl/ml α factor for 2 h, and then the expression of Sic1 was induced by YPG for 2 h. The medium was changed back to YPD. Yeast cells were harvested and lysed by the TCA lysis method as the indicated time interval. Extracts were subjected to immunoblot analysis (IB) with anti-FLAG, and the membrane was stained with Coomassie as a loading control. (G) Deletion of Lag2 decreases the stability of Cln2 in vivo. The indicated cells were cultured in YPD medium to an OD₆₀₀=0.4, and then the expression of Cln2 was inducted in YPG medium for 5 h. The medium was changed back to YPD. Turnover of Cln2 was examined with the same method as shown in (F).

Figure 7

Lag2 dose dependently inhibits the binding of Cdc34 to SCF^Cdc4 complex in vitro. (A) The indicated recombinant proteins were mixed and incubated on ice for 1 h. Binding of Cdc34 and SCF^Cdc4 complex was detected by GST pull-down analysis and followed immunoblot analysis (IB) with anti-Cdc53 and anti-GST. (B) Ni²⁺-agarose beads containing Cdc53/His₆-Rbx1 complex was mixed with the indicated amount of His₆-Lag2 at 4°C for 1 h. After washing, bound proteins were separated by SDS/PAGE and subjected to immunoblot analysis (IB) with anti-Cdc53 and anti-His.