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).