Apg12p conjugates with Apg7p via a thioester bond. (A) Apg12p was coimmunoprecipitated with Apg7p. pAPG7myc-314 plasmid (CEN) was transformed into apg7
Δ cells producing HA-tagged Apg12p to express c-myc–tagged Apg7p. A transformant was designated as the YIT702 strain. The pRS314 vector was used as a control. Cell lysates were prepared as described in MATERIALS AND METHODS. A c-myc–tagged Apg7p was immunoprecipitated with Agarose beads conjugated with anti–c-myc mAb (9E10). The immunoprecipitates were subjected to SDS-PAGE on a 10% gel and transferred to a polyvinylidene difluoride membrane. Proteins were detected by Western blotting with αApg7N antibody (for Apg7p) and anti-HA mouse mAb (16B12; for HA-tagged Apg12p). (B) The coimmunoprecipitation of Apg12p with Apg7p is sensitive to DTT. A cell lysate of the YIT702 strain was prepared as described above. The lysate was boiled with (DTT+) or without 1 mM DTT (DTT−). Immunoprecipitation and Western blotting were performed as described above. (C) The higher molecular weight band of Apg7p in cells overexpressing Apg7p and Apg12p is sensitive to β-mercaptoethanol. Cells grown to early logarithmic phase in MVD medium were harvested and converted to spheroplasts in spheroplasting solution. The spheroplasts were harvested in 1.3 M sorbitol as a cushion, lysed with a 4× SDS sample buffer (Ausubel et al., 1995
) with a protease-inhibitor mixture (Sigma). The lysate was boiled for 5 min in the presence (βME+) or absence (βME−) of 3% β-mercaptoethanol. SDS-PAGE on a 7% gel and Western blotting wereperformed as described above. pYO324/pAPG12HA-426: strain YIT704 cells; pAPG7myc-314/pAPG12HA-316: strain YIT702 cells; pAPG7myc-324/pAPG12HA-426: strain YIT703 cells.
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