FIGURE 4:

Fzo1 degradation induced by vacuole dysfunction is Rsp5- and Mdm30-dependent. (A) Whole cell extracts from wild-type (WT) and mdm30Δ yeast expressing Fzo1-HA grown in the absence or presence of concanamycin A for 4 h were analyzed by Western blot with anti-HA and anti-Pgk1 antibodies. (B) Whole cell extracts from wild-type (WT), mdm30Δ, doxycycline-repressible RSP5 (tet_p-RSP5), and mdm30Δ tet_p-RSP5 yeast expressing Fzo1-HA grown in the absence or presence of concanamycin A for 4 h were analyzed as in A. All cells were treated with 20 μg/ml doxycycline for 16 h before ConcA addition to inhibit RSP5 expression. (C) Schematic of Fzo1 showing the location of the heptad repeats (HRN, HR1, and HR2), GTPase domain, and PY motif (LPHY). (D) Whole cell extracts from wild-type (WT) and mdm30Δ yeast expressing wild-type Fzo1-HA or PY motif mutated Fzo1-HA (fzo1-PY) grown in the absence or presence of concanamycin A for 4 h were analyzed by Western blot as in A.