Fig. 1.

Sdh5 is required for respiration and interacts with Sdh1. (A) Mitochondria, mitoplasts generated by hypotonic swelling, and 1% Triton X-100–solubilized mitochondria from a strain expressing Sdh5-HA were treated with (+) or without (−) proteinase K and analyzed by immunoblotting with an untreated mitochondria control (UT). Mge1, Tim10, and Fzo1 are matrix, intermembrane space, and outer membrane proteins, respectively. (B) Soluble and membrane fractions of purified mitochondria (4) as in (A) were immunoblotted. Aco1, soluble matrix protein; Sdh1, membrane-associated matrix protein. (C) Serial dilutions of WT and sdh5Δ strains containing empty vector (EV) or a plasmid expressing Sdh5-HA were spotted on glucose or glycerol medium and grown at 30°C for 2 or 3 days, respectively. (D) Oxygen consumption in WT, sdh5Δ, and sdh1Δ strains grown to mid-log phase in raffinose media (±SD, n = 3 biological replicates). Similar results were obtained in glucose medium. (E) Tandem purification eluates (4) from a WT strain and a strain expressing Sdh5-His₆/HA were resolved with SDS-PAGE and visualized by silver staining (top panel) or immunoblot with antibodies to Sdh1 and HA (lower panels). (F) Immunoblot of purified mitochondria from WT, sdh1Δ, or sdh2Δ strains expressing Sdh5-HA. Porin, mitochondrial loading control.