Fig. 1.

Cqd1 is a mitochondrial inner membrane protein exposing its C-terminus to the intermembrane space. (A) Schematic representation of Cqd1. Violet, mitochondrial targeting sequence (MTS; amino acids 1–15); grey, predicted transmembrane domain (amino acids 125–141); turquoise, conserved protein kinase-like domain (amino acids 213–421). (B) Cqd1 exposes its C-terminus to the intermembrane space. Mitochondria isolated from a Cqd1–3xHA-expressing strain were treated with isotonic buffer, subjected to swelling by incubation in hypotonic buffer to disrupt the outer membrane (SW), or lysed using a Triton X-100 containing buffer (TX). Proteinase K (PK) was added as indicated. Samples were analyzed by SDS-PAGE and immunoblotting. Tom70 was used as a marker for the outer membrane, Tim50 for the inner membrane and Hep1 for the matrix. (C) Cqd1 is an integral membrane protein. Isolated mitochondria were subjected to alkaline extraction to separate soluble and membrane proteins. Soluble proteins present in the supernatant (S) and membrane proteins in the pellet (P) were analyzed by SDS-PAGE and immunoblotting. Blots are representative of at least three repeats.