Figure 3.

A part of Eno2p colocalizes with mitochondria. (A) Western analyses of mitochondrially associated proteins. After the treatment indicated below the picture, the mitochondrial pellets or the total material were analyzed, as indicated. The antibodies used were Aco1p, aconite hydratase, 85 kDa, matrix (MM) localization; Tom70p, 70 kDa, outer membrane (MOM) associated; and CCH1HLp, Cytochrome c heme lyase, 30 kDa, inner membrane space (IMS) protein. Aco1p and CCH1HLp are known to be resistant to carbonate treatment (Diekert et al. 2001). (B,C) In vitro assay of mitochondrial localization using reticulocyte lysate-synthesized [³⁵S]-labeled proteins incubated with isolated organelles. (B) Autoradiographs of gel-fractionated proteins after treatment of mitochondria as indicated. (I) Input; (SW) swelling; (NaCl) treatment with 200 mM of salt; (Na₂CO₃) washing with 200 mM carbonate; (Triton X-100) treatment with 10% detergent; (P) pellet; (S) supernatant. (C) Autoradiograph of the gel-fractionated proteins after treatment of the mitochondria with 50 μg/mL of proteinase K (PK). Mitochondrially imported preMsk1p was used as a reference. (D) Analysis of Eno2p localization in yeast cells by confocal microscopy. Eno2p was YFP-labeled, mitochondria were localized by mito-tracker red. Zones of colocalization are indicated by white arrows on the merge image. (E) Assay for Eno1/2p interaction with immobilized phospholipids. (LPA) Lisophosphatidic acid; (LPC) lisophosphocholine; (PI) phosphatidyl-inositol; (PI3/4/5) phosphatidyl-inositol-3/4/5-phosphate; (PE) phosphatidylethanolamine; (PC) phosphatidylcholine; (S1P) sphingosine-1-phosphate; (PI3, 4/3,5/4,5P2) phosphatidyl-inositol-3,4/3,5/4,5-diphosphate; (PI3, 4,5P3) phosphatidyl-inositol-3,4,5-triphosphate; (PA) phosphatidic acid; (PS) phosphatidylserine.