Fig. 2. sAC is localized and generates cAMP in mitochondria.

A) Mitochondria isolated from mouse liver after to two rounds of Nycodenz gradient purification. Lanes 2, 3, 4, and 5 were from the first round. Fractions 4.2, 4.3, 4.4 and 4.5 were from a second round of purification of fraction 4. Tim 23, mitochondrial marker. PDI, protein disulfide isomerase, ER rmarker. GAPDH, cytoplasmic marker,. Hom, homogenate B) Expression of sAC_t-HA in total cell homogenate of transiently transfected COS cells and in crude non-treated (n.t) mitochondrial fractions detected by R21 and HA antibodies. PK, Proteinase K; T+PK (Triton X-100 and PK). Hsp60 and Tim23 are markers of the matrix and the inner membrane, respectively. C) cAMP levels in intact mouse liver mitochondria in the presence of bicarbonate (HCO₃⁻) or KH7. Values are pmol cAMP per mg mitochondrial protein (n=3). D) Mitoplasts purity tested using protein markers for the different mitochondrial compartments: Hsp60 (matrix), COXI (inner membrane), cytochrome c (Cyt c, intermembrane space), Tom40 (outer membrane). M (intact mitochondria), Mp (mitoplasts), P-Mp (post-mitoplast fraction). E) Residual cAMP levels after 50pmol cAMP were added to a reaction mixture containing sonicated mitoplasts (mitoplast) or no mitoplasts (control), with and without PDE plus calmodulin (PDE), IBMX, or KH7. n=3 for each reaction. ***, p<0.0001.