Suborganelle sensing of mitochondrial cAMP-dependent protein kinase activity

Abstract

A fluorescent sensor of protein kinase activity has been developed and used to characterize the compartmentalized location of cAMP-dependent protein kinase activity in mitochondria. The sensor functions via a phosphorylation-induced release of a quencher from a peptide-based substrate, producing a 150-fold enhancement in fluorescence. The quenching phenomenon transpires via interaction of the quencher with Arg residues positioned on the peptide substrate. Although the cAMP-dependent protein kinase is known to be present in mitochondria, the relative amount of enzyme positioned in the major compartments (outer membrane, intermembrane space, and the matrix) of the organelle is unclear. The fluorescent sensor developed in this study was used to reveal the relative matrix/intermembrane space/outer membrane (85:6:9) distribution of PKA in bovine heart mitochondria.

Figure 1

Structures of the coumarin derivatives 1 – 3 of the general form fluorophore-Aoc-GRTGRRFSYP-amide. The fluorescent quencher Acid Green 27 (4) was identified from a library of forty-seven dyes (Table S1).

Figure 2

Fluorescence change as a function of incubation time of the PKA-catalyzed phosphorylation of sensors (A) 1, (B) 2, (C) 3, and (D) a nonphosphorylatable Ala-for-Ser control peptide 5 (coumarin-Aoc-GRTGRRFAYP-amide). Experiments were conducted in the presence of fluorescent quencher (4) and 14-3-3 domain. See “Optimization of enzyme-dependent fold-change” in the Material and Methods section for experimental details.

Figure 3

Fluorescence change as a function of mole fraction of sensor 1. See “Job plot for determination of stoichiometry” in the Material and Methods section for experimental details.

Figure 4

Reaction rate (nM of phosphopeptide formation/min) as a function of PKA concentration (pM).

Figure 5

Strategy for assessing PKA activity on the outer membrane (blue), in the intermembrane space (red) and in the matrix (yellow). PKA activity of intact mitochondria (A) is due to enzyme present on the outer membrane and in the intermembrane space. Trypsinized (i) mitochondria (B) lack outer membrane proteins and thus only intermembrane space PKA is present. Sonicated (ii) mitochondria (C) furnishes enzyme from all three compartments and thus represents total mitochondrial PKA.

Figure 6

Assessment of mitochondrial purity and extent of trypsinolysis by Western blot analysis. (A) The mitochondrial preparation was examined for the presence of ER (calnexin), plasma membrane (Na+/K+-ATPase), and cytoplasmic (glyceraldehyde phosphate dehydrogenase) proteins. A minimal amount of cytoplasmic contamination is observed. (B) Trypsin digestion of intact mitochondria. Untreated mitochondria (lane 1) and mitochondria treated with trypsin for 1 h at 37 °C (lane 2) were analyzed by Western blot for Hsp 60 and Tom 20, matrix and outer membrane markers, respectively, and the PKA catalytic subunit. Complete loss of Tom 20 upon trypsin exposure implies extensive trypsinolysis of the outer membrane surface. Complete retention of Hsp 60 implies that the mitochondrial matrix is preserved upon exposure to trypsin.

Scheme 1

Protein kinase-catalyzed phosphorylation of a fluorescently quenched peptide generates a fluorescent response in the presence of the phosphoSer-binding 14-3-3 domain.