In situ measurements of the pH of mammalian peroxisomes using the fluorescent protein pHluorin.

摘要: Peroxisomes are metabolically active organelles that participate in the oxidation of long-chain fatty acids and biosynthesis bile acids, cholesterol, ether phospholipids. Even though maintenance a stable acid-base milieu is essential for proper peroxisomal function, determination pH (pHp) remains inconclusive, little known about its regulation. To measure intact peroxisomesin situ, we used peroxisome-specific carboxyl-terminal targeting sequence, SKL, to deliver pH-sensitive mutant green fluorescent protein (pHluorin-SKL) selectively into peroxisomes. Proper was verified by colocalization with marker catalase. were visualized imaging fluorescence microscopy, ratiometric measurements combined calibration using ionophores or null-point method estimate pHp. The pHp between 6.9 7.1, resembling cytosolic pH. Manipulation cells after permeabilization plasmalemma streptolysin O revealed changed parallel, suggesting membrane highly permeable H+ (equivalents). We conclude peroxisomes do not regulate their independently, but instead large permeability effectively connects them buffer reservoir cytoplasm homeostatic mechanisms control