The Distribution Of AMPK In Mitochondria And The Effect Of Mitochondrial AMPKα1on Cellular Autophagy

Background and objective：AMP-activated protein kinase (AMPK) serves as a fuel sensor that plays animportant role in regulating energy metabolism in all cells under physiological andpathological circumstances. AMPK is activated under stress, when cells sense energycrisis concurrent with decreases in ATP levels and increases in AMP or the ratio ofAMP to ATP. Such pathological conditions as metabolic syndrome and cancer areaccompanied by deregulated energy metabolism and suppression of AMPK, underwhich pharmacological activation of AMPK tends to tune it to a normal level. Assuch, AMPK emerges as a promising therapeutic target for these disorders. Theregulation of energy homeostasis by AMPK is closely related to the function ofmitochondrion, which acts as an energy plant and is involved in both physiologicaland pathological processes. Mounting evidence has demonstrated that mitochondriacan regulate AMPK activity and vice versa, AMPK also regulates mitochondrialfunction in various aspects. It has never been documented whether AMPK directlybinds to mitochondrion, thereby regulating cell function. The present study aims toaddress this question and ascribe specific cellular function to mitochondrial AMPK.Methods：1. Determine mitochondrial localization of AMPK. Biochemical fractionationfollowed by Western blot and immunofluorescence were used to localize AMPK tomitochondrion. To determin domains responsible for mitochondrial binding, differentmutants of AMPK α1subunit were expressed in HEK293T cells and their ability toassociate with mitochondria was examined by biochemical methods.2. Characterize function of mitochondrial AMPK. Toward this end,constitutively active mutant of AMPK α1subunit was engineered with amitochondrial targeting peptide followed by a Flag epitope at its aminoterminus inlentiviral vector and a lung adenocarcinoma cell line, A549, was infected with therecombinant virus expressing the AMPK mutant. The effect of mitochondrial AMPK on autophagy induction was then examined.Results：1. Endogenous AMPK including both α and β subunits is localized tomitochondria in addition to other subcellular compartments;2. The mitochondria binding site on AMPK α1subunit is localized to itscarboxyl terminal portion between aa392and548, overlapping with the bindingsite for the subunit, suggesting that the α1subunit directly associates withmitochondrion or indirectly through the subunit.3. Targeting active mutant of AMPK to mitochondria enhances autophagyinduced by glucose deprivation, suggesting that the mitochondrial AMPK plays arole in autophagy.Conclusion：1. AMPK exists in mitochondrion and The mitochondria binding site on AMPKα1subunit is localized to its carboxyl terminal portion between aa392and548.2. Mitochondriial AMPK plays an important role in regulating autophagy,sugestingAMPK can protect cells under no glucose.