SLC29A1 /ENT1 and SLC29A3 /ENT3 differentially regulate autophagy

Despite the well-established role of equilibrative nucleoside transporters (ENTs) in salvaging nucleosides for DNA synthesis, the presence of multiple ENT subfamilies within a single genome suggests putative, non-redundant functions in maintaining cellular homeostasis. In this study, we demonstrate that, in contrast to endolysosomal SLC29A3/ENT3, which promotes macroautophagy/autophagy, cell surface-localized SLC29A1/ENT1 is capable of inhibiting autophagy by suppressing PRKAA/AMPK phosphorylation. Consistent with this, silencing SLC29A1 induces autophagy, whereas silencing SLC29A3 suppresses it. Treatment with adenosine (Ado), a shared substrate of SLC29A1 and SLC29A3, triggers PRKAA/AMPK phosphorylation and autophagy in a concentration-dependent manner. This effect is PRKAA-dependent, as Ado fails to induce autophagy in prkaa-null cells. Mechanistically, elevated SLC29A1 expression promotes increased efflux and decreased intracellular retention of Ado, thereby attenuating PRKAA/AMPK activation and autophagic flux. However, this effect is contingent upon the metabolic state of the cells. Importantly, SLC29A1’s regulatory effect is tied to its transport function, as pharmacological inhibition of SLC29A1 transport enhances intracellular Ado accumulation, PRKAA/AMPK phosphorylation, and autophagy. Unlike SLC29A3, which modulates the MTOR pathway, SLC29A1 does not affect MTOR signaling. Instead, it promotes BECN1-BCL2 interaction, thereby inhibiting autophagosome formation. Notably, autophagy itself differentially regulates SLC29A1 and SLC29A3 expression, with compensatory upregulation observed when either is modulated. Finally, slc29a1−/− and slc29a3−/− mice display autophagic proficiency and deficiency, respectively. These findings underscore a dynamic and reciprocal regulatory relationship between SLC29A1 and SLC29A3 in autophagy, offering new avenues for therapeutic modulation in autophagy-related disorders.