<i>SLC29A1</i> /ENT1 and <i>SLC29A3</i> /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 <i>SLC29A3</i>/ENT3, which promotes macroautophagy/autophagy, cell surface-localized <i>SLC29A1</i>/ENT1 is capable of inhibiting autophagy by suppressing PRKAA/AMPK phosphorylation. Consistent with this, silencing <i>SLC29A1</i> induces autophagy, whereas silencing <i>SLC29A3</i> suppresses it. Treatment with adenosine (Ado), a shared substrate of <i>SLC29A1</i> and <i>SLC29A3</i>, triggers PRKAA/AMPK phosphorylation and autophagy in a concentration-dependent manner. This effect is PRKAA-dependent, as Ado fails to induce autophagy in <i>prkaa</i>-null cells. Mechanistically, elevated <i>SLC29A1</i> 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, <i>SLC29A1</i>'s regulatory effect is tied to its transport function, as pharmacological inhibition of <i>SLC29A1</i> transport enhances intracellular Ado accumulation, PRKAA/AMPK phosphorylation, and autophagy. Unlike <i>SLC29A3</i>, which modulates the MTOR pathway, <i>SLC29A1</i> does not affect MTOR signaling. Instead, it promotes BECN1-BCL2 interaction, thereby inhibiting autophagosome formation. Notably, autophagy itself differentially regulates <i>SLC29A1</i> and <i>SLC29A3</i> expression, with compensatory upregulation observed when either is modulated. Finally, <i>slc29a1</i>^-/- and <i>slc29a3</i>^-/- mice display autophagic proficiency and deficiency, respectively. These findings underscore a dynamic and reciprocal regulatory relationship between <i>SLC29A1</i> and <i>SLC29A3</i> in autophagy, offering new avenues for therapeutic modulation in autophagy-related disorders.