Functional Characterization of the P‐Type ATPases ATP13A3 and ATP13A4 as Plasma Membrane Polyamine Transporters

Polyamines are ubiquitous polycations involved in diverse cellular functions, including proliferation, differentiation, and survival. While endogenous biosynthesis can maintain the intracellular levels of polyamines, uptake becomes critical during high metabolic demand. Despite the evidence for ATP‐dependent polyamine uptake systems in the plasma membrane, their molecular characteristics remain unclear. We found that two human P5B ATPases—ATP13A3 and ATP13A4—were localized to the plasma membrane and directly mediated the uptake of putrescine and spermidine in an ATP‐dependent manner. Using ATP13A3 and ATP13A4 heterologously expressed in COS‐7 cells, we demonstrated that they mediate the saturable, ATP‐dependent uptake of polyamines with distinct kinetic profiles, consistent with the characteristics of known uptake systems in mammalian cells. Site‐directed mutagenesis of the conserved ATPase motif abolished uptake, confirming the requirement of ATP hydrolysis. The immunofluorescence analysis in COS‐7 cells transiently expressing FLAG‐tagged ATP13A3 or ATP13A4 revealed that both transporters localize to the plasma membrane. RNA interference in HepG2 cells to suppress endogenous ATP13A3 and ATP13A4 reduced polyamine uptake, supporting the physiological relevance of these transporters. It was also found that ATP13A3 is ubiquitously expressed and has moderate affinity for polyamines, whereas ATP13A4 is more specific tissue‐restricted and has higher affinity for polyamines, particularly spermidine. These findings establish ATP13A3 and ATP13A4 as the first P‐type ATPases shown to transport small organic solutes for cellular uptake at the plasma membrane. This study elucidates the roles of these P‐type ATPases in polyamine homeostasis. Furthermore, given the emerging associations of ATP13A3 and ATP13A4 with neurological and metabolic disorders, these transporters are promising therapeutic targets in polyamine‐related diseases.