ATP13A3 is a major component of the enigmatic mammalian polyamine transport system

Norin Nabil Hamouda,Chris Van Den Haute,Roeland Vanhoutte,Ragna Sannerud,Mujahid Azfar,Rupert Mayer,Álvaro Cortés Calabuig,Johannes V Swinnen,Patrizia Agostinis,Veerle Baekelandt,Wim Annaert,Francis Impens,Steven H.L Verhelst,Jan Eggermont,Shaun Martin,Peter Vangheluwe

doi:10.1074/jbc.ra120.013908

Abstract

Polyamines, such as putrescine, spermidine, and spermine, are physiologically important polycations, but the transporters responsible for their uptake in mammalian cells remain poorly characterized. Here, we reveal a new component of the mammalian polyamine transport system using CHO-MG cells, a widely used model to study alternative polyamine uptake routes and characterize polyamine transport inhibitors for therapy. CHO-MG cells present polyamine uptake deficiency and resistance to a toxic polyamine biosynthesis inhibitor methylglyoxal bis-(guanylhydrazone) (MGBG), but the molecular defects responsible for these cellular characteristics remain unknown. By genome sequencing of CHO-MG cells, we identified mutations in an unexplored gene, ATP13A3, and found disturbed mRNA and protein expression. ATP13A3 encodes for an orphan P5B-ATPase (ATP13A3), a P-type transport ATPase that represents a candidate polyamine transporter. Interestingly, ATP13A3 complemented the putrescine transport deficiency and MGBG resistance of CHO-MG cells, whereas its knockdown in WT cells induced a CHO-MG phenotype demonstrated as a decrease in putrescine uptake and MGBG sensitivity. Taken together, our findings identify ATP13A3, which has been previously genetically linked with pulmonary arterial hypertension, as a major component of the mammalian polyamine transport system that confers sensitivity to MGBG.

Highlights

The polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), are abundant aliphatic polycations that are required for various cell functions including proliferation, differentiation, apoptosis, protein post-translational modifications, and ion channel regulation [1, 2]
Because no obvious SPD or SPM phenotype was observed in Chinese hamster ovary (CHO)-MG, we evaluated PUT uptake and toxicity
We identified the orphan P5B-ATPase, ATP13A3, as a major factor in the mammalian polyamine transport system (PTS) that is responsible for the polyamine-transport deficiency phenotype of CHO-MG cells, a frequently used model to characterize the mammalian PTS and polyamine transport inhibitors with therapeutic potential [19,20,21,22]

Summary

Introduction

The polyamines, putrescine (PUT), spermidine (SPD), and spermine (SPM), are abundant aliphatic polycations that are required for various cell functions including proliferation, differentiation, apoptosis, protein post-translational modifications, and ion channel regulation [1, 2]. We assessed the cellular polyamine uptake capacity using boron dipyrromethene (BODIPY)-conjugated SPD and SPM (Fig. 1B) that are taken up by the PTS [30] and transported by ATP13A2 [28], but both CHO cell lines exhibited similar levels of BODIPY–SPD and BODIPY–SPM uptake (Fig. 1B).

Results

Conclusion