Dataset Information

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

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.

SUBMITTER: Hamouda NN

PROVIDER: S-EPMC7948421 | biostudies-literature |

REPOSITORIES: biostudies-literature

ACCESS DATA

Similar Datasets

Project description:Mammalian polyamine transporters have not thus far been biochemically characterized. Since essential carboxy groups in the polyamine carrier might participate in the transport process, the ability of two different carbodi-imides to affect [3H]spermidine uptake was assessed in Chinese hamster ovary cells. Both the hydrophobic 1,3-dicyclohexylcarbodi-imide (DCC) and the more polar 1-ethyl-3-(3-dimethylaminopropyl)carbodi-imide (EDC) irreversibly inhibited spermidine transport with EC50 values of 11 +/- 4 and 96 +/- 16 microM after 30 min at 22 degrees C respectively. Prior treatment with EDC in the absence of substrate decreased both the Vmax and K(m) for spermidine uptake in a time- and concentration-dependent manner. Spermidine-transport inactivation by EDC (1 mM) was temperature-dependent, with 60 and 90% inhibition observed after 10 min at 22 and 37 degrees C respectively. Spermine (10 microM) almost fully protected against spermidine-transport inactivation by EDC at 22 degrees C, and decreased the rate of inactivation at 37 degrees C by about 80%. Putrescine, spermidine and spermine were all effective in protecting against EDC-mediated inactivation of [3H]spermidine and [3H]putrescine uptake at 22 degrees C with EC50 values estimated at 10, 1 and less than 1 microM respectively. The nucleophile glycine ethyl ester (up to 50 mM) prevented the inhibition brought about by 1 mM EDC. Inhibition by 1 mM EDC was greater at pH 7.2 than at pH 5.8 (89 +/- 3 compared with 44 +/- 5%), whereas the converse was true for 100 microM DCC (81 +/- 3 compared with 92 +/- 5%). On the other hand, spermine did not protect against inactivation of spermidine uptake by DCC. Moreover, DCC, but not EDC, inhibited Na(+)-dependent amino acid uptake. The present data indicate that (i) EDC and DCC inhibit polyamine transport through distinct mechanisms, (ii) substrate binding occludes one or several carboxy groups lying in a polar environment of the carrier and (iii) these carboxyl residues might be activated by EDC to crosslink a neighbouring nucleophile side group, resulting in a conformation of the polyamine carrier which is inactive for transport.

Project description:In bacteria, limited phosphate availability promotes the synthesis of active uptake systems, such as the Pst phosphate transport system. To understand the mechanisms that facilitate phosphate accumulation in the cyanobacterium Nostoc punctiforme, phosphate transport systems were identified, revealing a redundancy of Pst phosphate uptake systems that exists across three distinct operons. Four separate PstB system components were identified. pstB1 was determined to be a suitable target for creating phenotypic mutations that could result in the accumulation of excessive levels of phosphate through its overexpression or in a reduction of the capacity to accumulate phosphate through its deletion. Using quantitative real-time PCR (qPCR), it was determined that pstB1 mRNA levels increased significantly over 64 h in cells cultured in 0 mM added phosphate and decreased significantly in cells exposed to high (12.8 mM) phosphate concentrations compared to the level in cells cultured under normal (0.8 mM) conditions. Possible compensation for the loss of PstB1 was observed when pstB2, pstB3, and pstB4 mRNA levels increased, particularly in cells starved of phosphate. The overexpression of pstB1 increased phosphate uptake by N. punctiforme and was shown to functionally complement the loss of PstB in E. coli PstB knockout (PstB-) mutants. The knockout of pstB1 in N. punctiforme did not have a significant effect on cellular phosphate accumulation or growth for the most part, which is attributed to the compensation for the loss of PstB1 by alterations in the pstB2, pstB3, and pstB4 mRNA levels. This study provides novel in vivo evidence that PstB1 plays a functional role in phosphate uptake in N. punctiforme IMPORTANCE: Cyanobacteria have been evolving over 3.5 billion years and have become highly adept at growing under limiting nutrient levels. Phosphate is crucial for the survival and prosperity of all organisms. In bacteria, limited phosphate availability promotes the synthesis of active uptake systems. The Pst phosphate transport system is one such system, responsible for the internalization of phosphate when cells are in phosphate-limited environments. Our investigations reveal the presence of multiple Pst phosphate uptake systems that exist across three distinct operons in Nostoc punctiforme and functionally characterize the role of the gene product PstB1 as being crucial for the maintenance of phosphate accumulation. We demonstrate that the genes pstB2, pstB3, and pstB4 show alterations in expression to compensate for the deletion of pstB1 The overall outcomes of this work provide insights as to the complex transport mechanisms that exist in cyanobacteria like N. punctiforme, allowing them to thrive in low-phosphate environments.

Dataset Information

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

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets