Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter

Sebastian Pilsyk; Marzena Sieńko; Urszula Perlińska-Lenart; Jerzy Brzywczy; Maciej Przemysław Golan; Joanna Stefania Kruszewska

doi:10.18388/abp.2018_2620

Sebastian Pilsyk Institute of Biochemistry and Biophysics PAS Pawinskiego str. 5a 02-106 Warsaw POLAND
Marzena Sieńko Institute of Biochemistry and Biophysics PAS Pawinskiego str. 5a 02-106 Warsaw POLAND
Urszula Perlińska-Lenart Institute of Biochemistry and Biophysics PAS Pawinskiego str. 5a 02-106 Warsaw POLAND
Jerzy Brzywczy Institute of Biochemistry and Biophysics PAS Pawinskiego str. 5a 02-106 Warsaw POLAND
Maciej Przemysław Golan Opta – Tech Co. Ltd. KEN Av. 36/u211 02-797 Warsaw, Poland
Joanna Stefania Kruszewska Institute of Biochemistry and Biophysics PAS Pawinskiego str. 5a 02-106 Warsaw POLAND

DOI: https://doi.org/10.18388/abp.2018_2620

Abstract

AstA (alternative sulfate transporter) belongs to a large, but poorly characterized, Dal5 family of allantoate permeases of the Major Facilitator Superfamily. The astA gene has been cloned from an IAM 2006 Japanese strain of Aspergillus nidulans by complementation of a sulfate permease-deficient mutant. In this study we show that conserved lysine residues in Central Cytoplasmic Loop (CCL) of the AstA protein may participate in anion selectivity, and control kinetic properties of the AstA transporter. A three-dimensional model containing four clustered lysine residues was created, showing a novel substrate-interacting structure in Major Facilitator Superfamily transporters. The assimilation constant (K_τ) of wild type AstA protein is 85 μM, while V_max/mg of DW of AstA is twice that of the main sulfate transporter SB per mg of dry weight (DW) of mycelium (1.53 vs. 0.85 nmol/min, respectively). Amino acid substitutions in CCL did not abolish sulfate uptake, but affected its kinetic parameters. Mutants affected in the lysine residues forming the postulated sulfate-interacting pocket in AstA were able to grow and uptake sulfate, indicating that CCL is not crucial for sulfate transportation. However, these mutants exhibited altered values of K_τ and V_max, suggesting that CCL is involved in control of the transporter activity.

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