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Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter

Piłsyk, Sebastian and Sieńko, Marzena and Brzywczy, Jerzy and Golan, Maciej and Perlinska-Lenart, Urszula and Kruszewska, Joanna S. (2018) Molecular characterization of central cytoplasmic loop in Aspergillus nidulans AstA transporter. Acta Biochimica Polonica, 65 (4). pp. 545-554. ISSN 1734-154X

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Official URL: https://ojs.ptbioch.edu.pl/index.php/abp/article/v...

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 (Kτ) of wild type AstA protein is 85 μM, while Vmax/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 Kτ and Vmax, suggesting that CCL is involved in control of the transporter activity.

Item Type:Article
Subjects:Q Science > QR Microbiology
Divisions:Laboratory of Fungal Glycobiology
ID Code:1650
Deposited By: Dr Sebastian Piłsyk
Deposited On:03 Dec 2018 12:51
Last Modified:17 Dec 2018 11:54

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