Second-site Suppression of a Nonfunctional Mutation within the Leishmania donovani Inosine-Guanosine Transporter

Abstract

LdNT2 is a member of the equilibrative nucleoside transporter family, which possesses several conserved residues located mainly within transmembrane domains. One of these residues, Asp 389 within LdNT2, was shown previously to be critical for transporter function without affecting ligand affinity or plasma membrane targeting. To further delineate the role of Asp 389 in LdNT2 function, second-site suppressors of the ldnt2-D389N null mutation were selected in yeast deficient in purine nucleoside transport and incapable of purine biosynthesis. A library of random mutants within the ldnt2-D389N background was screened in yeast for restoration of growth on inosine. Twelve different clones were obtained, each containing secondary mutations enabling inosine transport. One mutation, N175I, occurred in four clones and conferred augmented inosine transport capability compared with LdNT2 in yeast. N175I was subsequently introduced into an ldnt2-D389N construct tagged with green fluorescent protein and transfected into a ⌬ldnt1/⌬ldnt2 Leishmania donovani knockout. GFP-N175I/D389N significantly suppressed the D389N phenotype and targeted properly to the plasma membrane and flagellum. Most interestingly, N175I increased the inosine K m by 10-fold within the D389N background relative to wild type GFP-LdNT2. Additional substitutions introduced at Asn 175 established that only large, nonpolar amino acids suppressed the D389N phenotype, indicating that suppression by Asn 175 has a specific size and charge requirement. Because multiple suppressor mutations alleviate the constraint imparted by the D389N mutation, these data suggest that Asp 389 is a conformationally sensitive residue. To impart spatial information to the clustering of second-site mutations, a three-dimensional model was constructed based upon members of the major facilitator superfamily using threading analysis. The model indicates that Asn 175 and Asp 389 lie in close proximity and that the second-site suppressor mutations cluster to one region of the transporter.

Residues Met89 and Ser160 in the Human Equilibrative Nucleoside Transporter 1 Affect Its Affinity for Adenosine, Guanosine, S6-(4-Nitrobenzyl)-mercaptopurine Riboside, and Dipyridamole

“…It is possible that these TMDs are in proximity in the packing of the ␣-helices that form the tertiary structure of hENT1, and our observation of the synergistic effect of simultaneous mutation of Met89 and Ser160 on NBMPR affinity is a result of this. Indeed, while this work was under review, Arastu-Kapur et al proposed a putative helical packing model of the ENT family in which TMDs 2 and 4 are adjacent and part of the putative aqueous pore (Arastu-Kapur et al, 2004).…”

Identification of the Intracellular Gate for a Member of the Equilibrative Nucleoside Transporter (ENT) Family

“…Support for structural commonality between ENTs and the major facilitator superfamily (MFS) of transporters was recently provided by site-directed mutagenesis studies of mammalian and protozoa ENTs, suggesting a common evolutionary origin (10) and similar packing of transmembrane (TM) helices around a solvent-accessible permeant binding site (11). These observations allowed the construction, by computational approaches, of putative tertiary structures of several protozoan ENTs, including Plasmodium falciparum PfENT1 (12), Trypanosoma brucei TbNBT1 (13), and Leishmania donovani LdNT2 (14). and LdNT1.1 (15).…”

Genetic selection for a highly functional cysteine-less membrane protein using site saturation mutagenesis

“…Saccharomyces cerevisiae (yeast) was chosen as a heterologous expression system for the functional evaluation and genetic selection of cysteine-substituted variants of CfNT2 for several reasons. First, yeast have been successfully exploited as a heterologous expression system for genetic and biochemical characterization of a variety of human and protozoan equilibrative nucleoside transporters [23,[27][28][29]. Second, they have no endogenous purine nucleoside transport activity and when mutant for ade2 are also purine auxotrophs, enabling functional complementation with a purine nucleoside transporter to be evaluated merely by manipulating the purine content of the media.…”