Two high affinity nucleoside transporters in Leishmania donovani

Aronow, Bruce J.; Kaur, Kiran; McCartan, K; Ullman, Buddy

doi:10.1016/0166-6851(87)90066-1

Cited by 127 publications

(67 citation statements)

References 27 publications

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“…Transport Capabilities of the Second-site Suppressor Clones-Nucleoside transport measurements in yeast were performed by a previously described oil-stop method with some modifications (20). YPH499 transformants were grown overnight in SC trpϪ media after which they were diluted to an absorbance at 600 nm (A 600 ) of 0.25 per ml.…”

Section: Methodsmentioning

confidence: 99%

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

Arastu‐Kapur¹,

Arendt²,

Purnat

et al. 2005

Journal of Biological Chemistry

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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.

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Section: Methodsmentioning

confidence: 99%

“…Nucleoside transport measurements in L. donovani promastigotes were performed by the oil-stop method described previously (20 (21).…”

Section: Methodsmentioning

confidence: 99%

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

Arastu‐Kapur¹,

Arendt²,

Purnat

et al. 2005

Journal of Biological Chemistry

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“…Transport Assays-Nucleoside transport measurements were performed by a previously described oil-stop method (15 (16).…”

Section: Generation and Expression Of Site-directed Mutants-mutationsmentioning

confidence: 99%

Functional Analysis of an Inosine-Guanosine Transporter from Leishmania donovani

Arastu‐Kapur¹,

Ford²,

Ullman

et al. 2003

Journal of Biological Chemistry

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Equilibrative nucleoside transporters encompass two conserved, charged residues that occur within predicted transmembrane domain 8. To assess the role of these "signature" residues in transporter function, the Asp 389 and Arg 393 residues within the LdNT2 nucleoside transporter from Leishmania donovani were mutated and the resultant phenotypes evaluated after transfection into ⌬ldnt2 parasites. Whereas an R393K mutant retained transporter activity similar to that of wild type LdNT2, the R393L, D389E, and D389N mutations resulted in dramatic losses of transport capability. Tagging the wild type and mutant ldnt2 proteins with green fluorescent protein demonstrated that the D389N and D389E mutants targeted properly to the parasite cell surface and flagellum, whereas the expression of R393L at the cell surface was profoundly compromised. To test whether Asp 389 and Arg 393 interact, a series of mutants was generated, D389R/R393R, D389D/R393D, and D389R/ R393D, within the green fluorescent protein-tagged LdNT2 construct. Although all of these ldnt2 mutants were transport-deficient, D389R/R393D localized properly to the plasma membrane, while neither D389R/ R393R nor D389D/R393D could be detected. Moreover, a transport-incompetent D389N/R393N double ldnt2 mutant also localized to the parasite membrane, whereas a D389L/R393L ldnt2 mutant did not, suggesting that an interaction between residues 389 and 393 may be involved in LdNT2 membrane targeting. These studies establish genetically that Asp 389 is critical for optimal transporter function and that a positively charged or polar residue at Arg 393 is essential for proper expression of LdNT2 at the plasma membrane.

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“…Cells were washed twice in assay buffer (30 mM MES, pH 5.6, 2% glucose), and resuspended at 20 OD 600 /ml in the same buffer. Uptake of [2, H]-inosine (American Radiolabeled Chemicals, Inc., St. Louis, MO) was measured by the oil-stop method [25] …”

Section: Inosine Uptake Assays On Live Yeast Cellsmentioning

confidence: 99%

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

Arendt

Yates

et al. 2007

Analytical Biochemistry

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We describe an efficient method for generating highly functional membrane proteins with variant amino acids at defined positions that couples a modified site-saturation strategy with functional genetic selection. We applied this method to the production of a cysteine-less variant of the Crithidia fasciculata inosine-guanosine permease CfNT2, in order to facilitate biochemical studies using thiolspecific modifying reagents. Of ten endogenous cysteine residues in CfNT2, two cannot be replaced with serine or alanine without loss of function. High-quality single-and double-mutant libraries were produced by combining a previously reported site-saturation mutagenesis scheme based on the Quikchange method with a novel gel purification step that effectively eliminated template DNA from the products. Following selection for functional complementation in S. cerevisiae cells auxotrophic for purines, several highly functional non-cysteine substitutions were efficiently identified at each desired position, allowing the construction of cysteine-less variants of CfNT2 that retained wild-type affinity for inosine. This combination of an improved site-saturation mutagenesis technique and positive genetic selection provides a simple and efficient means to identify functional and perhaps unexpected amino acid variants at a desired position. Keywordscysteine-less; substituted cysteine accessibility method; membrane protein; site-saturation mutagenesis; genetic selection; Quikchange mutagenesis; equilibrative nucleoside transporter A detailed understanding of how a protein functions must include structural information at some level of resolution. While x-ray crystal structures of thousands of soluble proteins have been determined to date, crystals of membrane proteins that diffract to high resolution are notoriously difficult to obtain [1]. In the absence of any high-resolution structural information for many membrane protein families, biochemical methods provide a valuable means to intuit structure. Several of these methods make use of strategically placed cysteine residues because of their unique chemistry among the natural amino acids. For example, cysteines engineered into two transmembrane helices that can be cross-linked with bifunctional thiol-reactive reagents of varying lengths provide information about the distance between the two helices in the folded structure of the protein [2]. Also, the steric and chemical environment of a cysteine residue engineered at a position of interest can be probed by the substituted cysteine Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. [3][...

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Two high affinity nucleoside transporters in Leishmania donovani

Cited by 127 publications

References 27 publications

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

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

Functional Analysis of an Inosine-Guanosine Transporter from Leishmania donovani

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

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