Role of a conserved glycine triplet in the NSS amino acid transporter KAAT1

Abstract

K+-coupled amino acid transporter 1 (KAAT1) belongs to the NSS family of solute transporters and it is expressed in the midgut and in salivary glands of Manduca sexta larvae. As more than 80% of family members, KAATI shows a stretch of three glycines (G85-G87) that according to the structure of the prototype transporter LeuT, is located close to the access of the permeation pathway. In this work the role of the triplet has been investigated by alanine and cysteine scanning methods in protein heterologously expressed in Xenopus laevis oocytes. All the mutants were functional but the surface expression level was reduced for G85A and G87A mutants and unaffected for G86A mutant. All presented altered amino acid uptake and transport associated currents in the presence of each of the cations (Na+, K+, Li+) that can be exploited by the wt. G87A mutant induced increased uncoupled fluxes in the presence of all the cations. Cross-linking studies, performed by the treatment of cysteine mutants with the oxidative complex Cu(Il)(l,10-phenanthroline)3, showed that limiting the flexibility of the region by covalent blockage of position 87, causes a significant reduction of amino acid uptake. Na+ protected G87C mutant from oxidation, both directly and indirectly. The conserved glycine triplet in KAAT1 plays therefore a complex role that allows initial steps of cation interaction with the transporter.

Structural Adaptation of Cold-Active RTX Lipase fromPseudomonassp. Strain AMS8 Revealed via Homology and Molecular Dynamics Simulation Approaches

“…The most consistent fluctuation across temperatures occurred at residues 393–476, which reside in the noncatalytic domain whereby the RTX repeats are present. The flexibility of the domain may occur because of the presence of high glycine residues, which are known to introduce flexibility in protein structure because they lack side chains [25]. The increased flexibility of LipAMS8 (which originated from the low stability of the noncatalytic domain) may imply that the noncatalytic domain is the crucial part of the LipAMS8 molecular structure.…”

Dictyostelium Nramp1, structurally and functionally close to mammalian DMT1 transporter, mediates phagosomal iron efflux

“…Groups of 8–10 oocytes were incubated for 60 min in uptake solution, washed in ice-cold uptake solution devoid of FeCl 2 , dissolved in 10% SDS solution and counted in a liquid scintillation counter ( Giovanola et al, 2012 ). Figures show mean±s.e.m.…”

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8

“…The flexibility of the non-catalytic domain might occur because of the presence of high Gly residues. Gly residues have introduced flexibility in the protein structure because they lack the side chain [23]. Here, AMS8 lipase also rich with Gly residue at the non-catalytic domain since the RTX nonapeptide repeats sequence consists of Gly and Asp-rich nonapeptide that constitutes a specific calcium-binding motif.…”