Supposed nitrite transport in A. nidulans has been reported previously. Pombeiro-Sponchiado
et al (1993) discussed nitrite uptake in a wild-type strain. Nitrite uptake in this strain would
not be as a result of one individual transporter but of the combined permeases (Wang et al., 2008). Additionally, the nihB gene was suggested to be a nitrite permease or involved in post-translational modifications of a nitrite permease (Pombeiro-Spondachio. R. C. et al., 1993). As shown here NitA is the only nitrite permease and is located on chromosome 3, whereas nihB is located on chromosome 1. The toxicity as discussed by Pombeiro- Sponchiado et al (1993) of nitrite at higher concentrations may be due to the destruction/inactivation of PABA, required by the strain, by nitrite in the medium. Therefore, their observations were of PABA auxotrophic mutants which failed to grow in the presence of high concentrations of nitrite and therefore, lack of PABA, in the medium.
7.4 Summary
This study represents the first investigation into a member of the FNT proteins on a molecular level. By comparison to homologous proteins, this study has shown the conservation of amino acids providing a secondary structure model for future work; in addition it has probed further into the functionality of conserved residues. Though no substrate specific signature
sequences were identified by the bioinformatic approach, it is hoped that this will be achieved in the future once further sequences have been published.
No distinct roles have been found for conserved charge residues, though it is thought that D88 could be involved in the formation of salt bridges (pending the discovery of a salt bridge partner), due to the charge and possible side chain size restrictions at this point. Disappointingly charge status was not defined for histidine at position H210, though it is hoped that with further mutagenesis a role will be found for this highly conserved residue. It is thought that of the conserved asparagine residues, position 173 holds the greatest potential for a role in nitrite trafficking; from this study it seems that there are space constraints at this position. However due to the tetrameric nature of this protein, work continues to establish the quaternary structure of mutant proteins that so far appear to not be expressed i.e. to ensure that where we see lack of expression on BN-PAGE is not just a lack of transfer of monomeric, dimeric or trimeric proteins. It is hoped that expression characteristics will be fine tuned in this regard for the final publication of this work.
Chapter Eight
Future work and perspectives
8.1 NrtA
The work described in Chapters Three, Four and Five discussed a site-directed mutagenesis approach which was used to target conserved amino acids of interest in NrtA. Altering every residue to all other possible amino acids would provide a vast amount of data, potentially indicating crucial amino acids. However, this approach would also produce several hundred useless mutants. It is therefore optimal to target residues thought to be important on the basis of conservation and presumptive functional importance, as performed here using complementary computational analyses.
The main aims of this thesis, regarding NrtA, were to understand motifs primarily in the first half of the protein and the putative phosphorylation sites in the central loop domain. Further mutagenesis, particularly on the nitrate signature and further putative phosphorylation sites, may provide vital information about residues involved with translocation processes. The next step would logically be to characterise the second nitrate signature of NrtA. This work is currently ongoing in our laboratory but is incomplete and thus is not discussed in detail here. It has been hypothesised that the nitrate signature of NrtA is primarily responsible for maintenance of structure and that N168 may perform another role. Cysteine scanning in vitro mutagenesis is currently underway on all Tms which seem to line the pore (i.e. all but Tm 3, 6, 9 and 12). This should provide novel insight into this uncharacterised region. Current assumptions are largely based on the crystal structure and mutagenesis work on other MFS proteins i.e. GlpT and LacY. It is hoped that with known dimensions, a role for the first nitrate signature could be defined in more detail, as a result of its position in relation to the other helices. In LacY, six critical residues for function were identified by cysteine scanning mutagenesis in combination with other site-directed mutants (Frillingos et al., 1997; Frillingos et al., 1998). This mutagenesis helped to elucidate information as regards helical packing, tilt, and ligand induced conformational changes. In addition, it allowed the development of a model for proton coupled β-galactosidase transport in LacY (Frillingos et al., 1997; Frillingos et al., 1998). This method may also be useful for the analysis of NitA, particularly as so little is known about its critical residues. Perhaps, like MFS proteins, this approach could help in the development of a transport model for NitA. Future work on the NrtA protein should target residues thought to be important in different facets of ion
translocation, e.g. in membrane targeting, tertiary and quaternary structure formation, structural maintenance, proton coupling, and substrate binding.
A crystal structure would of-course, be the ‘holy-grail’ of this work, though it would not provide all the information as regards substrate translocation, but more of a snap-shot of the permease. However, it would certainly direct future studies, providing new questions as to the structure and function of this protein. While awaiting crystal structures of NrtA, further modelling based on GlpT shall be pursued by our laboratory in silico using mutageneses results described here and data from other studies to tailor the GlpT template to nitrate transport. This will facilitate further investigations on the structural and functional mechanisms of NrtA. In particular, residues for proton binding and translocation have yet to be identified in NrtA; from electrophysiological studies, protons are known to be important for energy provision and NrtA function (Zhou et al., 2000b). Cysteine, scanning mutagenesis may hold the key to identifying these residues. The crystal structure of membrane proteins is notoriously difficult to achieve due to the metastable nature of transporters (Kaback et al., 2001; Miller, 2003; Fleishman et al., 2006). However, the publication of crystal structures of LacY and GlpT has provided an opportunity to develop crystallography in other membrane proteins. Again, in LacY the structure with bound substrate confirmed the model developed previously for translocation and defined interactive residues (Abramson et al., 2003b; Huang
et al., 2003). Work on crystallography is underway for NitA and NrtA and orthologous
proteins in my laboratory.
None of the putative phosphorylation residues targeted in Chapter Five provided an answer to whether or not NrtA is phosphorylated. The loop region was thought to be the most likely candidate for phosphorylation events to trigger conformational change. In AtNrt1.1, phosphorylation occurs at residue T101, much earlier in the protein, on an independent loop region separate from the central domain (Liu & Tsay, 2003). A further investigation should identify further putative phosphorylation sites elsewhere in the NrtA protein and knock-out the target phosphorylation residues. It is common to use mass spectrometry to identify phosphorylation sites in proteins; this could also be an interesting option should purified protein be available for NrtA. Also, an understanding of down regulation in NrtA would be interesting as it has been shown in YNT1 as this could explain the protein’s environmental response in fluctuating nitrate concentrations (Navarro et al., 2006).
Putative ubiquitination sites were also investigated in Chapter Five. It was thought that this post-translational modification, if occurring on NrtA, could be involved in regulation of
conformational change or protein degradation. Computational approaches taken here were unsuccessful at identifying PEST sequences thought to be crucial, as in YNT1 (Navarro et al., 2006). A specific investigation into the degradation of unwanted protein could give further depth to the understanding of nitrate transport and the NrtA permease.
As discussed in Chapter One, membrane proteins employ different mechanisms to permit membrane insertion and folding. While aromatic residues are known in the LamB channel for roles in substrate translocation (Dutzler et al., 1996; Denker et al., 2005) these residues are also known for their ability to aid membrane protein insertion (Kelkar & Chattopadhyay, 2006; Shank et al., 2006). It is therefore interesting to note the high degree of aromatic residues in the first Tm of NrtA. Until thorough mutageneses of this region are completed, the function of this unusual domain shall remain a matter of speculation. This work is currently underway by another PhD student in the group. Since Tm 1 forms part of the pore, the role of these residues could be with anion co-ordination and partial binding as the ion translocates as in LamB (Denker et al., 2005), or the function could solely be with membrane insertion as suggested here.