Candida Infection Biology – fungal armoury, battlefields ... - FINSysB
Candida Infection Biology – fungal armoury, battlefields ... - FINSysB
Candida Infection Biology – fungal armoury, battlefields ... - FINSysB
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Poster number: 14<br />
Functional analysis of H-loop residues of Cdr1p, an ABC<br />
transporter of human <strong>fungal</strong> pathogen <strong>Candida</strong> albicans.<br />
Antresh Kumar 1 and Rajendra Prasad 1<br />
1 School of Life Sciences, Jawaharlal Nehru University, New Delhi, INDIA<br />
Nucleotide Binding Domains (NBDs) of multidrug transporter of <strong>Candida</strong> albicans,<br />
Cdr1p possess unique divergent amino acids in their conserved motifs. For<br />
example, NBD1 possesses divergent H-loop (IYQ) while the same motif is<br />
conserved (IHQ) in NBD2.<br />
We report here the contribution of these conserved and divergent H-loop motifs of<br />
Cdr1p in ATP catalysis and drug transport. For this, we mutagenized two residues<br />
of divergent (IYQ) and conserved H-loop region (IHQ) either by replacing them with<br />
alanines or replacing residues with equiposition residues of another H-loop. These<br />
mutations were introduced into GFP-tagged Cdr1p which was stably overexpressed<br />
at the PDR5 locus in a heterologous host S. cerevisiae mutant strain, AD1-8u - which<br />
lacks seven major ABC transporters.<br />
All the Cdr1p mutant variants of H-loop region were properly expressed and<br />
localized to the cell surface similar to wild-type protein. Alanine mutants of H-loop<br />
region, Y361A in NBD1 has no effect on Cdr1p function and showed normal R6G<br />
transport and ATPase activity whereas corresponding mutation (H1059A) in NBD2<br />
abolished R6G transport without any significant loss of ATPase activity. Moreover,<br />
cells expressing mutation like Q362A severely abrogated Cdr1p function while<br />
Q1060A mutation in NBD2 showed no effect on Cdr1p function and measured<br />
normal transport and ATPase activity similar to wild type. Though, Arginine<br />
substitution of glutamine residue present in H-loop region of Cdr1p (Q362R and<br />
Q1060R) displayed deferential effect on Cdr1 transport. These results suggest that<br />
not only the conserved residue like H1059 but divergent Q362 is also crucial for<br />
Cdr1p function.<br />
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