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Advisor(s)
Abstract(s)
Nodulin 26-like intrinsic proteins (NIPs) of the plant aquaporin family majorly facilitate the
transport of physiologically relevant solutes. The present study intended to investigate how substrate
selectivity in grapevine NIPs is influenced by the aromatic/arginine (ar/R) selectivity filter within
the pore and the possible underlying mechanisms. A mutational approach was used to interchange
the ar/R residues between grapevine NIPs (VvTnNIP1;1 withUniversidade de Lisboa, VvTnNIP6;1,
and VvTnNIP2;1 with VvTnNIP5;1). Their functional characterization by stopped-flow spectroscopy
in Saccharomyces cerevisiae revealed that mutations in residues of H2/H5 helices in VvTnNIP1;1 and
VvTnNIP6;1 caused a general decline in membrane glycerol permeability but did not impart the
expected substrate conductivity in the mutants. This result suggests that ar/R filter substitution could
alter the NIP channel activity, but it was not su cient to interchange their substrate preferences.
Further, homology modeling analyses evidenced that variations in the pore radius combined with
the di erences in the channel’s physicochemical properties (hydrophilicity/hydrophobicity) may
drive substrate selectivity. Furthermore, yeast growth assays showed that H5 residue substitution
alleviated the sensitivity of VvTnNIP2;1 and VvTnNIP5;1 to As, B, and Se, implying importance
of H5 sequence for substrate selection. These results contribute to the knowledge of the overall
determinants of substrate selectivity in NIPs
Description
Keywords
nodulin 26-like intrinsic proteins grapevine ar/R selectivity filter site-directed mutagenesis substrate selectivity homology modeling
Pedagogical Context
Citation
Int. J. Mol. Sci. 2020, 21, 6697
Publisher
MDPI