Substitution of a single amino acid residue in the aromatic/arginine selectivity filter alters the transport profiles of tonoplast aquaporin homologs

作者：Azad Abul Kalam^*; Yoshikawa Naoki; Ishikawa Takahiro; Sawa Yoshihiro; Shibata Hitoshi

来源：Biochimica et Biophysica Acta-Biomembranes, 2012, 1818(1): 1-11.

DOI：10.1016/j.bbamem.2011.09.014

• Aquaporin
• ar/R selectivity filter
• Transport selectivity
• TgTIP
• TIP homolog
• Water channel activity

Aquaporins are integral membrane proteins that facilitate the transport of water and some small solutes across cellular membranes. X-ray crystallography of aquaporins indicates that four amino acids constitute an aromatic/arginine (ar/R) pore constriction known as the selectivity filter. On the basis of these four amino acids, tonoplast aquaporins called tonoplast intrinsic proteins (TIPs) are divided into three groups in Arabidopsis. Herein, we describe the characterization of two group I TIP1s (TgTIP1:1 and TgTIP1:2) from tulip (Tulipa gesneriana). TgTIP1;1 and TgTIP1:2 have a novel isoleucine in loop E (LE2 position) of the ar/R filter; the residue at LE2 is a valine in all group I TIPs from model plants. The homologs showed mercury-sensitive water channel activity in a fast kinetics swelling assay upon heterologous expression in Pichia pastoris. Heterologous expression of both homologs promoted the growth of P. pastoris on ammonium or urea as sole sources of nitrogen and decreased growth and survival in the presence of H(2)O(2). TgTIP1;1- and TgTIP1;2-mediated H(2)O(2) conductance was demonstrated further by a fluorescence assay. Substitutions in the ar/R selectivity filter of TgTIP1;1 showed that mutants that mimicked the ar/R constriction of group I TIPs could conduct the same substrates that were transported by wild-type TgTIP1;1. In contrast, mutants that mimicked group II TIPs showed no evidence of urea or H(2)O(2) conductance. These results suggest that the amino acid residue at LE2 position is critical for the transport selectivity of the TIP homologs and group I Ill%26apos;s might have a broader spectrum of substrate selectivity than group II TIPs.

• 出版日期2012-1