Increased Permeability of the Aquaporin SoPIP2;1 by Mercury and Mutations in Loop A.

Andreas Kirscht,Urban Johanson,Per Kjellbom,Sabeen Survery

doi:10.3389/fpls.2016.01249

Abstract

Aquaporins (AQPs) also referred to as Major intrinsic proteins, regulate permeability of biological membranes for water and other uncharged small polar molecules. Plants encode more AQPs than other organisms and just one of the four AQP subfamilies in Arabidopsis thaliana, the water specific plasma membrane intrinsic proteins (PIPs), has 13 isoforms, the same number as the total AQPs encoded by the entire human genome. The PIPs are more conserved than other plant AQPs and here we demonstrate that a cysteine residue, in loop A of SoPIP2;1 from Spinacia oleracea, is forming disulfide bridges. This is in agreement with studies on maize PIPs, but in contrast we also show an increased permeability of mutants with a substitution at this position. In accordance with earlier findings, we confirm that mercury increases water permeability of both wild type and mutant proteins. We report on the slow kinetics and reversibility of the activation, and on quenching of intrinsic tryptophan fluorescence as a potential reporter of conformational changes associated with activation. Hence, previous studies in plants based on the assumption of mercury as a general AQP blocker have to be reevaluated, whereas mercury and fluorescence studies of isolated PIPs provide new means to follow structural changes dynamically.

Highlights

Aquaporins (AQPs) are common in all domains of life and facilitate permeation of a wide range of small polar molecules through biological membranes (Abascal et al, 2014)
The isolated wild type (WT) protein was incubated for various length of time with different reducing agents prior to analysis by SDS-PAGE, in order to monitor any effect of potentially disrupted disulfide bridges on the migration pattern in acrylamide gels (Figure 1)
Dimeric bands were still visible in both mutants, they were significantly weaker as compared to the WT protein, which was expected from the destabilizing effect of reducing agents on dimers of the WT protein

Summary

Introduction

Aquaporins (AQPs) are common in all domains of life and facilitate permeation of a wide range of small polar molecules through biological membranes (Abascal et al, 2014). Members belonging to the plant subfamily of plasma membrane intrinsic proteins (PIPs) are permeable to water and show a more strict amino acid sequence conservation than AQPs in other subfamilies (Danielson and Johanson, 2010). Function and abundance of these proteins is tightly regulated and the high number of isoforms suggests a highly redundant system for water homeostasis (Johanson et al, 2001; Alexandersson et al, 2005, 2010). Structure and regulation of one particular member of the PIP subfamily from spinach (Spinacia oleracea), SoPIP2;1, which constitutes a dominating integral protein of the plasma membrane has been thoroughly studied (Johansson et al, 1996, 1998; Kukulski et al, 2005; Törnroth-Horsefield et al, 2006; Nyblom et al, 2009; Frick et al, 2013)

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