A conserved cysteine residue is involved in disulfide bond formation between plant plasma membrane aquaporin monomers

Abstract

AQPs (aquaporins) are conserved in all kingdoms of life and facilitate the rapid diffusion of water and/or other small solutes across cell membranes. Among the different plant AQPs, PIPs (plasma membrane intrinsic proteins), which fall into two phylogenetic groups, PIP1 and PIP2, play key roles in plant water transport processes. PIPs form tetramers in which each monomer acts as a functional channel. The intermolecular interactions that stabilize PIP oligomer complexes and are responsible for the resistance of PIP dimers to denaturating conditions are not well characterized. In the present study, we identified a highly conserved cysteine residue in loop A of PIP1 and PIP2 proteins and demonstrated by mutagenesis that it is involved in the formation of a disulfide bond between two monomers. Although this cysteine seems not to be involved in regulation of trafficking to the plasma membrane, activity, substrate selectivity or oxidative gating of ZmPIP1s (Zm is Zea mays), ZmPIP2s and hetero-oligomers, it increases oligomer stability under denaturating conditions. In addition, when PIP1 and PIP2 are co-expressed, the loop A cysteine of ZmPIP1;2, but not that of ZmPIP2;5, is involved in the mercury sensitivity of the channels.

Functional and molecular characteristics of rice and barley NIP aquaporins transporting water, hydrogen peroxide and arsenite

“…In mammalian aquaporins, a cysteine located exactly three-residues before the 2nd NPA motif in loop E was revealed to be the mercury-sensitive residue (Jung et al 1994). In plant PIPs, on one hand, a cysteine in loop A was involved in the disulfide bond formation between monomers and, in particular, the mercury-sensitivity in the case of maize PIP (Bienert et al 2012). On the other hand, mercury did not inhibit but increased water permeability via a non-cysteine mechanism in spinach SoPIP2;1 (Frick et al 2013).…”

Selective Regulation of Maize Plasma Membrane Aquaporin Trafficking and Activity by the SNARE SYP121

“…No signal was observed in eluted fractions from mock oocytes or oocytes expressing Venus:ZmSYP121 alone ( Figure 5A, lanes c and d). The 6His:ZmPIP2;5 dimeric form was due to the presence of a disulfide bond linking two Zm-PIP2;5 monomers (Bienert et al, 2012) and was detected as no reducing agent was added in the solubilization buffer. Interestingly, a YFP signal corresponding to Venus:ZmSYP121 was observed in the fraction coming from oocytes coexpressing 6His:ZmPIP2;5 and Venus:ZmSYP121 ( Figure 5B, lane b), demonstrating that both proteins were copurified.…”

A New LxxxA Motif in the Transmembrane Helix3 of Maize Aquaporins Belonging to the Plasma Membrane Intrinsic Protein PIP2 Group Is Required for Their Trafficking to the Plasma Membrane

“…2C; Bienert et al, 2012). This model showed that the TM3 was located on the outer surface of the ZmPIP2;5 tetramer.…”

“…Maize mesophyll protoplasts were transiently transformed with genetic constructs encoding the chimeric ZmPIPs fused downstream of the monomeric yellow fluorescent protein (mYFP; see the "Materials and Methods"). Previous studies demonstrated that tagging ZmPIPs with GFP variants at their N terminus does not affect their activity (Fetter et al, 2004;Besserer et al, 2012;Bienert et al, 2012), nor does it prevent N-terminal trafficking motifs to be functional (Zelazny et al, 2009). In plant cells, PM, ER, and cytosol are sometimes difficult to distinguish because of the presence of the central vacuole that occupies most of the cell volume and pushes all other cell compartments against the PM.…”