Trafficking of plant plasma membrane aquaporins: multiple regulation levels and complex sorting signals.

Aquaporins are small channel proteins which facilitate the diffusion of water and small neutral molecules across biological membranes. Compared with animals, plant genomes encode numerous aquaporins, which display a large variety of subcellular localization patterns. More specifically, plant aquaporins of the plasma membrane intrinsic protein (PIP) subfamily were first described as plasma membrane (PM)-resident proteins, but recent research has demonstrated that the trafficking and subcellular localization of these proteins are complex and highly regulated. In the past few years, PIPs emerged as new model proteins to study subcellular sorting and membrane dynamics in plant cells. At least two distinct sorting motifs (one cytosolic, the other buried in the membrane) are required to direct PIPs to the PM. Hetero-oligomerization and interaction with SNAREs (soluble N-ethylmaleimide-sensitive factor protein attachment protein receptors) also influence the subcellular trafficking of PIPs. In addition to these constitutive processes, both the progression of PIPs through the secretory pathway and their dynamics at the PM are responsive to changing environmental conditions.

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.

Aquaporins play important roles in maintaining plant water status under challenging environments. The regulation of aquaporin density in cell membranes is essential to control transcellular water flows. This work focuses on the maize (Zea mays) plasma membrane intrinsic protein (ZmPIP) aquaporin subfamily, which is divided into two sequence-related groups (ZmPIP1s and ZmPIP2s). When expressed alone in mesophyll protoplasts, ZmPIP2s are efficiently targeted to the plasma membrane, whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). A protein domain-swapping approach was utilized to demonstrate that the transmembrane domain3 (TM3), together with the previously identified N-terminal ER export diacidic motif, account for the differential localization of these proteins. In addition to protoplasts, leaf epidermal cells transiently transformed by biolistic particle delivery were used to confirm and refine these results. By generating artificial proteins consisting of a single transmembrane domain, we demonstrated that the TM3 of ZmPIP1;2 or ZmPIP2;5 discriminates between ER and plasma membrane localization, respectively. More specifically, a new LxxxA motif in the TM3 of ZmPIP2;5, which is highly conserved in plant PIP2s, was shown to regulate its anterograde routing along the secretory pathway, particularly its export from the ER.

Selective regulation of maize plasma membrane aquaporin trafficking and activity by the SNARE SYP121.

Plasma membrane intrinsic proteins (PIPs) are aquaporins facilitating the diffusion of water through the cell membrane. We previously showed that the traffic of the maize (Zea mays) PIP2;5 to the plasma membrane is dependent on the endoplasmic reticulum diacidic export motif. Here, we report that the post-Golgi traffic and water channel activity of PIP2;5 are regulated by the SNARE (for soluble N-ethylmaleimide-sensitive factor protein attachment protein receptor) SYP121, a plasma membrane resident syntaxin involved in vesicle traffic, signaling, and regulation of K(+) channels. We demonstrate that the expression of the dominant-negative SYP121-Sp2 fragment in maize mesophyll protoplasts or epidermal cells leads to a decrease in the delivery of PIP2;5 to the plasma membrane. Protoplast and oocyte swelling assays showed that PIP2;5 water channel activity is negatively affected by SYP121-Sp2. A combination of in vitro (copurification assays) and in vivo (bimolecular fluorescence complementation, Förster resonance energy transfer, and yeast split-ubiquitin) approaches allowed us to demonstrate that SYP121 and PIP2;5 physically interact. Together with previous data demonstrating the role of SYP121 in regulating K(+) channel trafficking and activity, these results suggest that SYP121 SNARE contributes to the regulation of the cell osmotic homeostasis.

Expression and characterization of plasma membrane aquaporins in stomatal complexes of Zea mays.

Stomata, the microscopic pores on the surface of the aerial parts of plants, are bordered by two specialized cells, known as guard cells, which control the stomatal aperture according to endogenous and environmental signals. Like most movements occurring in plants, the opening and closing of stomata are based on hydraulic forces. During opening, the activation of plasma membrane and tonoplast transporters results in solute accumulation in the guard cells. To re-establish the perturbed osmotic equilibrium, water follows the solutes into the cells, leading to their swelling. Numerous studies have contributed to the understanding of the mechanism and regulation of stomatal movements. However, despite the importance of transmembrane water flow during this process, only a few studies have provided evidence for the involvement of water channels, called aquaporins. Here, we microdissected Zea mays stomatal complexes and showed that members of the aquaporin plasma membrane intrinsic protein (PIP) subfamily are expressed in these complexes and that their mRNA expression generally follows a diurnal pattern. The substrate specificity of two of the expressed ZmPIPs, ZmPIP1;5 and ZmPIP1;6, was investigated by heterologous expression in Xenopus oocytes and yeast cells. Our data show that both isoforms facilitate transmembrane water diffusion in the presence of the ZmPIP2;1 isoform. In addition, both display CO2 permeability comparable to that of the CO2 diffusion facilitator NtAQP1. These data indicate that ZmPIPs may have various physiological roles in stomatal complexes.

The LxxxA motif in the third transmembrane helix of the maize aquaporin ZmPIP2;5 acts as an ER export signal.

The subcellular localization of aquaporins belonging to the plasma membrane intrinsic protein (PIP) subfamily is highly regulated. In maize (Zea mays), ZmPIP1s are retained in the endoplasmic reticulum (ER) whereas ZmPIP2s are able to reach the plasma membrane (PM). We recently identified a new sorting determinant which is buried within the third transmembrane domain (TM3) of ZmPIP2;5. The Leu127 and Ala131 are required for the localization of ZmPIP2;5 in the PM and for its exit from the ER. However, when inserted into ZmPIP1;2, these amino acids were not sufficient to export the protein out of the ER. Here, we show that, when inserted into a truncated version of ZmPIP1;2 consisting only of its TM3 region, Leu127 and Ala131 of ZmPIP2;5 are able to partially bring the protein to the PM, demonstrating the active anterograde sorting function of this motif.

Insights into plant plasma membrane aquaporin trafficking.

Plasma membrane intrinsic proteins (PIPs) are plant aquaporins that facilitate the diffusion of water and small uncharged solutes through the cell membrane. Deciphering the network of interacting proteins that modulate PIP trafficking to and activity in the plasma membrane is essential to improve our knowledge about PIP regulation and function. This review highlights the most recent advances related to PIP subcellular routing and dynamic redistribution, identifies some key molecular interacting proteins, and indicates exciting directions for future research in this field. A better understanding of the mechanisms by which plants optimize water movement might help in identifying new molecular players of agronomical relevance involved in the control of cellular water uptake and drought tolerance.