Protein motifs required for maize aquaporin trafficking to the plasma membrane

Aquaporins of the Plasma membrane Intrinsic Protein (PIP) subfamily are molecular actors of primary physiological importance. PIPs are subdivided in two groups, PIP1s and PIP2s. When expressed alone in maize (Zea mays; Zm) cells, ZmPIP2s reach the plasma membrane (PM) whereas ZmPIP1s are retained in the endoplasmic reticulum (ER). However, upon coexpression, ZmPIP1s interact with ZmPIP2s and are relocalized to the PM. An N-terminal diacidic motif is required for some ZmPIP2s to leave the ER but not all isoforms contain this motif, indicating that additional signals are necessary for the ER export of ZmPIPs. This work aimed at uncovering new sorting signals responsible for the anterograde routing of ZmPIPs along the secretory pathway. First, a microparticle-mediated DNA delivery technique was optimized for the transient transformation of maize leaf epidermal cells. This technique allowed cells within their native tissue to be transformed and proved to be a powerful system to study the subcellular localization of aquaporins. To identify new trafficking signals in ZmPIPs, a domain-swapping approach between ER-retained ZmPIP1s and PM-localized ZmPIP2s was undertaken. An LxxxA motif in the transmembrane domain 3 emerged as a critical governor of the progression of ZmPIP2;5 in the secretory pathway, as early as its export from the ER. The influence of N-terminal acidic sequences on the trafficking of ZmPIP2;6 and ZmPIP2;7 was analyzed. ZmPIP2;7 was not efficiently exported from the ER, but this process fully relied on its N-terminal DxE6 motif. On the other hand, mutation of the DxDE33 motif of ZmPIP2;6, which was well targeted to the PM, impaired Golgi export. Altogether, this work contributed to a better understanding of the subcellular journey of plant aquaporins.