Mapping the membrane orientation of auxin homeostasis regulators PIN5 and PIN8 in Arabidopsis thaliana root cells reveals their divergent topology.

PIN proteins establish the auxin concentration gradient, which coordinates plant growth. PIN1-4 and 7 localized at the plasma membrane (PM) and facilitate polar auxin transport while the endoplasmic reticulum (ER) localized PIN5 and PIN8 maintain the intracellular auxin homeostasis. Although an antagonistic activity of PIN5 and PIN8 proteins in regulating the intracellular auxin homeostasis and other developmental events have been reported, the membrane topology of these proteins, which might be a basis for their antagonistic function, is poorly understood. In this study we optimized digitonin based PM-permeabilizing protocols coupled with immunocytochemistry labeling to map the membrane topology of PIN5 and PIN8 in Arabidopsis thaliana root cells. Our results indicate that, except for the similarities in the orientation of the N-terminus, PIN5 and PIN8 have an opposite orientation of the central hydrophilic loop and the C-terminus, as well as an unequal number of transmembrane domains (TMDs). PIN8 has ten TMDs with groups of five alpha-helices separated by the central hydrophilic loop (HL) residing in the ER lumen, and its N- and C-terminals are positioned in the cytoplasm. However, the topology of PIN5 comprises nine TMDs. Its N-terminal end and the central HL face the cytoplasm while its C-terminus resides in the ER lumen. Overall, this study shows that PIN5 and PIN8 proteins have a divergent membrane topology while introducing a toolkit of methods for studying membrane topology of integral proteins including those localized at the ER membrane.

The Nuts and Bolts of PIN Auxin Efflux Carriers.

The plant-specific proteins named PIN-FORMED (PIN) efflux carriers facilitate the direction of auxin flow and thus play a vital role in the establishment of local auxin maxima within plant tissues that subsequently guide plant ontogenesis. They are membrane integral proteins with two hydrophobic regions consisting of alpha-helices linked with a hydrophilic loop, which is usually longer for the plasma membrane-localized PINs. The hydrophilic loop harbors molecular cues important for the subcellular localization and thus auxin efflux function of those transporters. The three-dimensional structure of PIN has not been solved yet. However, there are scattered but substantial data concerning the functional characterization of amino acid strings that constitute these carriers. These sequences include motifs vital for vesicular trafficking, residues regulating membrane diffusion, cellular polar localization, and activity of PINs. Here, we summarize those bits of information striving to provide a reference to structural motifs that have been investigated experimentally hoping to stimulate the efforts toward unraveling of PIN structure-function connections.