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.

Arabidopsis flippase ALA3 is required for adjustment of early subcellular trafficking in plant response to osmotic stress.

To compensate for their sessile lifestyle, plants developed several responses to exogenous changes. One of the previously investigated and not yet fully understood adaptations occurs at the level of early subcellular trafficking, which needs to be rapidly adjusted to maintain cellular homeostasis and membrane integrity under osmotic stress conditions. To form a vesicle, the membrane needs to be deformed, which is ensured by multiple factors, including the activity of specific membrane proteins, such as flippases from the family of P4-ATPases. The membrane pumps actively translocate phospholipids from the exoplasmic/luminal to the cytoplasmic membrane leaflet to generate curvature, which might be coupled with recruitment of proteins involved in vesicle formation at specific sites of the donor membrane. We show that lack of the AMINOPHOSPHOLIPID ATPASE3 (ALA3) flippase activity caused defects at the plasma membrane and trans-Golgi network, resulting in altered endocytosis and secretion, processes relying on vesicle formation and movement. The mentioned cellular defects were translated into decreased intracellular trafficking flexibility failing to adjust the root growth on osmotic stress-eliciting media. In conclusion, we show that ALA3 cooperates with ARF-GEF BIG5/BEN1 and ARF1A1C/BEX1 in a similar regulatory pathway to vesicle formation, and together they are important for plant adaptation to osmotic stress.

Arabidopsis Flippases Cooperate with ARF GTPase Exchange Factors to Regulate the Trafficking and Polarity of PIN Auxin Transporters.

Cell polarity is a fundamental feature of all multicellular organisms. PIN auxin transporters are important cell polarity markers that play crucial roles in a plethora of developmental processes in plants. Here, to identify components involved in cell polarity establishment and maintenance in plants, we performed a forward genetic screening of PIN2:PIN1-HA;pin2 Arabidopsis (Arabidopsis thaliana) plants, which ectopically express predominantly basally localized PIN1 in root epidermal cells, leading to agravitropic root growth. We identified the regulator of PIN polarity 12 (repp12) mutation, which restored gravitropic root growth and caused a switch in PIN1-HA polarity from the basal to apical side of root epidermal cells. Next Generation Sequencing and complementation experiments established the causative mutation of repp12 as a single amino acid exchange in Aminophospholipid ATPase3 (ALA3), a phospholipid flippase predicted to function in vesicle formation. repp12 and ala3 T-DNA mutants show defects in many auxin-regulated processes, asymmetric auxin distribution, and PIN trafficking. Analysis of quintuple and sextuple mutants confirmed the crucial roles of ALA proteins in regulating plant development as well as PIN trafficking and polarity. Genetic and physical interaction studies revealed that ALA3 functions together with the ADP ribosylation factor GTPase exchange factors GNOM and BIG3 in regulating PIN polarity, trafficking, and auxin-mediated development.