The Arabidopsis SAC9 enzyme is enriched in a cortical population of early endosomes and restricts PI(4,5)P2 at the plasma membrane

Abstract

Membrane lipids, and especially phosphoinositides, are differentially enriched within the eukaryotic endomembrane system. This generates a landmark code by modulating the properties of each membrane. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] specifically accumulates at the plasma membrane in yeast, animal, and plant cells, where it regulates a wide range of cellular processes including endocytic trafficking. However, the functional consequences of mispatterning PI(4,5)P2 in plants are unknown. Here, we functionally characterized the putative phosphoinositide phosphatase SUPPRESSOR OF ACTIN9 (SAC9) in Arabidopsis thaliana (Arabidopsis). We found that SAC9 depletion led to the ectopic localization of PI(4,5)P2 on cortical intracellular compartments, which depends on PI4P and PI(4,5)P2 production at the plasma membrane. SAC9 localizes to a subpopulation of trans-Golgi Network/early endosomes that are enriched in a region close to the cell cortex and that are coated with clathrin. Furthermore, it interacts and colocalizes with Src Homology 3 Domain Protein 2 (SH3P2), a protein involved in endocytic trafficking. In the absence of SAC9, SH3P2 localization is altered and the clathrin-mediated endocytosis rate is reduced. Together, our results highlight the importance of restricting PI(4,5)P2 at the plasma membrane and illustrate that one of the consequences of PI(4,5)P2 misspatterning in plants is to impact the endocytic trafficking.

The phosphoinositide signature guides the final step of plant cytokinesis

“…To identify the molecular processes leading to exclusion of PI(4,5)P 2 from the leading zone in late cytokinesis, we followed the subcellular localization of the plant-specific enzyme SAC9 which participates in the depletion of PI(4,5)P 2 at the plasma membrane during endocytosis ( 11 ). We observed that mCit-SAC9 is enriched at the phragmoplast leading zone (index > 1, Fig.…”

“…2a to 2c, yellow arrow, Extended Data 4, 5), suggesting a role of SAC9 in PI(4,5)P 2 depletion at the leading zone. To test this hypothesis, we analyzed the localization of a catalytically inactive variant of SAC9 (tdTOM-SAC9 C459A ), for which the signal in the cytoplasm is reduced compared with mCIT-SAC9, allowing imaging at higher resolution ( 11 ). Like mCit-SAC9, tdTOM-SAC9 C459A is enriched at the leading zone while PI(4,5)P 2 is depleted, leading to a clear mutual exclusion between the enzyme and its substrate along the cell plate (Fig.…”

“…We found that SAC9 progressively disappears from the fully attached cell plate upon its unilateral attachment to the mother cell. Consequently, SAC9 does not localize to the plasma membrane in the resulting interphasic cells ( 11 ). SAC9 and its substrate are mutually co-excluded spatially.…”

“…In order to test the relationship between the SAC9 localization and function and the patterning of its PI(4,5)P 2 substrate, we mutated the cysteine in the conserved C-x(5)-R-[T/S] catalytic motif found in all SAC domain-containing phosphoinositide phosphatase ( 11 ). Here, we showed that while sac9-3 is complemented with SAC9pro:mCIT-SAC9, SAC9pro:mCIT-SAC9 C459A was not sufficient to rescue the cytokinesis phenotypes observed in sac9-3 .…”

“…This landmark is set up by enzymes involved in phosphoinositide metabolism, in particular phosphatases and kinases that locally convert phosphoinositide pools (10). At the plasma membrane, phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is enriched, whereas PI(4,5)P2 is excluded from the endocytic pathway (11). This spatial distribution of PI(4,5)P2 allows the polar recruitment of proteins to orchestrate key processes, including membrane trafficking (12)(13)(14) and cytoskeleton remodeling (15)(16)(17).…”